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.     

Pathway-specific genetic attenuation of glutamate release alters select features of competition-based visual circuit refinement

A hallmark of mammalian neural circuit development is the refinement of initially imprecise connections by competitive activity-dependent processes. In the developing visual system retinal ganglion cell (RGC) axons from the two eyes undergo activity-dependent competition for territory in the dorsal lateral geniculate nucleus (dLGN). The direct contributions of synaptic transmission to this process, however, remain unclear. We used a genetic approach to reduce glutamate release selectively from ipsilateral-projecting RGCs and found that their release-deficient axons failed to exclude competing axons from the ipsilateral eye territory in the dLGN. Nevertheless, the release-deficient axons consolidated and maintained their normal amount of dLGN territory, even in the face of fully active competing axons. These results show that during visual circuit refinement glutamatergic transmission plays a direct role in excluding competing axons from inappropriate target regions, but they argue that consolidation and maintenance of axonal territory are largely insensitive to alterations in synaptic activity levels.     

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.     

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.     

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     

A quantitative study of synaptic interconnections in the dorsal lateral geniculate nucleus of the cat

Summary 2,700 synaptic contacts have been classified according to criteria described in an accompanying paper and the results summarized in tabular form. Only about 20% of the synaptic contacts in laminae A and A1 are formed by axons identifiable as retinogeniculate fibers. About 1/4 of these retinogeniculate synapses are axo-axonal. Approximately 45% of the contacts in these laminae are formed by axons tentatively identifiable as corticogeniculate fibers; about 35% by presumed intrageniculate fibers. Close to one half of the synapses occur in encapsulated synaptic zones, where grapelike dendritic appendages are related mainly to intrageniculate and retinogeniculate axons, and about half lie in interstitial zones, where corticogeniculate and some intrageniculate axons contact distal dendritic segments.Regions of the nucleus receiving from peripheral parts of the retina have relatively more corticogeniculate synapses, and have fewer intrageniculate synapses in the encapsulated zones than do regions receiving from the central parts of the retina.Most of the tissue in lamina B resembles the interstitial zones of laminae A and A1 and over 2/3 of the contacts in lamina B may prove to be corticogeniculate. The retinogeniculate fibers in this lamina are associated with relatively few other axons in simple, small encapsulated zones.Supported by Grant NB 06662 from the USPHS. The skillful technical assistance given by Mrs. E. Langer during the course of this work is gratefully acknowledged.     

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.     

Synaptic patterns in the dorsal lateral geniculate nucleus of the monkey

Summary Synaptic junctions are found in all parts of the nucleus, being almost as densely distributed between cell laminae as within these laminae.In addition to the six classical cell laminae, two thin intercalated laminae have been found which lie on each side of lamina 1. These laminae contain small neurons embedded in a zone of small neural processes and many axo-axonal synapses occur there.Three types of axon form synapses in all cell laminae and have been called RLP, RSD and F axons. RLP axons have large terminals which contain loosely packed round synaptic vesicles, RSD axons have small terminals which contain closely packed round vesicles and F axons have terminals intermediate in size containing many flattened vesicles.RLP axons are identified as retinogeniculate fibers. Their terminals are confined to the cell laminae, where they form filamentous contacts upon large dendrites and asymmetrical regular synaptic contacts (with a thin postsynaptic opacity) upon large dendrites and F axons. RSD axons terminate within the cellular laminae and also between them. They form asymmetrical regular synaptic contacts on small dendrites and on F axons. F axons, which also occur throughout the nucleus, form symmetrical regular contacts upon all portions of the geniculate neurons and with other F axons. At axo-axonal junctions the F axon is always postsynaptic.Supported by Grant R 01 NB 06662 from the USPHS and by funds of the Neurological Sciences Group of the Medical Research Council of Canada. Most of the observations were made while R. W. Guillery was a visiting professor in the Department of Physiology at the University of Montreal. We thank the Department of Physiology for their support and Mr. K. Watkins, Mrs. E. Langer and Mrs. B. Yelk for their skillful technical assistance.     

Rearrangement of Retinogeniculate Projection Patterns after Eye-Specific Segregation in Mice

It has been of interest whether and when the rearrangement of neuronal circuits can be induced after projection patterns are formed during development. Earlier studies using cats reported that the rearrangement of retinogeniculate projections could be induced even after eye-specific segregation has occurred, but detailed and quantitative characterization of this rearrangement has been lacking. Here we delineate the structural changes of retinogeniculate projections in the C57BL/6 mouse in response to monocular enucleation (ME) after eye-specific segregation. When ME was performed after eye-specific segregation, rearrangement of retinogeniculate axons in the dorsal lateral geniculate nucleus (dLGN) was observed within 5 days. Although this rearrangement was observed both along the dorsomedial-ventrolateral and outer-inner axes in the dLGN, it occurred more rapidly along the outer-inner axis. We also examined the critical period for this rearrangement and found that the rearrangement became almost absent by the beginning of the critical period for ocular dominance plasticity in the primary visual cortex. Taken together, our findings serve as a framework for the assessment of phenotypes of genetically altered mouse strains as well as provide insights into the mechanisms underlying the rearrangement of retinogeniculate projections.     

Parvalbumin,calbindin, and calretinin mark distinct pathways during development of monkey dorsal lateral geniculate nucleus

Immunocyochemical labeling was applied to follow the developmental changes in the calcium-binding proteins parvalbumin (PV), calbindin D28k (CaB), and calretinin (CaR) during fetal and infant development of Macaca monkey dorsal lateral geniculate nucleus (LGN). For all three proteins, LGN cell body and retinal ganglion cell (RGC) axon labeling patterns changed temporally and spatially over development, and many of these were LGN laminar specific. CaR+ and CaB+ cells were present at the youngest age studied, fetal day 55 (F55). After lamination of the LGN occurred between F90 and F115, CaR+ and CaB+ neurons were specific markers for the S, intercalated, and interlaminar layers. Double label immunocytochemistry showed that all CaR+ cells contained CaB, and none contained GABA. CaR+ cell bodies decreased in number soon after birth so that adult LGN contained only a very small number of CaR+ cells. These patterns and cell counts indicated that a downregulation of CaR had occurred in the CaB+ population. Although CaB+ cell density in S and interlaminar zones declined in the adult, cell counts indicated that this is due to dilution of a stable population into a much larger nucleus during development. PV+ cells appeared at F85 only within the putative magnocellular (M) and parvocellular (P) layers, and PV remained a marker for these layers throughout development. Fetal PV cells also contained GABA, indicating that they were LGN interneurons. After birth, GABA−/PV+ cell numbers increased dramatically throughout the whole nucleus so that by the end of the first year, P and M layers were filled with PV+ cells. Their number and size indicated that these were the LGN projection neurons. Beginning at F66, bundles of PV+ axons occupied the anterior-middle LGN and filled the optic tract. Up to F101, PV+ synaptic terminals were restricted to P layers, but after F132 labeling in M layers was heavier than in P layers. Axonal labeling for CaR began at F125. Prenatally CaR+ terminals were present mainly in P layers, whereas by postnatal 9 weeks labeling in M layers much exceeded P layers. Axonal labeling for CaB was present at F132, but CaB+ terminals were observed only after birth with labeling always heavier in M than P layers. By postnatal 9 weeks, PV, CaR, and CaB were colocalized in the same axons and terminals. These experiments indicated that during development and in the adult LGN, both CaR and CaB were markers for the LGN neurons in the S and intercalated pathway. CaR was present transiently while CaB persisted into adulthood. PV was a M and P layer marker first for interneurons and later for projection cells. The complex temporal developmental patterns found in this study suggested that viewing PV, CaB, and CaR simply as calcium-buffering proteins severely underestimates their functional roles during visual system maturation. © 1996 John Wiley & Sons, Inc.     

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.     

An instructive role for retinal waves in the development of retinogeniculate connectivity

A central hypothesis of neural development is that patterned activity drives the refinement of initially imprecise connections. We have examined this hypothesis directly by altering the frequency of spontaneous waves of activity that sweep across the mammalian retina prior to vision. Activity levels were increased in vivo using agents that elevate cAMP. When one eye is made more active, its layer within the LGN is larger despite the other eye having normal levels of activity. Remarkably, when the frequency of retinal waves is increased in both eyes, normally sized layers form. Because relative, rather than absolute, levels of activity between the eyes regulate the amount of LGN territory devoted to each eye, we conclude that activity acts instructively to guide binocular segregation during development.     

Unequal competition between axons for neuronal targets.

Precise wiring of the nervous system depends not only on a matching between neurons and their synaptic targets, but also upon competition between neurons for particular targets. Neurons in adult leeches regenerate synaptic connections with their usual neuronal targets in the central nervous system, selecting only those targets with which they connect during embryogenesis. Thus during development axons of nociceptive (N) sensory cells make contacts on the cell bodies of certain neurons in adjacent ganglia but not upon those same types of cells in their own ganglion. After injury the N cell axons accurately regenerate contacts on the appropriate target cells. An abnormal feature observed after injury is that N cell axons sprout and grow to make contacts upon cell bodies within their own ganglion. This is a consequence of the normal innervation of those cells having been removed, thereby eliminating the source of competition. Similar competition during embryogenesis may guide the formation of selective connections.     

Fine structure of the first optic ganglion (lamina) of the cockroach, Periplaneta americana

The structural organization of the first optic ganglion (lamina) of the cockroach (Periplaneta americana) was investigated by the use of light and electron microscopy. Each compound eye of the cockroach is composed of up to 2000 visual units (ommatidia) of the fused rhabdom type. The ommatidia themselves consist of eight receptor cells which terminate as axons in either the first or second optic ganglion. Three different short visual fibre types end in two separate strata in the lamina, and one long fibre type ends in the second optic ganglion. Monopolar second-order neurons with wide field branching patterns in the middle stratum of the first synaptic region have postsynaptic contacts with short visual fibres. Horizontal fibre elements with branching patterns at different levels of the lamina apparently form three horizontal plexuses with presynaptic and/or postsynaptic connections to first- and secondorder neurons. The lack of well-organized fibre cartridges containing a constant number of first and second order neurons in each fascicle and the presence of only unistratified wide field monopolar cells could represent, as compared to other insect orders, a primitive stage in the development of the first optic ganglion.     

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.     

Rapid plasticity of visual responses in the adult lateral geniculate nucleus

Compared to the developing visual system, where neuronal plasticity has been well characterized at multiple levels, little is known about plasticity in the adult, particularly within subcortical structures. We made intraocular injections of 2-amino-4-phosphonobutyric acid (APB) in adult cats to block visual responses in On-center retinal ganglion cells and examined the consequences on visual responses in the lateral geniculate nucleus (LGN) of the thalamus. In contrast to current views of retinogeniculate organization, which hold that On-center LGN neurons should become silent with APB, we find that ～50% of On-center neurons rapidly develop Off-center responses. The time course of these emergent responses and the actions of APB in the retina indicate the plasticity occurs within the LGN. These results suggest there is greater divergence of retinogeniculate connections than previously recognized and that functionally silent, nonspecific retinal inputs can serve as a substrate for rapid plasticity in the adult.     

Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish

In adult crayfish, Procambarus clarkii, motoneurons to a denervated abdominal superficial flexor muscle regenerate long-lasting and highly specific synaptic connections as seen from recordings of excitatory postsynaptic potentials, even when they arise from the ganglion of another crayfish. To confirm the morphological origins of these physiological connections we examined the fine structure of the allotransplanted tissue that consisted of the third abdominal ganglion and the nerve to the superficial flexor muscle (the fourth ganglion and the connecting ventral nerve cord were also included). Although there is considerable degeneration, the allotransplanted ganglia display intact areas of axon tracts, neuropil, and somata. Thus in both short (6–8 weeks) and long (24–30 weeks) term transplants approximately 20 healthy somata are present and this is more than the five axons regenerated to the host muscle. The principal neurite and dendrites of these somata receive both excitatory and inhibitory synaptic inputs, and these types of synaptic contacts also occur among the dendritic profiles of the neuropil. Axon tracts in the allotransplanted ganglia and ventral nerve cord consist largely of small diameter axons; most of the large axons including the medial and lateral giant axons are lost. The transplanted ganglia have many blood vessels and blood lacunae ensuring long-term survival. The transplanted superficial flexor nerve regenerates from the ventral to the dorsal surface of the muscle where it has five axons, each consisting of many profiles rather than a single profile. This indicates sprouting of the individual axons and accounts for the enlarged size of the regenerated nerve. The regenerated axons give rise to normal-looking synaptic terminals with well-defined synaptic contacts and presynaptic dense bars or active zones. Some of these synaptic terminals lie in close proximity to degenerating terminals, suggesting that they may inhabit old sites and in this way ensure target specificity. The presence of intact somata, neuropil, and axon tracts are factors that would contribute to the spontaneous firing of the transplanted motoneurons. © 1996 John Wiley & Sons, Inc.     

Axonal sprouts of the hypoglossal nerve implanted in the superior cervical ganglion of adult rats establish synaptic contacts under long-lasting GABA effect. An experimental degeneration study

Experimental degeneration was used in this study to determine if the hypoglossal nerve implanted already in the superior cervical ganglion of adult rat under GABA treatment has established morphologically-identifiable synapses with the dendrites of principal ganglion cells. The implanted hypoglossal nerve trunk was cut in a re-operation, and the ganglionic samples were studied by electron microscopy after 0, 6, 12, 24 and 48 h survival times. First signs of degenerative changes were found in the myelinated and non-myelinated axons alike, 6 h after axotomy. The fine-structural signs of degeneration resembled those of the preganglionic nerve fibres. Degenerating nerve terminals establishing synaptic contacts with the dendrites of the principal ganglion cells were also seen, indicating that the axonal sprouts of the implanted hypoglossal nerve established synaptic contacts with the ganglion cells. It remained, however, to be elucidated whether or not these synapses of the hypoglossal nerve are functionally active contacts while the preganglionic innervation is also present within the ganglion.     

Novel mouse model reveals distinct activity-dependent and -independent contributions to synapse development

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.     

Dynamics of retinal waves are controlled by cyclic AMP

Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.