A fibronectin-like molecule is present in the developing cat cerebral cortex and is correlated with subplate neurons.

The subplate is a transient zone of the developing cerebral cortex through which postmitotic neurons migrate and growing axons elongate en route to their adult positions within the cortical plate. To learn more about the cellular interactions that occur in this zone, we have examined whether fibronectins (FNs), a family of molecules known to promote migration and elongation in other systems, are present during the fetal and postnatal development of the cat's cerebral cortex. Three different anti-FN antisera recognized a single broad band with an apparent molecular mass of 200-250 kD in antigen-transfer analyses (reducing conditions) of plasma-depleted (perfused) whole fetal brain or synaptosome preparations, indicating that FNs are present at these ages. This band can be detected as early as 1 mo before birth at embryonic day 39. Immunohistochemical examination of the developing cerebral cortex from animals between embryonic day 46 and postnatal day 7 using any of the three antisera revealed that FN-like immunoreactivity is restricted to the subplate and the marginal zones, and is not found in the cortical plate. As these zones mature into their adult counterparts (the white matter and layer 1 of the cerebral cortex), immunostaining gradually disappears and is not detectable by postnatal day 70. Previous studies have shown that the subplate and marginal zones contain a special, transient population of neurons (Chun, J. J. M., M. J. Nakamura, and C. J. Shatz. 1987. Nature (Lond.). 325:617-620). The FN-like immunostaining in the subplate and marginal zone is closely associated with these neurons, and some of the immunostaining delineates them. Moreover, the postnatal disappearance of FN-like immunostaining from the subplate is correlated spatially and temporally with the disappearance of the subplate neurons. When subplate neurons are killed by neurotoxins, FN-like immunostaining is depleted in the lesioned area. These observations show that an FN-like molecule is present transiently in the subplate of the developing cerebral cortex and, further, is spatially and temporally correlated with the transient subplate neurons. The presence of FNs within this zone, but not in the cortical plate, suggests that the extracellular milieu of the subplate mediates a unique set of interactions required for the development of the cerebral cortex.

Involvement of subplate neurons in the formation of ocular dominance columns.

During development of the mammalian visual system, axon terminals of lateral geniculate nucleus (LGN) neurons, initially intermixed within layer 4 of the visual cortex, gradually segregate according to eye preference to form ocular dominance columns. In addition to LGN axons and layer 4 neurons, subplate neurons may also participate in interactions leading to column formation. Deletion of subplate neurons before the formation of ocular dominance columns prevents the segregation of LGN axons within layer 4. Thus, interactions between LGN axons and layer 4 neurons are not sufficient; subplate neurons are also required for formation of ocular dominance columns in the visual cortex.

Siamese cat: altered connections of visual cortex.

In siamese cats, each side of the brain receives a retinal input serving part of the ipsilateral visual field as well as the normal contralateral field representation. Both corticothalamic and cortico-cortical projections are systematically rearranged, but while one is retinotopically appropriate, the other fails to make a distinction between ipsilateral and contralateral fields. Different rules appear to govern the development of these two sets of connections.

Inhibition of ocular dominance column formation by infusion of NT-4/5 or BDNF.

During the development of the visual system of higher mammals, axons from the lateral geniculate nucleus (LGN) become segregated into eye-specific patches (the ocular dominance columns) within their target, layer 4 of the primary visual cortex. This occurs as a consequence of activity-dependent synaptic competition between axons representing the two eyes. The possibility that this competition could be mediated through neurotrophin-receptor interactions was tested. Infusion of neurotrophin-4/5 (NT-4/5) or brain-derived neurotrophic factor (BDNF) into cat primary visual cortex inhibited column formation within the immediate vicinity of the infusion site but not elsewhere in the visual cortex. Infusion of nerve growth factor, neurotrophin 3 (NT-3), or vehicle solution did not affect column formation. These observations implicate TrkB, the common receptor for BDNF and NT-4/5, in the segregation of LGN axons into ocular dominance columns in layer 4. Moreover, they suggest that in addition to their better known roles in the prevention of cell death, neurotrophins may also mediate the activity-dependent control of axonal branching during development of the central nervous system.

Transient morphological features of identified ganglion cells in living fetal and neonatal retina.

The function and morphology of retinal ganglion cells in the adult mammalian visual system has been well studied, but little is known about how the adult state is achieved. To address this question, the morphological changes that retinal ganglion cells undergo during development were studied. Ganglion cells were first identified by retrograde labeling with rhodamine latex microspheres deposited in retinorecipient targets in fetal and early postnatal cats. The structure of ganglion cells was then revealed by intracellular injection of Lucifer yellow in living retinas removed and maintained in vitro. As early as 2 weeks before birth, a morphologically diverse assortment of ganglion cells is present, some of which resemble the alpha, beta, and gamma classes found in the adult. However, in contrast to the adult, developing ganglion cells exhibit several transient features, including excessive axonal and dendritic branching and exuberant somatic and dendritic spines. These morphological features indicate that there is a transient network of connectivity that could play an important role in the final determination of retinal ganglion cell form and function.

Subplate neurons pioneer the first axon pathway from the cerebral cortex.

During the development of the nervous system, growing axons must traverse considerable distances to find their targets. In insects, this problem is solved in part by pioneer neurons, which lay down the first axonal pathways when distances are at a minimum. Here the existence of a similar kind of neuron in the developing mammalian telencephalon is described. These are the subplate cells, the first postmitotic neurons of the cerebral cortex. Axons from subplate neurons traverse the internal capsule and invade the thalamus early in fetal life, even before the neurons of cortical layers 5 and 6, which will form the adult subcortical projections, are generated. During postnatal life, after the adult pattern of axonal projections is firmly established, most subplate neurons disappear. These observations raise the possibility that the early axonal scaffold formed by subplate cells may prove essential for the establishment of permanent subcortical projections.

Prenatal tetrodotoxin infusion blocks segregation of retinogeniculate afferents.

In the adult mammalian visual system, ganglion cell axons from the two eyes are segregated from each other into separate layers within their principal target, the lateral geniculate nucleus. The involvement of spontaneously generated action potential activity in the process of segregation was investigated during the fetal period in which segregation normally occurs in the cat, between embryonic day 45 (E45) and birth (E65). Tetrodotoxin, which blocks the voltage-sensitive sodium channel, was used to prevent action potentials. Fetuses received continuous intracranial infusions of tetrodotoxin from osmotic minipumps implanted in utero on E42. After a 2-week infusion, intraocular injections of anterograde tracers revealed that tetrodotoxin prevented segregation. The contralateral projection filled the lateral geniculate nucleus uniformly, and the ipsilateral projection expanded to occupy most of what would normally be contralaterally innervated layer A. Thus, in the fetus, long before the onset of vision, spontaneous action potential activity is likely to be present in the visual system and to contribute to the segregation of the retinogeniculate pathway.

Synaptic activity and the construction of cortical circuits.

Vision is critical for the functional and structural maturation of connections in the mammalian visual system. Visual experience, however, is a subset of a more general requirement for neural activity in transforming immature circuits into the organized connections that subserve adult brain function. Early in development, internally generated spontaneous activity sculpts circuits on the basis of the brain's "best guess" at the initial configuration of connections necessary for function and survival. With maturation of the sense organs, the developing brain relies less on spontaneous activity and increasingly on sensory experience. The sequential combination of spontaneously generated and experience-dependent neural activity endows the brain with an ongoing ability to accommodate to dynamically changing inputs during development and throughout life.

Activity-dependent cortical target selection by thalamic axons.

Connections in the developing nervous system are thought to be formed initially by an activity-independent process of axon pathfinding and target selection and subsequently refined by neural activity. Blockade of sodium action potentials by intracranial infusion of tetrodotoxin in cats during the early period when axons from the lateral geniculate nucleus (LGN) were in the process of selecting visual cortex as their target altered the pattern and precision of this thalamocortical projection. The majority of LGN neurons, rather than projecting to visual cortex, elaborated a significant projection within the subplate of cortical areas normally bypassed. Those axons that did project to their correct target were topographically disorganized. Thus, neural activity is required for initial targeting decisions made by thalamic axons as they traverse the subplate.

Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina.

The development of orderly connections in the mammalian visual system depends on action potentials in the optic nerve fibers, even before the retina receives visual input. In particular, it has been suggested that correlated firing of retinal ganglion cells in the same eye directs the segregation of their synaptic terminals into eye-specific layers within the lateral geniculate nucleus. Such correlations in electrical activity were found by simultaneous recording of the extracellular action potentials of up to 100 ganglion cells in the isolated retina of the newborn ferret and the fetal cat. These neurons fired spikes in nearly synchronous bursts lasting a few seconds and separated by 1 to 2 minutes of silence. Individual bursts consisted of a wave of excitation, several hundred micrometers wide, sweeping across the retina at about 100 micrometers per second. These concerted firing patterns have the appropriate spatial and temporal properties to guide the refinement of connections between the retina and the lateral geniculate nucleus.

Competition in retinogeniculate patterning driven by spontaneous activity.

When contacts are first forming in the developing nervous system, many neurons generate spontaneous activity that has been hypothesized to shape appropriately patterned connections. In Mustela putorius furo, monocular intraocular blockade of spontaneous retinal waves of action potentials by cholinergic agents altered the subsequent eye-specific lamination pattern of the lateral geniculate nucleus (LGN). The projection from the active retina was greatly expanded into territory normally belonging to the other eye, and the projection from the inactive retina was substantially reduced. Thus, interocular competition driven by endogenous retinal activity determines the pattern of eye-specific connections from retina to LGN, demonstrating that spontaneous activity can produce highly stereotyped patterns of connections before the onset of visual experience.

Requirement for cholinergic synaptic transmission in the propagation of spontaneous retinal waves.

Highly correlated neural activity in the form of spontaneous waves of action potentials is present in the developing retina weeks before vision. Optical imaging revealed that these waves consist of spatially restricted domains of activity that form a mosaic pattern over the entire retinal ganglion cell layer. Whole-cell recordings indicate that wave generation requires synaptic activation of neuronal nicotinic acetylcholine receptors on ganglion cells. The only cholinergic cells in these immature retinas are a uniformly distributed bistratified population of amacrine cells, as assessed by antibodies to choline acetyltransferase. The results indicate that the major source of synaptic input to retinal ganglion cells is a system of cholinergic amacrine cells, whose activity is required for wave propagation in the developing retina.

Functional requirement for class I MHC in CNS development and plasticity.

Class I major histocompatibility complex (class I MHC) molecules, known to be important for immune responses to antigen, are expressed also by neurons that undergo activity-dependent, long-term structural and synaptic modifications. Here, we show that in mice genetically deficient for cell surface class I MHC or for a class I MHC receptor component, CD3zeta, refinement of connections between retina and central targets during development is incomplete. In the hippocampus of adult mutants, N-methyl-D-aspartate receptor-dependent long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is absent. Specific class I MHC messenger RNAs are expressed by distinct mosaics of neurons, reflecting a potential for diverse neuronal functions. These results demonstrate an important role for these molecules in the activity-dependent remodeling and plasticity of connections in the developing and mature mammalian central nervous system (CNS).

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.

Redistribution of synaptic vesicle antigens is correlated with the disappearance of a transient synaptic zone in the developing cerebral cortex.

To examine the distribution of synaptic vesicle antigens during development of the cerebral cortex, antibodies against synapsin I and p65 were used on sections of cat cerebral cortex between E40 and adulthood. In the adult, the layers of the cerebral cortex are immunoreactive for each of these antigens, while the white matter is free of staining. In contrast, the fetal and neonatal pattern of immunostaining is reversed: the cortical plate (future cortical layers) is devoid of immunoreactivity, while the marginal (future layer 1) and the intermediate zones (future white matter) are stained. Electron microscopic immunohistochemistry shows that immunolabeling is associated with presynaptic nerve terminals in the adult and during development. These observations suggest that during development the white matter is a transient synaptic neuropil and that a global redistribution of synapses takes place as the mature pattern of connections within the cerebral cortex emerges.

Transient period of correlated bursting activity during development of the mammalian retina.

The refinement of early connections in the visual pathway requires electrical activity in the retina before the onset of vision. Using a multielectrode array, we have shown that the spontaneous activity of cells in the neonatal ferret retina is correlated by patterns of periodically generated traveling waves. Here, we examine developmental changes in the characteristics of the waves and show that retinal ganglion cells participate in these patterns of activity, which are seen during the same period as synaptic modification in the lateral geniculate nucleus; that the waves subside gradually as the connectivity in the lateral geniculate nucleus stabilizes; and that their spatial structure allows for refinement of the retinotopic map, as well as for eye-specific segregation in the lateral geniculate nucleus.

Enhancement of transmission at the developing retinogeniculate synapse.

Axon terminals from retinal ganglion cells in the left and right eyes initially overlap with each other in the lateral geniculate nucleus of the neonatal ferret, then segregate into eye-specific layers via an activity-dependent process. Brain slices were used to show that, during this period of reorganization, retinal terminals within the lateral geniculate nucleus evoke excitatory postsynaptic currents composed of both NMDA and non-NMDA receptor-mediated currents. The amplitude of these currents could be enhanced for several tens of minutes to more than an hour by several bursts of high frequency synaptic stimulation, and the induction of enhancement appears to depend on NMDA receptor activation. Synaptic enhancement such as this could provide one of the physiological mechanisms by which retinal terminals segregate into eye-specific layers during development.

Thalamic relay of spontaneous retinal activity prior to vision.

Before vision, retinal ganglion cells produce spontaneous waves of action potentials. A crucial question is whether this spontaneous activity is transmitted to lateral geniculate nucleus (LGN) neurons. Using a novel in vitro preparation, we report that LGN neurons receive periodic barrages of postsynaptic currents from the retina that drive them to fire bursts of action potentials. Groups of LGN neurons are highly correlated in their firing. Experiments in wild-type and NMDAR1 knockout mice show that NMDA receptor activation is not necessary for firing. The transmission of the highly correlated retinal activity to the LGN supports the hypothesis that retinal waves drive retinogeniculate synaptic remodeling. Because LGN neurons are driven to fire action potentials, this spontaneous activity could also act more centrally to influence synaptic modification within the developing visual cortex.

Regulation of class I MHC gene expression in the developing and mature CNS by neural activity.

To elucidate molecular mechanisms underlying activity-dependent synaptic remodeling in the developing mammalian visual system, we screened for genes whose expression in the lateral geniculate nucleus (LGN) is regulated by spontaneously generated action potentials present prior to vision. Activity blockade did not alter expression in the LGN of 32 known genes. Differential mRNA display, however, revealed a decrease in mRNAs encoding class I major histocompatibility complex antigens (class I MHC). Postnatally, visually driven activity can regulate class I MHC in the LGN during the final remodeling of retinal ganglion cell axon terminals. Moreover, in the mature hippocampus, class I MHC mRNA levels are increased by kainic acid-induced seizures. Normal expression of class I MHC mRNA is correlated with times and regions of synaptic plasticity, and immunohistochemistry confirms that class I MHC is present in specific subsets of CNS neurons. Finally, beta2-microglobulin, a cosubunit of class I MHC, and CD3zeta, a component of a receptor complex for class I MHC, are also expressed by CNS neurons. These observations indicate that class I MHC molecules, classically thought to mediate cell-cell interactions exclusively in immune function, may play a novel role in neuronal signaling and activity-dependent changes in synaptic connectivity.

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.