Biomedical subjects
C J Shatz
Publications and source records attributed to C J Shatz.
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.
Neuronal death, a tradition of dying.
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How are specific connections formed between thalamus and cortex?
During development precise thalamocortical connections are established, with reciprocal connections forming correctly in a laminar pattern as well as between the correct thalamic and cortical areas. Recent evidence suggests that both spatial and temporal cues may account for this specificity.
Synapses formed by identified retinogeniculate axons during the segregation of eye input.
The synaptic organization of identified retinogeniculate axons was studied during the prenatal development of eye-specific layers in the LGN of the cat. During this period, retinogeniculate axons undergo stereotyped morphological changes. Retinogeniculate axons originating from one eye and passing through LGN territory destined to be solely innervated by the other eye (inappropriate territory) initially give rise to many side branches. As the eye-specific layers emerge, these axons elaborate extensive terminal arbors within territory appropriate to their eye of origin and concurrently retract their side branches from inappropriate territory (Sretavan and Shatz, 1986). These transient side branches may therefore represent a morphological substrate for the observed functional convergence of inputs from the two eyes onto common LGN neurons during prenatal development (Shatz and Kirkwood, 1984). This possibility was investigated by examining whether identified axons and their side branches form synapses in inappropriate territory. Three retinogeniculate axons from two fetuses aged embryonic day 53 (E53) and E57 were filled with HRP in an in vitro preparation, prior to being processed for electron microscopy (EM). The HRP-filled axons, originating from the contralateral eye, were first reconstructed at the light microscope level. The portion of axon passing through the center of ipsilaterally innervated layer A1 was then serially sectioned and reconstructed by EM. Two sets of 450 serial EM sections revealed that all three contralateral axons established synaptic contacts in ipsilateral territory. Many of these synapses were made by side branches and a few were even formed by the main axon trunks. Both side branches and trunks formed mainly en passant asymmetrical contacts that were associated with spherical synaptic vesicles and that were apposed to immature dendritic elements and dendritic shafts. For comparison, a portion of the same E53 axon within the future contralateral layer A was also serially sectioned and reconstructed for EM. Within this contralateral zone, the E53 axon formed synaptic contacts similar to those established in the ipsilateral region, except that in the appropriate zone they contained significantly more synaptic vesicles. These results demonstrate that axons from the contralateral eye can establish synapses in territory simultaneously innervated by the ipsilateral eye, both via side branches and by means of contacts along the main axon trunk. Thus, the development of eye-specific layers is accompanied by the formation and subsequent elimination of synapses that almost certainly represent a morphological substrate for the known transient functional convergence of inputs from the two eyes.
Pathfinding and target selection by developing geniculocortical axons.
During development of the mammalian cerebral cortex, thalamic axons must grow into the telencephalon and select appropriate cortical targets. In order to begin to understand the cellular interactions that are important in cortical target selection by thalamic axons, we have examined the morphology of axons from the lateral geniculate nucleus (LGN) as they navigate their way to the primary visual cortex. The morphology of geniculocortical axons was revealed by placing the lipophilic tracer Dil into the LGN of paraformaldehyde-fixed brains from fetal and neonatal cats between embryonic day 26 (E26; gestation is 65 d) and postnatal day 7 (P7). This morphological approach has led to three major observations. (1) As LGN axons grow within the intermediate zone of the telencephalon toward future visual cortex (E30-40), many give off distinct interstitial axon collaterals that penetrate the subplate of nonvisual cortical areas. These collaterals are transient and are not seen postnatally. (2) There is a prolonged period during which LGN axons are restricted to the visual subplate prior to their ingrowth into the cortical plate; the first LGN axons arrive within visual subplate by E36 but are not detected in layer 6 of visual cortex until about E50. (3) Within the visual subplate, LGN axons extend widespread terminal branches. This represents a marked change in their morphology from the simple growth cones present earlier as LGN axons navigate en route to visual cortex. The presence of interstitial collaterals suggests that there may be ongoing interactions between LGN axons and subplate neurons along the entire intracortical route traversed by the axons. From the extensive branching of LGN axons within the visual subplate during the waiting period, it appears that they are not simply "waiting." Rather, LGN axons may participate in dynamic cellular interactions within the subplate long before they contact their ultimate target neurons in layer 4. These observations confirm the existence of a prolonged waiting period in the development of thalamocortical connections and provide important morphological evidence in support of the previous suggestion that interactions between thalamic axons and subplate neurons are necessary for cortical target selection.
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.
Morphology of pioneer and follower growth cones in the developing cerebral cortex.
In the developing nervous systems of both invertebrates and vertebrates, neurons must develop precise sets of axonal connections. One strategy used by both orders of animals is to generate a special class of neurons whose axons "pioneer" the first pathways between these cells and their targets. In the developing mammalian telencephalon, the subplate neurons (which are among the first neurons to be generated in development) extend axons to long-distance subcortical targets before the neurons of the deep cortical layers 5 and 6 have been generated. The axons of layer 5 and 6 neurons later follow a similar pathway to form permanent subcortical projections to the thalamus and tectum, and thereafter the vast majority of subplate neurons die. These results have generated the hypothesis that subplate axons may actually be required for the axons of layer 5 and 6 neurons to innervate their appropriate subcortical targets. The complexity of growth cones has previously been correlated with axonal decision making: differences in growth cone morphologies have been noted in comparisons of leading versus following axons (LoPresti, Macagno, and Levinthal, 1973; Nordlander, 1987; Yaginuma, Homma, Kunzi, and Oppenheim, 1991), and at choice points along axon pathways (Raper, Bastiani, and Goodman, 1983; Tosney and Landmesser, 1985; Caudy and Bentley, 1986a,b; Bovolenta and Mason, 1987; Holt, 1989; Bovolenta and Dodd, 1990; Yaginuma et al., 1991). Thus, as a first step toward addressing the question of whether the axons of deep-layer neurons simply follow subplate axons to their targets, we have studied the morphology of cortical growth cones at various points along the corticothalamic pathway and at different stages of development. We examined the brains of fetal ferrets and cats at ages ranging from embryonic days (E) 24 to E50, using the fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) to reveal the axons and growth cones of cortical neurons. Growth cones were drawn, and quantitative measurements of their complexity were made by counting filopodia and calculating their surface area. No morphological differences were found among growth cones at different points along the corticothalamic pathway at a given age. However, growth cones belonging to early-generated cells (likely to be subplate neurons) are significantly larger and more complex than are the growth cones of later-generated cortical neurons. This evidence is consistent with the suggestion that subplate growth cones actively pioneer the corticothalamic pathway, and that the axons of layer 5 and 6 neurons follow it.
Remodeling of retinal ganglion cell dendrites in the absence of action potential activity.
The dendrites of ganglion cells in the retina have an excess number of spines and branches that are normally lost during the first postnatal month of development. We investigated whether this dendritic remodeling can be prevented when the action potential activity of ganglion cells is abolished by chronic intraocular injections of tetrodotoxin (TTX) during the first 4 or 5 postnatal weeks in the cat. Dendritic tree morphologies of alpha and beta ganglion cells from TTX-treated, non-TTX-treated (contralateral eye), and normal control retinae were compared after intracellular filling with Lucifer yellow. Qualitative observations and quantitative measurements indicate that TTX treatment does not prevent the normally occurring loss of spines and dendritic branches. Indeed, the dendritic trees of both alpha and beta cells in TTX injected eyes actually have even fewer spines and branches than normal cells at equivalent ages. However, because the total dendritic lengths of these cells are also reduced after TTX blockade, spine density is indistinguishable from untreated animals at the same age. In addition, although dendritic field areas are not altered with treatment, the complexity of the dendritic trees is reduced. These observations suggest that dendritic remodeling can occur in the absence of ganglion cell action potential activity. Thus, the factors that influence the dendritic and axonal development of retinal ganglion cells must differ, because similar TTX treatment during the period of axonal remodeling does have profound effects on the final pattern of terminal arborizations.
Changing patterns of synaptic input to subplate and cortical plate during development of visual cortex.
1. The development of excitatory activation in the visual cortex was studied in fetal and neonatal cats. During fetal and neonatal life, the immature cerebral cortex (the cortical plate) is sandwiched between two synaptic zones: the marginal zone above, and an area just below the cortical plate, the subplate. The subplate is transient and disappears by approximately 2 mo postnatal. Here we have investigated whether the subplate and the cortical plate receive functional synaptic inputs in the fetus, and when the adultlike pattern of excitatory synaptic input to the cortical plate appears during development. 2. Extracellular field potential recording to electrical stimulation of the optic radiation was performed in slices of cerebral cortex maintained in vitro. Laminar profiles of field potentials were converted by the current-source density (CSD) method to identify the spatial and temporal distribution of neuronal excitation within the subplate and the cortical plate. 3. Between embryonic day 47 (E47) and postnatal day 28 (P28; birth, E65), age-related changes occur in the pattern of synaptic activation of neurons in the cortical plate and the subplate. Early in development, at E47, E57, and P0, short-latency (probably monosynaptic) excitation is most obvious in the subplate, and longer latency (presumably polysynaptic) excitation can be seen in the cortical plate. Synaptic excitation in the subplate is no longer apparent at P21 and P28, a time when cell migration is finally complete and the cortical layers have formed. By contrast, excitation in the cortical plate is prominent in postnatal animals, and the temporal and spatial pattern has changed. 4. The adultlike sequence of synaptic activation in the different cortical layers can be seen by P28. It differs from earlier ages in several respects. First, short-latency (probably monosynaptic) excitation can be detected in cortical layer 4. Second, multisynaptic, long-lasting activation is present in layers 2/3 and 5. 5. Our results show that the subplate zone, known from anatomic studies to be a synaptic neurophil during development, receives functional excitatory inputs from axons that course in the developing white matter. Because the only mature neurons present in this zone are the subplate neurons, we conclude that subplate neurons are the principal, if not the exclusive, recipients of this input. The results suggest further that the excitation in the subplate in turn is relayed to neurons of the cortical plate via axon collaterals of subplate neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
Requirement for subplate neurons in the formation of thalamocortical connections.
The neurons of layer 4 in the adult cerebral cortex receive their major ascending inputs from the thalamus. In development, however, thalamic axons arrive at the appropriate cortical area long before their target layer 4 neurons have migrated into the cortical plate. The axons accumulate and wait in the zone below the cortical plate, the subplate, for several weeks before invading the cortical plate. The subplate is a transient zone that contains the first postmitotic neurons of the telencephalon. These neurons mature well before other cortical neurons, and disappear by cell death after the thalamic axons have grown into the overlying cortical plate. The close proximity of growing thalamocortical axons and subplate neurons suggests that they might be involved in interactions important for normal thalamocortical development. Here we show that early in development the deletion of subplate neurons located beneath visual cortex prevents axons from the lateral geniculate nucleus of the thalamus from recognizing and innervating visual cortex, their normal target. In the absence of subplate neurons, lateral geniculate nucleus axons continue to grow in the white matter past visual cortex despite the presence of their target layer 4 neurons. Thus the transient subplate neurons are necessary for appropriate cortical target selection by thalamocortical axons.
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 functional. 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.
Impulse activity and the patterning of connections during CNS development.
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Nerve growth factor receptor immunoreactivity is transiently associated with the subplate neurons of the mammalian cerebral cortex.
Nerve growth factor and its receptor (NGFR) are known to be present in diverse embryonic and neonatal central nervous system tissues, including the cerebral cortex. However, the identity of the cortical cells expressing NGFR immunoreactivity has not been established. We have used immunolabeling coupled with [3H]thymidine autoradiography to identify such cells in ferret and cat brain. Polyclonal antibodies raised against a synthetic peptide corresponding to a conserved amino acid sequence of the NGFR were used for this purpose. Western (immunologic) blot analyses show that these antibodies specifically recognize NGFR and precursor proteins. In both species, NGFR immunoreactivity is primarily associated with the early generated and transient subplate neuron population of the developing neocortex, as indicated by the following evidence: the immunoreactive cells (i) are located directly beneath the developing cortical plate, (ii) frequently have the inverted pyramid shape characteristic of subplate neurons, and (iii) can be labeled by an injection of [3H]thymidine on embryonic day (E) 28, a time when only subplate neurons are being generated. Intense NGFR immunostaining is seen on the cell bodies of these neurons as early as E30, several days after their last round of cell division, and this immunostaining remains strong for approximately 3 weeks. The NGFR immunoreactivity begins to decline around E52 and has disappeared from the region altogether by E60, at which time subplate neurons begin to die. The cellular localization and timing of expression suggest that the NGFR may play a role in the maintenance of subplate neurons and in the maturation of the cerebral cortex.
Pioneer neurons and target selection in cerebral cortical development.
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Functional synaptic circuits in the subplate during fetal and early postnatal development of cat visual cortex.
Among the first postmitotic cells of the cerebral cortex is a special population located below the cortical plate: the subplate neurons. These neurons reach a high degree of morphological maturity during fetal life, well before the neurons of the cortical layers have matured, yet nearly all of these cells die after birth in the cat. Subplate neurons are also known to receive synaptic contacts. Here we have investigated whether these contacts are functional by making intracellular recordings from subplate neurons in cortical slices maintained in vitro. Subplate neurons were identified based on their location and morphology by injecting them with biocytin following the intracellular recordings. At all ages studied between embryonic day 50 and postnatal day 9, electrical stimulation of the optic radiations elicited EPSPs and synaptic and antidromic spikes in subplate neurons, indicating that some of the synapses seen at the ultrastructural level are indeed capable of synaptic transmission. The spiking patterns of 39 morphologically identified subplate neurons were examined by injecting depolarizing current, which revealed that a large majority gave only a single spike or a brief train of spikes in response to maintained depolarization, in contrast to the regular spiking pattern found in many neurons of adult cortex. Biocytin injections into subplate neurons revealed that they are a morphologically heterogeneous population with respect to their dendritic branching patterns; roughly half were inverted pyramids, the classic subplate neuron morphology. The axonal processes of subplate neurons were remarkable in that many not only arborized within the subplate, but also entered the cortical plate and terminated in the marginal zone. At early postnatal ages, these axons also gave off collaterals within cortical layer 4. The results of this study indicate that subplate neurons participate in synaptic microcircuits during development. While the presynaptic identity of the input to subplate neurons is not known conclusively, it is likely that geniculocortical axons, which wait in close proximity to subplate neurons, contribute significantly. The pattern of axonal branching of subplate neurons also implies that information conferred to subplate neurons may be relayed, in turn, to the neurons of cortical layer 4. Finally with the death of subplate neurons, the geniculocortical axons leave the subplate and invade the cortical plate to innervate directly the neurons of layer 4. Thus, subplate neurons may function as a crucial, but transient synaptic link between waiting geniculocortical axons and their ultimate target cells in the cortex.
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.