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How are specific connections formed between thalamus and cortex?
How are specific connections formed between thalamus and cortex? Carla j. Shatz University
During
development
with reciprocal between that
of California,
precise thalamocortical
connections
the correct
forming
thalamic
Opinion
in a laminar
The mammalian neocortex is an extraordinary computa tional machine that performs a myriad of complex functions, each of which is highly dependent on the speciiic pattern of neural connections it receives from lower structures of the nervous system such as the thalamus. For example, primary visual cortex receives input from the lateral geniculate nucleus (LGN), an Important structure within the visual thalamus, while primary auditory cortex receives input from the medial geniculate nucleus (MGN) of the thalamus. Indeed, as shown in Fig. 1, there is a precise set of connections from each subdivision of the thalamus to speciIic subregions of the cerebral cortex in the adult. Throughout the cerebral cortex, the connections from the thalamus terminate chiefly in cortical
USA
are established, pattern
areas. Recent evidence
cues may account
in Neurobiology
Introduction
California,
connections
correctly
and cortical
both spatial and temporal
Current
Auditory
Berkeley,
as well as suggests
for this specificity.
1992, 2:7882
layer 4. In turn, there is an equally specific reciprocal connection back to the appropriate thalamic nucleus by the neurons of layer 6 of the cerebral cortex. Thus, the cortex is divided tangentially into many functionally dIstinct areas. In this review I will look at recent evidence suggesting how the precise sets of connections between thalamus and cortex are formed during development in both the tangential and radial dimensions. Intrinsically
specified or a result of dynamic
interactions? The importance of the identity of the thalamic nucleus that sends its input to a speciiic region of cortex for the functioning of that cortical region was underscored in a
Y
+i$$& Cortex
. Visual
Fig.1.A diagram illustrating the specific, reciprocal sets of connections between the thalamus and cerebral cortex in the adult mammalian brain. As examples, connections from two thalamic nuclei, the lateral geniculate nucleus (LCN), which subserves vision, and the medial geniculate nucleus (MCN), which subserves audition, are shown. Each projects to layer 4 neurons within its own cortical area. In turn, neurons of cortical layer 6 project back to the appropriate thalamic nucleus. WM, white matter. Abbreviations LGN-lateral 78
geniculate nucleus; MCN-medial @
geniculate nucleus.
Current Biology Ud ISSN 0959-4388
How are specific connections formed between thalamus and cortex ? Shatz
series of experiments by SW and colleagues [ 11. They surgically forced visual axons arising from the retina to grow into the MGN (auditory thalamus) during development. The surgically manipulated animals were then permitted to grow up, and the physiological properties of neurons in the primary auditory cortex were examined. Many neurons in ‘auditory cortex’ responded to visual stimuli in a similar, if not always identical, fashion to those in normal visual cortex. The results indicate that the basic computational machinery of the cerebral cortex is vety similar throughout and that many of the functional properties of cortical neurons arise due to the modality of information conveyed by each thalamic nucleus. A major question for developmental neurobiology is how the precision of thalamocortical connectivity is established during development. One idea is that each cortical region is intrinsically specified during development to receive input.5 from the appropriate thalamic nucleus [ 21. For example, neurons belonging to each cortical area might express Merent cell surface properties, and the appropriate thalamic neurons might recognize these differences. Such an idea is attractive because studies very early in the development of thalamocortical connections suggest that even the initial pattern of connections between thalatnus and cortex appears to be precise [3,4], and does not exhibit the extensive relining and remod eling of axon projections known to occur elsewhere in nervous system development (for reviews, see [ 5-71). The results of several recent experiments, however, make it hard to support this hypothesis. For instance, Cepko and colleagues [8*] have used retrovirus labeling and polymerase chain reaction technology to trace cell lineage in the developing cerebral cortex of rodents. They have found that clonally related neurons can undergo a surprising amount of intermixing during migration. Thus, specification of regional identity, at least at the level of single autonomous clones, seems highly unlikely. Nor does it seem very likely that entire assemblies of cells within a given region of cortex contain rigid intrinsic cues that promote the formation of specific sets of thalamocortical connections. In recent elegant experiments in rodents, Schlaggar and O’Leary [!+I transplanted visual cortex to somatosensory cortex during early development and examined the consequences on the formation of thalamocortical connections. Not only were the appropriate connections formed between somatosensory thalamus and the transplanted piece of visual cortex, but the cortex actually took on the histological and cytoarchitectural characteristics normally uniquely associated with somatosensoly cortex (e.g. the ‘barrels’). The possibility that thalamocortical connections form as a consequgnce of the matching of prespeci6ed cues between an area of cortical neurons and the appropriate set of thalamic axons seems even less likely in view of a new line of exciting experiments in which thalamus and cortex from developing rodent brain are co-cultured and the specilicity of thalamocortical connections assessed in vitro [10,11**,12**]. Even in vitro, reciprocal functional connections can form between thalamus and cortex, and remarkably they do so in the correct laminar pattern. Thalamic axons terminate within layer 4 and the cortical projection back to the thalamus arises from layer 6 neu-
rons. Despite the fact that the laminar specificity of thalamocortical connections is preserved in these co-cultures, the tangential specificity is not. Evidently connections can form between the LGN of the thalamus and any cortical area [12**]. This observation is again consistent with the idea that different regions of the cerebral cortex do not themselves contain unique (and stable) cues for rigidity specifying their areal identity. Moreover, the experiments suggest that there may be significant differences between the cellular mechanisms that specify the tangential (e.g. cortical areas) and radial (e.g. cortical layers) domains of cerebral cortex during development. It is interesting to note that while no clear examples have yet revealed distinct molecular differences between neocortical areas, there are known differences between the cortical layers in the expression of certain POU genes J[131. There is also a marked difference in the way.,@ which axonal connections of many types form .wiwii; the cortex. For instance, the patterning of axon,qutgro+th from layer 3 cortical neurons differs in the &dial and tangential dimensions. The horizontal (tangential) sequence of ax onal outgrowth within a cortical layer involves extensive growth followed by remodeling and pruning, thought to require neural activity [14], whereas the vertical outgrowth is highly directed, with axons specifically avoiding growth into inappropriate cortical layers, such as layer 4, from the outset [15*3. If unique markers intrinsic to each cortical area are not present during development, then how do thalamocortical axons select the appropriate cortical targets? Some clues have come from a direct examination of the formation of this pathway in vivo. In the visual system of higher mammals such as cats and primates, pathway tracing studies have shown that axons from the LGN grow out at very early times in development and arrive below the forming visual cortex even before many of their target neurons within cortical layer 4 have become postmitotic and migrated to their positions within the forming cortical plate (which will eventually become the six-layered structure of the adult cerebral cortex) [3,4]. The LGN axons accumulate beneath the cortical plate where they ‘wait’ in a zone called the subplate [16,17] for several weeks (cat) to months (primate) before growing into the cortex, when the layer 4 neurons finally arrive. Recent studies of the development of thalamocortic~ connections in rodents suggest a similar scenario, except that there may be little or no waiting period, as the timing mismatch between the formation of layer 4 neurons and the arrival of thalamic axons is negligible [WI.
During development, the subplate zone contains not only the waiting thalamocortical axons, but also a special transient population of subplate neurons. These neurons are among the first postmitotic cells of the cerebral cortex [ 16,171. They receive functional synaptic inputs [ 191, some from the thalamus (K Herrmann et al.: Sot Neurosci Abstr 1991, 16:8!9!9), and become immunoreactive for certain neurotransmitters and peptides [20,21] long before the neurons of the cortical plate. Although many disappear by programmed cell death shortly after birth [22,23-l, earlier in development these neurons may be crucial for the normal formation of thalamocortical connections.
79
80
Development
Visual cortex
One observation consistent with this suggestion is that in both the cat and rat, the axons of subplate neurons are the first to grow down from the cortex to the thalamus [24,25e,26*]; only later do the neurons of cortical layer 6 contribute axons (see Fig. 2). Evidence suggests that subplate neurons beneath each cortical area send axons to the appropriate thalamic nucleus. In fact, axon tracing studies suggest that subplate axons and thalamic axons probably meet and grow past each other in the internal capsule, the gateway between thalamus and cortex [ 24,26*,27*]. This observation raises the possibility that subplate axons may be required for the formation of connections between thalamus and cortex. If so, then deleting subplate neurons should perturb the development of these connections. The optimal experiment would be to remove subplate neurons beneath the visual cortex for example, well before their axons had formed a complete pathway back to the LGN; technically this experiment has not yet proved possible. If the deletions are performed just after LGN axons have first arrived beneath the visual subplate, but well before they have grown in (see Fig. 2), there is a surprising outcome. LGN axons fail to accumulate and do not wait below the cortical plate. Instead, they grow past the visual cortex and remain within the white matter, never entering the cortex even up to one month later [28-l. The results of this experiment have several implications for the mechanisms that underlie the formation of specific sets of thalamocortical connections. First, they suggest that subplate neurons are somehow involved in the process bywhich thalamic axons select and grow into the appropriate cortical area. Second, they point out that the process of pathway formation and target selection are not necessarily one and the same, as many LGN axons were
Fig.2. A hypothetical diagram illustrating the sets of connections present between thalamus and cortex at early times in development. Axons from thalamic nuclei such as the lateral geniculate nucleus (LCN) grow through the internal capsule until they reach the visual cortex, where they ‘wait’ in the subplate in the vicinity of subplate neurons. The axons of visual subplate neurons, in turn, have already pioneered the pathway out of the cortex and have arrived at the LGN, where they also wait. The cortical plate is still forming and only consists of neurons belonging to deeper cortical layers (5 and 6). (Mitotic figures in the ventricular zone, and postmitotic cells migrating along radial glia, are shown in the right of the diagram.) The axons of layer 6 cortical neurons are still growing en route to their thalamic target nuclei. It is conceivable that the axons of thalamic and subplate neurons meet in the internal capsule and fasciculate together as they grow to their appropriate targets. The later growing layer 6 axons could then follow a preformed axonal pathway to the correct thalamic target. Erased on observations from [24,26*,27*1.
already in their correct positions beneath the visual cortex at the time of the deletion of subplate neurons, and yet failed to subsequently grow into the cortex. Third, the results suggest that the cortical plate by itself does not contain sufficient information to promote the normal formation of thalamocortical connections. How might subplate neurons be involved in the process of target selection and ingrowth? One possibility is that the subplate neurons beneath distinct cortical areas are different. But this suggestion would simply move the site of specificity from the cortical plate to the subplate, and while simple conceptually, it is fraught with all the problems discussed earlier in the context of the cortical plate itself. Another possibility is that both spatial and temporal cues are involved in the formation of specific thalamocortical connections. Subplate axons from more posterior regions grow and reach the internal capsule later than those from more anterior regions [24], and axons from more anterior medial thalamic nuclei (e.g. MGN) reach the internal capsule before those from more posterior and lateral locations (e.g. LGN). Consequently, when LGN axons enter the cerebral hemisphere travelling towards the visual cortex, they grow past the auditory subplate, which is already filled with earlier-arriving MGN axons (A Ghosh and C Shatz, unpublished data). Thus, dynamic interactions with the subplate may prevent later-arriving axons from stopping there and force those axons to grow until they arrive in a region free of other thalamocortical axons. If so, then it should be possible to re-route thalamic axons from the MGN, for example, into an inappropriate cortical area such as visual cortex by deleting subplate neurons beneath auditory cortex early in development. The MGN axons could then be forced to ‘compete’ with later growing thalamic axons for available subplate
How are specific
neurons posterior to the lesion area. If MGN axons can grow into foreign cortex, the result would complement the transplantation experiments which show that visual cortex can accept innervation from somatosensofy tierents [P-l. It would also be entirely consistent with the co-culture experiments [10,11**,12**] in the sense that speciiicity between thalamus and cortex is lost in co-culture because all timing relationships are gone. Conclusion In thisreview, I have considered the general question of how the precise sets of connections between thalamus and cortex are formed during development. Experimental evidence suggests that the mechanisms that underlie the laminar specificity of thalamocortical connections may be quite different from those responsible for tangential (areal) specilicity. Despite the fact that the pattern of projection from each thalamic nucleus to the appropriate cortical area appears very precise even at the outset of development, such precision need not require the existence of unique cues restricted to each cortical area. Rather, it seems possible that a series of dynamic interactions involving broader spatial cues, along with temporal gradients of axon growth, could account for the precision in the development of thalamocortical development. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: - . of special interest .. of outstanding interest 1.
SURM, GARRAGH~PE, ROE AW: Experimentally Induced Visual Projections into Auditory Thalamus and Cortex. Science 1988, 242:1437-1441.
2.
RAKICP: Specification of Cerebral 1988, 241:17&176.
3.
RAKICP: Prenatal Development of the Visual System in the Rhesus Monkey. Phil Trans R Sot Lond B 1977, 278:245-260.
4.
SHATZCJ, LGKIN MB: Relationship Between the Geniculocortical A&rents and Their Cortical Target Cells During Development of the Cat’s Primary Visual Cortex. J Neuro xi 1986, 6:3655-3668.
5.
MCCONNELL SK: Development and Decision-Making in the Mammalian Cerebral Cortex. Brain Res Rev 1988, 13:1-23.
6.
O’IEARYDDM: Do Cortical Areas Emerge from a Protocortex? Trends Neuraui 1989, 12:400X16.
7.
SHAZ CJ: Impulse Activity and the Patterning of Connections During CNS Development. Neuron 1990, 5:74%756.
Cortical
Areas.
AUSTIN CP, CEPKO CL: Cellular Migration Patterns in tbe Developing Mouse Cerebral Cortex. Development 1990, 110:713-732. A thorough lineage tracing study in which a retrotirus was used to label clonally related cells in the developing mouse cortex and the subsequent location of labeled cells within the cortex examined. Results show that members of a given clone can become wideiy dispersed during migration and end up in distant cortical locations, suggesting that the areal identity of cortex cannot arise due to a simple restriction of migration of clonally related cells. SCHIAGGARBL, O’L~RY DDM: Potential of Visual Cortex to Develop an Array of Functional Units Unique to Somatosensory Cortex. Science 1991, 252:15561560.
formed
between
thalamus
and cortex?
Shatz
This creative transplantation experiment demonstrates that visual cortex transplanted to the site of somatosensoty cortex can accept innervation from somatosensoly thalamus and form barrels in uiYg. 10.
YAMAMOTO N, KUROTANI T, TOYAMAK: Neural Connections Between Lateral Geniculate Nucleus and Visual Cortex In Vitro. Science 1989, 245:192-194.
of BOL? J, NOVAKN, GOTZ M, BONHOEFFERT: Formation Target-Specific Neuronal Projections in Organotypic Slice Cultures from Rat Visual Cortex. Nature 1990, 346:359-362. Here the authors show convincingly that co-culturing thakunus and cortex in vitro leads to the formation of laminar specific interconnections between the thalamus and layers 4 and 6 of the cortex. 11. ..
12. ..
MOLNAR 2, BL~KEMOREC: Lack of Regional
13.
HE X, TREKY MN, SIMMONS DM,/INGRAHAM H& SWANSON LW, ROSENFELD MG: Expression of a Large Family of F’OU-Domain Regulatory Genes in Mammalian Brain Development. Nature 1989, 340:3542.
14.
CAUAWAV EM, KATZ LC: Emergence and Refinement of Clustered Horizontal Connections in Cat Striate Cortex. J Neuroxi 1990, lo:11341153.
Specificity for Connections Formed Between Thalamus and Cortex in CoCulture. Nature 1991, 351:47w77.’ The authors address the important question of whether the specificity of connections between thalamic nuclei and corticaLtarget areas can be reconstituted in t&r0 By co-culturing LGN with visual or frontal cortex, the authors find that thalamic neurons exhibitobvious preference for one cortical area over the other. “* : t
KATZ IC: Specificity in the Development of Vertical Con15. . nections in Cat Striate Cortex. EurJ Neurosci 1991, 31-9. An intracellular labeling study examining the morphological development of axonal projections from cortical layer 3 neurons which shows that the vertical (interlaminar) connections formed by growing axons are specific at the outset and do not undergo extensive collateral remodeling. This contrasts with the horizontal (interlaminar) axonal connections from the same neurons (see [ 141). 16.
KOSTOV~C I, RAKICP: Cytology and Time of Origin of Interstitial Neurons in tbe White Matter in Infant and Adult Human and Monkey Telencephalon. J Neurocylo 1980, 9:213-242.
17.
IJJSKINME%, SHATZCJ: Studies
Science
a. .
9. ..
connections
of the Earliest-Generated Cells of the Cat’s Visual Cortex: Co-generation of Subplate and Marginal Zones. J Neurosci 1985, 5:1062-1075.
CA’IXIANO SM, ROBERTSON RT, KILL~CK!ZY HB: Early Ingrowth of Thalamocortical AlTerems to the Neocortex of the Prenatal Rat. Proc Nat1 Acud Sci USA 1991, 88:29993003. An anatomical study of the timecourse and pattern of development of the thalamocortical projection in rodents using the vety sensitive tracing technique of Dii labeling The results indicate that thalamwortical axons in rodents may not wait in the subplate before growing into the cortical plate. 18. .
F!UAKJF E, MCCONNELL SK, SHAIZ CJ: Functional Synaptic Circuits in the Subplate During Fetal and Early Postnatal Development of Cat Visual Cortex. J Neurosci 1990, 10:260-2613. Intracellular recordings from subplate neurons in cortical slices in vitro during electrical stimulation of the white matter demonstrate that they receive synaptic inputs, implying the presence of a functional microcircuit in fetal telencephalon. 19. .
20.
CHUNJJM, SHA’IZCJ: The Earliest-Generated Neurons of the Cat Cerebral Cortex: Characterization by MAP2 and Neurotransmitter Immunohistochemistry During Fetal Life. / Neurosci 1989, 91648-1667.
21.
ANTONINI A, SHATZCJ: Relation Between Putative Transmitter Phenotypes and Connectivity of Subplate Neurons During Cerebral Cortical Development. Eur J Neurasci 1990, 2:744-761.
22.
CHUN JJM, SHA’IZ CJ: Interstitial Cells of the Adult Neocortical White Matter are the Remnant of the Early Generated Subplate Neuron Population. J Comp New-d 1989, 282:555-569.
81
82
Development 23. .
JA Distribuof Alx-50 Immunoreactive Cells in tion and Morphology the Developing Visual Cortex of Kittens. J Neurccytol1990, 19662471. Ah-50 is an antigen associated with cortical neurons in Alzheimer’s disease and its presence is loosely correlated with cell death in other sys terns. Here the authors demonstrate that subplate neurons become immunoreactive during the period in which they undergo programmed cell death. 24.
QUE LL, DECAWS VALVERDEF, LOPEZ-iVkKAR4
MCCONNEU.SK, GHO~H A SHATZ CJ: Subplate Neurons Pioneer the First Axon Pathways from the Cerebral Cortex. Science 1989, 245:278281.
25. .
Ktht GJ, SBATZ CJ, MCCONNEU SK: Morphology of Pioneer and Follower Growth Cones in the Developing Cerebral Cortex. J Neumbiol 1991, 22:629-642. The authors use DR labeling to examine the morphology of growth cones from cortical neurons en route to the thalamus. Early growth cones (presumably from subplate neurons) are much larger and more complex than those from later growing (presumably cortical plate) neurons. DE CARGOS JA, O’LEARYDDM: Growth and Targeting of Subplate Axons and Establishment of Major Cortical Pathways. J Neurosci 1991, in press. This study examines the earliest axons to grow out of cerebral cortex in the rodent somatosensory system by means of Dil labeling. Results suggest that, as in highei mammals, subplate axons in rodents pioneer the early pathway from cortex to thalamus. The authors argue, how26. .
ever, that subplate neuron axons do not pioneer the entire pathway into the spinal cord, rather, layer 5 pyramidal axons appear to be the first cortical axons to grow into the descending corticospinal tract. CJ: Pathtinding and Target Selection. J Neumci 1991, 11: in press. ;n this DiI labeling study of the development of the geniculocortic~ projection in the cat, the authors reaffirm and extend previous observations that thalamocortical axons ‘wait’ in the subplate for almost 3 weeks before growing into the cortical plate. Moreover, LGN axons branch extensively within the subplate, suggesting that there may be dynamic interactions with subplate neurons. 27.
GHOSH A, Smm
GHOSH A, ANTom 4 MCCONNELLSK, SHAR CJ: Requirement for Subplate Neurons in the Formation of Thalamocortical Connections. Nature 1990, 3473179181. In this experiment, subplate neurons were ablated using injections of kainic acid, and the consequences for the development of the genicu locortical projection subsequently examined by means of DiI labeling. In the absence of subplate neurons, LGN axons failed to grow into the cortical plate and instead remained within the white matter, traveling for long distances away from the visual cortex. This experiment suggests that subplate neurons participate in the process of target recognition and ingrowth by thalamic axons.
28. ..
CJ Shatz, Division of Neurobiology, Department of Molecular and Cell Biology, ISA 221, University of California, Berkeley, California 94720, USA
During
development
with reciprocal between that
of California,
precise thalamocortical
connections
the correct
forming
thalamic
Opinion
in a laminar
The mammalian neocortex is an extraordinary computa tional machine that performs a myriad of complex functions, each of which is highly dependent on the speciiic pattern of neural connections it receives from lower structures of the nervous system such as the thalamus. For example, primary visual cortex receives input from the lateral geniculate nucleus (LGN), an Important structure within the visual thalamus, while primary auditory cortex receives input from the medial geniculate nucleus (MGN) of the thalamus. Indeed, as shown in Fig. 1, there is a precise set of connections from each subdivision of the thalamus to speciIic subregions of the cerebral cortex in the adult. Throughout the cerebral cortex, the connections from the thalamus terminate chiefly in cortical
USA
are established, pattern
areas. Recent evidence
cues may account
in Neurobiology
Introduction
California,
connections
correctly
and cortical
both spatial and temporal
Current
Auditory
Berkeley,
as well as suggests
for this specificity.
1992, 2:7882
layer 4. In turn, there is an equally specific reciprocal connection back to the appropriate thalamic nucleus by the neurons of layer 6 of the cerebral cortex. Thus, the cortex is divided tangentially into many functionally dIstinct areas. In this review I will look at recent evidence suggesting how the precise sets of connections between thalamus and cortex are formed during development in both the tangential and radial dimensions. Intrinsically
specified or a result of dynamic
interactions? The importance of the identity of the thalamic nucleus that sends its input to a speciiic region of cortex for the functioning of that cortical region was underscored in a
Y
+i$$& Cortex
. Visual
Fig.1.A diagram illustrating the specific, reciprocal sets of connections between the thalamus and cerebral cortex in the adult mammalian brain. As examples, connections from two thalamic nuclei, the lateral geniculate nucleus (LCN), which subserves vision, and the medial geniculate nucleus (MCN), which subserves audition, are shown. Each projects to layer 4 neurons within its own cortical area. In turn, neurons of cortical layer 6 project back to the appropriate thalamic nucleus. WM, white matter. Abbreviations LGN-lateral 78
geniculate nucleus; MCN-medial @
geniculate nucleus.
Current Biology Ud ISSN 0959-4388
How are specific connections formed between thalamus and cortex ? Shatz
series of experiments by SW and colleagues [ 11. They surgically forced visual axons arising from the retina to grow into the MGN (auditory thalamus) during development. The surgically manipulated animals were then permitted to grow up, and the physiological properties of neurons in the primary auditory cortex were examined. Many neurons in ‘auditory cortex’ responded to visual stimuli in a similar, if not always identical, fashion to those in normal visual cortex. The results indicate that the basic computational machinery of the cerebral cortex is vety similar throughout and that many of the functional properties of cortical neurons arise due to the modality of information conveyed by each thalamic nucleus. A major question for developmental neurobiology is how the precision of thalamocortical connectivity is established during development. One idea is that each cortical region is intrinsically specified during development to receive input.5 from the appropriate thalamic nucleus [ 21. For example, neurons belonging to each cortical area might express Merent cell surface properties, and the appropriate thalamic neurons might recognize these differences. Such an idea is attractive because studies very early in the development of thalamocortical connections suggest that even the initial pattern of connections between thalatnus and cortex appears to be precise [3,4], and does not exhibit the extensive relining and remod eling of axon projections known to occur elsewhere in nervous system development (for reviews, see [ 5-71). The results of several recent experiments, however, make it hard to support this hypothesis. For instance, Cepko and colleagues [8*] have used retrovirus labeling and polymerase chain reaction technology to trace cell lineage in the developing cerebral cortex of rodents. They have found that clonally related neurons can undergo a surprising amount of intermixing during migration. Thus, specification of regional identity, at least at the level of single autonomous clones, seems highly unlikely. Nor does it seem very likely that entire assemblies of cells within a given region of cortex contain rigid intrinsic cues that promote the formation of specific sets of thalamocortical connections. In recent elegant experiments in rodents, Schlaggar and O’Leary [!+I transplanted visual cortex to somatosensory cortex during early development and examined the consequences on the formation of thalamocortical connections. Not only were the appropriate connections formed between somatosensory thalamus and the transplanted piece of visual cortex, but the cortex actually took on the histological and cytoarchitectural characteristics normally uniquely associated with somatosensoly cortex (e.g. the ‘barrels’). The possibility that thalamocortical connections form as a consequgnce of the matching of prespeci6ed cues between an area of cortical neurons and the appropriate set of thalamic axons seems even less likely in view of a new line of exciting experiments in which thalamus and cortex from developing rodent brain are co-cultured and the specilicity of thalamocortical connections assessed in vitro [10,11**,12**]. Even in vitro, reciprocal functional connections can form between thalamus and cortex, and remarkably they do so in the correct laminar pattern. Thalamic axons terminate within layer 4 and the cortical projection back to the thalamus arises from layer 6 neu-
rons. Despite the fact that the laminar specificity of thalamocortical connections is preserved in these co-cultures, the tangential specificity is not. Evidently connections can form between the LGN of the thalamus and any cortical area [12**]. This observation is again consistent with the idea that different regions of the cerebral cortex do not themselves contain unique (and stable) cues for rigidity specifying their areal identity. Moreover, the experiments suggest that there may be significant differences between the cellular mechanisms that specify the tangential (e.g. cortical areas) and radial (e.g. cortical layers) domains of cerebral cortex during development. It is interesting to note that while no clear examples have yet revealed distinct molecular differences between neocortical areas, there are known differences between the cortical layers in the expression of certain POU genes J[131. There is also a marked difference in the way.,@ which axonal connections of many types form .wiwii; the cortex. For instance, the patterning of axon,qutgro+th from layer 3 cortical neurons differs in the &dial and tangential dimensions. The horizontal (tangential) sequence of ax onal outgrowth within a cortical layer involves extensive growth followed by remodeling and pruning, thought to require neural activity [14], whereas the vertical outgrowth is highly directed, with axons specifically avoiding growth into inappropriate cortical layers, such as layer 4, from the outset [15*3. If unique markers intrinsic to each cortical area are not present during development, then how do thalamocortical axons select the appropriate cortical targets? Some clues have come from a direct examination of the formation of this pathway in vivo. In the visual system of higher mammals such as cats and primates, pathway tracing studies have shown that axons from the LGN grow out at very early times in development and arrive below the forming visual cortex even before many of their target neurons within cortical layer 4 have become postmitotic and migrated to their positions within the forming cortical plate (which will eventually become the six-layered structure of the adult cerebral cortex) [3,4]. The LGN axons accumulate beneath the cortical plate where they ‘wait’ in a zone called the subplate [16,17] for several weeks (cat) to months (primate) before growing into the cortex, when the layer 4 neurons finally arrive. Recent studies of the development of thalamocortic~ connections in rodents suggest a similar scenario, except that there may be little or no waiting period, as the timing mismatch between the formation of layer 4 neurons and the arrival of thalamic axons is negligible [WI.
During development, the subplate zone contains not only the waiting thalamocortical axons, but also a special transient population of subplate neurons. These neurons are among the first postmitotic cells of the cerebral cortex [ 16,171. They receive functional synaptic inputs [ 191, some from the thalamus (K Herrmann et al.: Sot Neurosci Abstr 1991, 16:8!9!9), and become immunoreactive for certain neurotransmitters and peptides [20,21] long before the neurons of the cortical plate. Although many disappear by programmed cell death shortly after birth [22,23-l, earlier in development these neurons may be crucial for the normal formation of thalamocortical connections.
79
80
Development
Visual cortex
One observation consistent with this suggestion is that in both the cat and rat, the axons of subplate neurons are the first to grow down from the cortex to the thalamus [24,25e,26*]; only later do the neurons of cortical layer 6 contribute axons (see Fig. 2). Evidence suggests that subplate neurons beneath each cortical area send axons to the appropriate thalamic nucleus. In fact, axon tracing studies suggest that subplate axons and thalamic axons probably meet and grow past each other in the internal capsule, the gateway between thalamus and cortex [ 24,26*,27*]. This observation raises the possibility that subplate axons may be required for the formation of connections between thalamus and cortex. If so, then deleting subplate neurons should perturb the development of these connections. The optimal experiment would be to remove subplate neurons beneath the visual cortex for example, well before their axons had formed a complete pathway back to the LGN; technically this experiment has not yet proved possible. If the deletions are performed just after LGN axons have first arrived beneath the visual subplate, but well before they have grown in (see Fig. 2), there is a surprising outcome. LGN axons fail to accumulate and do not wait below the cortical plate. Instead, they grow past the visual cortex and remain within the white matter, never entering the cortex even up to one month later [28-l. The results of this experiment have several implications for the mechanisms that underlie the formation of specific sets of thalamocortical connections. First, they suggest that subplate neurons are somehow involved in the process bywhich thalamic axons select and grow into the appropriate cortical area. Second, they point out that the process of pathway formation and target selection are not necessarily one and the same, as many LGN axons were
Fig.2. A hypothetical diagram illustrating the sets of connections present between thalamus and cortex at early times in development. Axons from thalamic nuclei such as the lateral geniculate nucleus (LCN) grow through the internal capsule until they reach the visual cortex, where they ‘wait’ in the subplate in the vicinity of subplate neurons. The axons of visual subplate neurons, in turn, have already pioneered the pathway out of the cortex and have arrived at the LGN, where they also wait. The cortical plate is still forming and only consists of neurons belonging to deeper cortical layers (5 and 6). (Mitotic figures in the ventricular zone, and postmitotic cells migrating along radial glia, are shown in the right of the diagram.) The axons of layer 6 cortical neurons are still growing en route to their thalamic target nuclei. It is conceivable that the axons of thalamic and subplate neurons meet in the internal capsule and fasciculate together as they grow to their appropriate targets. The later growing layer 6 axons could then follow a preformed axonal pathway to the correct thalamic target. Erased on observations from [24,26*,27*1.
already in their correct positions beneath the visual cortex at the time of the deletion of subplate neurons, and yet failed to subsequently grow into the cortex. Third, the results suggest that the cortical plate by itself does not contain sufficient information to promote the normal formation of thalamocortical connections. How might subplate neurons be involved in the process of target selection and ingrowth? One possibility is that the subplate neurons beneath distinct cortical areas are different. But this suggestion would simply move the site of specificity from the cortical plate to the subplate, and while simple conceptually, it is fraught with all the problems discussed earlier in the context of the cortical plate itself. Another possibility is that both spatial and temporal cues are involved in the formation of specific thalamocortical connections. Subplate axons from more posterior regions grow and reach the internal capsule later than those from more anterior regions [24], and axons from more anterior medial thalamic nuclei (e.g. MGN) reach the internal capsule before those from more posterior and lateral locations (e.g. LGN). Consequently, when LGN axons enter the cerebral hemisphere travelling towards the visual cortex, they grow past the auditory subplate, which is already filled with earlier-arriving MGN axons (A Ghosh and C Shatz, unpublished data). Thus, dynamic interactions with the subplate may prevent later-arriving axons from stopping there and force those axons to grow until they arrive in a region free of other thalamocortical axons. If so, then it should be possible to re-route thalamic axons from the MGN, for example, into an inappropriate cortical area such as visual cortex by deleting subplate neurons beneath auditory cortex early in development. The MGN axons could then be forced to ‘compete’ with later growing thalamic axons for available subplate
How are specific
neurons posterior to the lesion area. If MGN axons can grow into foreign cortex, the result would complement the transplantation experiments which show that visual cortex can accept innervation from somatosensofy tierents [P-l. It would also be entirely consistent with the co-culture experiments [10,11**,12**] in the sense that speciiicity between thalamus and cortex is lost in co-culture because all timing relationships are gone. Conclusion In thisreview, I have considered the general question of how the precise sets of connections between thalamus and cortex are formed during development. Experimental evidence suggests that the mechanisms that underlie the laminar specificity of thalamocortical connections may be quite different from those responsible for tangential (areal) specilicity. Despite the fact that the pattern of projection from each thalamic nucleus to the appropriate cortical area appears very precise even at the outset of development, such precision need not require the existence of unique cues restricted to each cortical area. Rather, it seems possible that a series of dynamic interactions involving broader spatial cues, along with temporal gradients of axon growth, could account for the precision in the development of thalamocortical development. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: - . of special interest .. of outstanding interest 1.
SURM, GARRAGH~PE, ROE AW: Experimentally Induced Visual Projections into Auditory Thalamus and Cortex. Science 1988, 242:1437-1441.
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SHAZ CJ: Impulse Activity and the Patterning of Connections During CNS Development. Neuron 1990, 5:74%756.
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AUSTIN CP, CEPKO CL: Cellular Migration Patterns in tbe Developing Mouse Cerebral Cortex. Development 1990, 110:713-732. A thorough lineage tracing study in which a retrotirus was used to label clonally related cells in the developing mouse cortex and the subsequent location of labeled cells within the cortex examined. Results show that members of a given clone can become wideiy dispersed during migration and end up in distant cortical locations, suggesting that the areal identity of cortex cannot arise due to a simple restriction of migration of clonally related cells. SCHIAGGARBL, O’L~RY DDM: Potential of Visual Cortex to Develop an Array of Functional Units Unique to Somatosensory Cortex. Science 1991, 252:15561560.
formed
between
thalamus
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Shatz
This creative transplantation experiment demonstrates that visual cortex transplanted to the site of somatosensoty cortex can accept innervation from somatosensoly thalamus and form barrels in uiYg. 10.
YAMAMOTO N, KUROTANI T, TOYAMAK: Neural Connections Between Lateral Geniculate Nucleus and Visual Cortex In Vitro. Science 1989, 245:192-194.
of BOL? J, NOVAKN, GOTZ M, BONHOEFFERT: Formation Target-Specific Neuronal Projections in Organotypic Slice Cultures from Rat Visual Cortex. Nature 1990, 346:359-362. Here the authors show convincingly that co-culturing thakunus and cortex in vitro leads to the formation of laminar specific interconnections between the thalamus and layers 4 and 6 of the cortex. 11. ..
12. ..
MOLNAR 2, BL~KEMOREC: Lack of Regional
13.
HE X, TREKY MN, SIMMONS DM,/INGRAHAM H& SWANSON LW, ROSENFELD MG: Expression of a Large Family of F’OU-Domain Regulatory Genes in Mammalian Brain Development. Nature 1989, 340:3542.
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CAUAWAV EM, KATZ LC: Emergence and Refinement of Clustered Horizontal Connections in Cat Striate Cortex. J Neuroxi 1990, lo:11341153.
Specificity for Connections Formed Between Thalamus and Cortex in CoCulture. Nature 1991, 351:47w77.’ The authors address the important question of whether the specificity of connections between thalamic nuclei and corticaLtarget areas can be reconstituted in t&r0 By co-culturing LGN with visual or frontal cortex, the authors find that thalamic neurons exhibitobvious preference for one cortical area over the other. “* : t
KATZ IC: Specificity in the Development of Vertical Con15. . nections in Cat Striate Cortex. EurJ Neurosci 1991, 31-9. An intracellular labeling study examining the morphological development of axonal projections from cortical layer 3 neurons which shows that the vertical (interlaminar) connections formed by growing axons are specific at the outset and do not undergo extensive collateral remodeling. This contrasts with the horizontal (interlaminar) axonal connections from the same neurons (see [ 141). 16.
KOSTOV~C I, RAKICP: Cytology and Time of Origin of Interstitial Neurons in tbe White Matter in Infant and Adult Human and Monkey Telencephalon. J Neurocylo 1980, 9:213-242.
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IJJSKINME%, SHATZCJ: Studies
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CA’IXIANO SM, ROBERTSON RT, KILL~CK!ZY HB: Early Ingrowth of Thalamocortical AlTerems to the Neocortex of the Prenatal Rat. Proc Nat1 Acud Sci USA 1991, 88:29993003. An anatomical study of the timecourse and pattern of development of the thalamocortical projection in rodents using the vety sensitive tracing technique of Dii labeling The results indicate that thalamwortical axons in rodents may not wait in the subplate before growing into the cortical plate. 18. .
F!UAKJF E, MCCONNELL SK, SHAIZ CJ: Functional Synaptic Circuits in the Subplate During Fetal and Early Postnatal Development of Cat Visual Cortex. J Neurosci 1990, 10:260-2613. Intracellular recordings from subplate neurons in cortical slices in vitro during electrical stimulation of the white matter demonstrate that they receive synaptic inputs, implying the presence of a functional microcircuit in fetal telencephalon. 19. .
20.
CHUNJJM, SHA’IZCJ: The Earliest-Generated Neurons of the Cat Cerebral Cortex: Characterization by MAP2 and Neurotransmitter Immunohistochemistry During Fetal Life. / Neurosci 1989, 91648-1667.
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ANTONINI A, SHATZCJ: Relation Between Putative Transmitter Phenotypes and Connectivity of Subplate Neurons During Cerebral Cortical Development. Eur J Neurasci 1990, 2:744-761.
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CHUN JJM, SHA’IZ CJ: Interstitial Cells of the Adult Neocortical White Matter are the Remnant of the Early Generated Subplate Neuron Population. J Comp New-d 1989, 282:555-569.
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Development 23. .
JA Distribuof Alx-50 Immunoreactive Cells in tion and Morphology the Developing Visual Cortex of Kittens. J Neurccytol1990, 19662471. Ah-50 is an antigen associated with cortical neurons in Alzheimer’s disease and its presence is loosely correlated with cell death in other sys terns. Here the authors demonstrate that subplate neurons become immunoreactive during the period in which they undergo programmed cell death. 24.
QUE LL, DECAWS VALVERDEF, LOPEZ-iVkKAR4
MCCONNEU.SK, GHO~H A SHATZ CJ: Subplate Neurons Pioneer the First Axon Pathways from the Cerebral Cortex. Science 1989, 245:278281.
25. .
Ktht GJ, SBATZ CJ, MCCONNEU SK: Morphology of Pioneer and Follower Growth Cones in the Developing Cerebral Cortex. J Neumbiol 1991, 22:629-642. The authors use DR labeling to examine the morphology of growth cones from cortical neurons en route to the thalamus. Early growth cones (presumably from subplate neurons) are much larger and more complex than those from later growing (presumably cortical plate) neurons. DE CARGOS JA, O’LEARYDDM: Growth and Targeting of Subplate Axons and Establishment of Major Cortical Pathways. J Neurosci 1991, in press. This study examines the earliest axons to grow out of cerebral cortex in the rodent somatosensory system by means of Dil labeling. Results suggest that, as in highei mammals, subplate axons in rodents pioneer the early pathway from cortex to thalamus. The authors argue, how26. .
ever, that subplate neuron axons do not pioneer the entire pathway into the spinal cord, rather, layer 5 pyramidal axons appear to be the first cortical axons to grow into the descending corticospinal tract. CJ: Pathtinding and Target Selection. J Neumci 1991, 11: in press. ;n this DiI labeling study of the development of the geniculocortic~ projection in the cat, the authors reaffirm and extend previous observations that thalamocortical axons ‘wait’ in the subplate for almost 3 weeks before growing into the cortical plate. Moreover, LGN axons branch extensively within the subplate, suggesting that there may be dynamic interactions with subplate neurons. 27.
GHOSH A, Smm
GHOSH A, ANTom 4 MCCONNELLSK, SHAR CJ: Requirement for Subplate Neurons in the Formation of Thalamocortical Connections. Nature 1990, 3473179181. In this experiment, subplate neurons were ablated using injections of kainic acid, and the consequences for the development of the genicu locortical projection subsequently examined by means of DiI labeling. In the absence of subplate neurons, LGN axons failed to grow into the cortical plate and instead remained within the white matter, traveling for long distances away from the visual cortex. This experiment suggests that subplate neurons participate in the process of target recognition and ingrowth by thalamic axons.
28. ..
CJ Shatz, Division of Neurobiology, Department of Molecular and Cell Biology, ISA 221, University of California, Berkeley, California 94720, USA