Termination and initial branch formation of SNAP-25-deficient thalamocortical fibres in heterochronic organotypic co-cultures

We are interested in the role of neural activity mediated through regulated vesicular release in the stopping and early branching of the thalamic projections in the cortex. Axon outgrowth, arrival at the cortical subplate, side-branch formation during the waiting period and cortical plate innervation of embryonic thalamocortical projections occurs without major abnormalities in the absence of regulated release in Snap25?(-/-) null mutant mice [Washbourne et al. (2002) Nat. Neurosci. 5:19-26; Molnár et al. (2002) J. Neurosci. 22:10313-10323]. The fact that Snap25?(-/-) null mutant mice die at birth limited our previous experiments to the prenatal period. We therefore investigated the behaviour of thalamic projections in co-culture paradigms by using heterochronic thalamic [embryonic day (E)16-E18] and cortical [postnatal day (P)0-P3] explants, in which the stopping and branching behaviour has been previously documented. Our current co-culture experiments established that thalamic projections from E16-E18 Snap25(+/+) or Snap25?(-/-) explants behaved in an identical fashion in P0-P3 Snap25?(+/+) cortical explants after 7 days in vitro. Thalamic projections from Snap25?(-/-) explants developed similar patterns of fibre ingrowth to the cortex, and stopped and formed branches at a similar depth in the Snap25(+/+) cortical slice as in control cultures. These results imply that thalamic projections can reach their ultimate target cells in layer 4, stop, and start to develop branches in the absence of regulated vesicular transmitter release from their own terminals.     

Mechanisms controlling the guidance of thalamocortical axons through the embryonic forebrain

Thalamocortical axons must cross a complex cellular terrain through the developing forebrain, and this terrain has to be understood for us to learn how thalamocortical axons reach their destinations. Selective fasciculation, guidepost cells and various diencephalic and telencephalic gradients have been implicated in thalamocortical guidance. As our understanding of the relevant forebrain patterns has increased, so has our knowledge of the guidance mechanisms. Our aim here is to review recent observations of cellular and molecular mechanisms related to: the growth of thalamofugal projections to the ventral telencephalon, thalamic axon avoidance of the hypothalamus and extension into the telencephalon to form the internal capsule, the crossing of the pallial-subpallial boundary, and the growth towards the cerebral cortex. We shall review current theories for the explanation of the maintenance and alteration of topographic order in the thalamocortical projections to the cortex. It is now increasingly clear that several mechanisms are involved at different stages of thalamocortical development, and each contributes substantially to the eventual outcome. Revealing the molecular and cellular mechanisms can help to link specific genes to details of actual developmental mechanisms.     

The organization of thalamic connections

Connections ascending to the thalamus. Contrary to classical opinion, all thalamic nuclei receive extrathalamic afferents. Segregation or convergence within a topographically defined nucleus represent two modalities of thalamic afferents. In addition, certain topographically organized thalamic afferents possess "privileged" or primary "targets" in the thalamic nucleus while others possess supplementary "targets" in other thalamic nuclei (see cerebellar, pallidal and spinothalamic projections). Ascending connections from several brain stem structures can converge on the same nucleus or diverge to several thalamic nuclei. Thalamic connections with the telencephalon. Methods for determining axonal transport have demonstrated that all thalamic nuclei, with the exception of the reticular nucleus and the ventral part of the lateral geniculate body, project towards the cerebral cortex. Four nuclear complexes can be recognized in the cat as a function of the different modalities of localization, concentration and lamination of the projections towards the cortex and the central grey nuclei. In general, the thalamocortical connections have reciprocal ipsilateral corticothalamic projections originating in the infragranular layers of the cerebral cortex. The reticular nucleus and the ventral part of the lateral geniculate body, which is not projected to the cerebral cortex, are exceptions. Each cortical area receives a "privileged" connection from a thalamic nucleus and a supplementary connection- from one or several other thalamic nuclei. The "privileged" connections usually pass to the fourth and third layers of the neocortex, and sometimes also to the first layer. In contrast, the supplementary connections pass to different superficial or deep cortical layers. Each nucleus is formed of subunits which possess different hodologic and topographic characteristics as a function of the nucleus considered. Convergence or divergence of thalamocortical and corticothalamic projections on the different thalamic nuclei, as well as the laminar distribution of efferents in the cerebral cortex, are related strictly to the hodologic organization of different cellular subunits constituting the nuclei. Concentration or diffusion of thalamic projections on cerebral cortex is related more to the single or multiple projection of cell populations belonging to a thalamic nucleus than to widespread collateralization of thalamocortical axons.     

Thalamic connections of the cortex of the superior temporal sulcus in the rhesus monkey

The thalamocortical connections of the superior temporal sulcus (STS) were studied by means of the WGA-HRP retrograde tracing technique. The results indicate that the distribution of thalamic projections varies along the rostral-caudal dimension of the STS. Thus the rostral portion of the upper bank receives input primarily from the medialmost portion of the medial pulvinar (PM) nucleus. The middle region of the upper bank receives projections from medial and central portions of the PM nucleus, and also from the oral pulvinar, limitans, suprageniculate, medial geniculate, and dorsomedial nuclei. The cortex of the caudal portion of the upper bank has basically similar thalamic input; however, the projections from the PM nucleus originate in central and lateral portions. Additionally, there are projections from the lateral pulvinar (PL), ventroposterolateral, central lateral, parafascicular, and paracentral nuclei. In contrast to the dorsal bank, the cortex of the ventral bank of the STS receives somewhat different and less extensive thalamic input. The rostral portion of the lower bank receives projections only from the ventromedial sector of the PM nucleus, whereas the middle portion of the lower bank receives projections from the PL and the inferior pulvinar nuclei as well as from the PM nucleus. The upper bank of the STS, on the basis of physiological and anatomical studies (Jones and Powell, '70; Seltzer and Pandya, '78; Gross et al., '81; Baylis et al., '87), has been shown to contain multimodal areas. The present data indicate that the multimodal region of the STS has a preferential relationship with the central sector of the PM nucleus.     

Thalamic projections to sensorimotor cortex in the newborn macaque.

In the present experiments thalamocortical projections to different functional areas of the newborn (or prematurely delivered) macaque's sensorimotor cortex were labeled using retrogradely transported fluorescent dyes. Several dyes were used in each animal to (1) enable the direct comparison of the soma distributions of different thalamocortical projections within thalamic space, and (2) identify by double labeling neurons shared between these distributions. The projection patterns in the newborn macaque were compared with those of the mature animal reported by Darian-Smith et al. (J. Comp. Neurol. 1990;298:000-000). The main observations were (1) all thalamocortical projections to the sensorimotor cortex of the mature macaque are well established by embryonic days 146-150, as was shown by labeling these pathways in infants delivered by cesarean section, (2) a significant number of thalamocortical neurons in the newborn were double-labeled following dye injections into different pre- or postcentral areas, and where the margins of the dye uptake zones were separated by 3-8 mm, and (3) extensive projections from the anterior pulvinar nucleus to the motor and premotor cortex, and to the supplementary motor cortex were labeled in the newborn macaque. Both the exuberant terminal arborizations, and the precentral pulvinar projections were diminished by the 6th postnatal month, and absent in the mature macaque. The role of epigenetic determinants of these postnatal events is briefly considered.     

The insular auditory field receives input from the lemniscal subdivision of the auditory thalamus in mice

The insular cortex plays important roles in vocal communication, but the origin of auditory input to the insular cortex has not been fully clarified. Here we studied the auditory thalamic input to the insular cortex using mice as a model system. An insular auditory field (IAF) has recently been identified in mice. By using retrograde neuronal tracing, we identified auditory thalamic neurons projecting to the IAF, primary auditory cortex (AI), and anterior auditory field (AAF). After mapping the IAF, AAF, and AI by using optical imaging, we injected a distinct fluorescent tracer into each of the three fields at frequency‐matched locations. Tracer injection into the IAF resulted in retrogradely labeled cells localized ventromedially in the lemniscal division, i.e., the ventral subdivision of the medial geniculate body (MGv). Cells retrogradely labeled by injections into the AAF were primarily found in the medial half of the MGv, whereas those from AI injections were located in the lateral half, although some of these two subsets were intermingled within the MGv. Interestingly, retrogradely labeled cells projecting to the IAF showed virtually no overlap with those projecting to the AAF or the AI. Dual tracer injections into two sites responding to low‐ and high‐frequency tones within each of the three auditory fields demonstrated topographic organizations in all three thalamocortical projections. These results indicate that the IAF receives thalamic input from the MGv in a topographic manner, and that the MGv–IAF projection is parallel to the MGv–AAF and MGv–AI projections. J. Comp. Neurol. 522:1373–1389, 2014. © 2013 Wiley Periodicals, Inc.     

Dorsal telencephalon-specific RA-GEF-1 knockout mice develop heterotopic cortical mass and commissural fiber defect

Neural migration defects lead to various types of human malformations of cortical development including subcortical band heterotopia, which shows formation of a secondary cortical plate beneath the primary cortex and is typically caused by mutation of the DCX ( doublecortin ) gene. Subcortical band heterotopia is usually associated with mental retardation and epilepsy. We previously discovered RA-GEF-1 as a guanine nucleotide exchange factor (GEF) for Rap1 small GTPase. Here we have analysed its in-vivo role in formation of the adult cerebral cortex by using telencephalon-specific RA-GEF-1 conditional knockout (cKO) mice, generated by mating RA-GEF-1 ^{flox / flox} mice with Emx1-cre knockin mice. RA-GEF-1 cKO mice showed severe defects in their brain structures including an ectopic cortical mass underlying a relatively normal cortex. The ectopic cortical mass lacked the normal six-layered lamination but preserved the subcortical connectivity as revealed by retrograde tracing. Further, RA-GEF-1 cKO mice exhibited a lower threshold for the induction of epileptic seizures. These phenotypes have a resemblance to those of human subcortical band heterotopia. In addition, the agenesis of anterior commissures, the dorsal hippocampus commissure, the corpus callosum and the enlargement of the lateral ventricles were observed in cKO mice. Our findings suggest a crucial function of RA-GEF-1 in neural migration.     

Laminar specific alterations of thalamocortical projections in organotypic cultures following layer 4 disruption in ferret somatosensory cortex

The developing neocortex influences the growth of thalamocortical projections. Layer 4 in particular receives the majority of input from the thalamus and is important in instructing thalamic afferents to terminate. Previous in vivo experiments demonstrated that disruption of layer 4 during corticogenesis in ferret somatosensory cortex by application of methylazoxy methanol acetate (MAM) prevents proper termination of thalamic afferents in appropriate cortical regions. To further explore the role of layer 4 in thalamocortical development, we prepared organotypic cocultures consisting of normal gestational day 0 (P0) ferret thalamus paired with normal, embryonic day 33 (E33), or E38 MAM-treated cortex obtained from ferrets at either P0 or P7. Injection of MAM on E33 disrupts layer 4 formation, whereas similar injections on E38 interfere with layer 2 formation. The cocultures grew together for a number of days, then discrete injections of either fluorescent dextrans or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were made into the thalamic piece. The labeled thalamic afferents that grew into the cortical slice were analysed and the sites of their terminations quantified after 3, 5, or 7-10 days in culture (DIC). Our results varied somewhat with the amount of time in culture, but the preponderance of thalamic fibers in normal cortex terminated in layer 4, whereas their counterparts in E33 MAM-treated cortex grew beyond the cortical plate and many fibers terminated inappropriately within lower cortical layers or white matter. Terminal distribution of thalamic fibers in E38 MAM-treated cortex looked similar to normal. These results demonstrate that the cells of layer 4 provide thalamic afferents with important positional and termination cues.     

VGluT2 immunochemistry identifies thalamocortical terminals in layer 4 of adult and developing visual cortex

A vesicular glutamate transporter, VGluT2, has been suggested to be the transporter utilized in the thalamocortical pathway. We examined the reliability of this marker in identifying and discriminating thalamic terminals in adult and developing ferret visual cortex. We studied brain sections stained for the transporter protein and/or anterogradely filled thalamocortical or intracortical axons, by using light, confocal, and electron microscopy. Under light microscopy, VGluT2 immunoreactivity (ir) in adult animals [past postnatal day (P)90] and in neonatal animals as early as P27 formed a dense band in layer 4 and appeared as scattered puncta in layers 6 and 1. Confocal dual-labeling analyses of P46 and adult striate cortices indicated that VGluT2 was present in thalamocortical axons, suggesting that thalamic projections utilize this transporter during postnatal development as well as adulthood. In contrast, extracellularly filled intracortical axons failed to colocalize with VGluT2-ir, suggesting that no significant terminal population originating in cortex contained VGluT2 in layer 4. Electron microscopic analysis revealed that, in adult layer 4, VGluT2-ir was present in large terminals, forming asymmetric synapses. Similar to anterogradely labeled thalamocortical terminals, VGluT2-ir synaptic terminals were different from their unlabeled counterparts in terms of terminal area (0.6 vs. 0.3 microm), synaptic length (486 vs. 353 nm), and preference for synapsing on spines (77% vs. 59%). Moreover, no significant differences were found between VGluT2-ir and anterogradely labeled thalamocortical terminals. Comparable similarities were also demonstrated at P46. These results indicate that thalamocortical terminals in layer 4 of visual cortex utilize VGluT2 and suggest that this marker can be used to identify thalamic axons specifically in adult and developing animals.     

Corticothalamic neurons and thalamocortical terminal fields: an investigation in rat using horseradish peroxidase and autoradiography.

Subsequent to thalamic injections in rats of horseradish peroxidase (HRP) alone or HRP and [3H]leucine in combination, the cells of origin of the corticothalamic projections and the terminal fields of the thalamocortical projections were identified. HRP-labeled corticothalamic neurons were uniformly found in layers V and VI. They were medium to small in size and always pyramidal in shape with the larger neurons being found in layer V. On the other hand, 3 different patterns for the distribution of thalamocortical terminal fields were observed. The autoradiographic material indicated that in prefrontal cortex the bulk of thalamocortical fibers terminate in layer III while in motor cortex they terminate primarily in layer V. A third pattern was shared by temporal, occipital and parietal cortex where the bulk of thalamocortical fibers terminate preferentially in layer IV. The data derived from the rats which had received thalamic injections of HRP and [3H]leucine in combination indicated that the connections between cortex and thalamus are in general reciprocal. These results are discussed with regard to earlier studies using classical or more recently developed neuroanatomical methods.     

Development of thalamocortical projections in the South American gray short-tailed opossum (Monodelphis domestica)

We determined the time-course and general pattern of thalamocortical development of Monodelphis domestica by tracing projections with carbocyanine dye in fixed postnatal brains between postnatal day 2 (P2) and P30. By P2, the first neurons have migrated to form the preplate of the lateral cortex and have sent out axons into the intermediate zone. By P3, fibers from the preplate of more dorsal cortex have entered the intermediate zone, and, by P5, they reach the primitive internal capsule. Crystal placements in the dorsal thalamus at P2–P3 reveal thalamic axons extending down through the diencephalon and growing out through the internal capsule among groups of back-labelled cells that already project into the thalamus. Thalamic axons arrive at the cortex after the arrival of cells of the true cortical plate has split the preplate into marginal zone and subplate. Axons from the ventral part of the dorsal thalamus reach the lateral cortex by P5: Dorsal thalamic fibers arrive at the extreme dorsal cortex by P9. The deeper layers of the cortex appear to mature relatively earlier in Monodelphis than in eutherian mammals, and the subplate becomes less distinct. Thalamic fibers and their side branches proceed into the cortex without an obvious period of waiting in the subplate, but they do not penetrate the dense cortical plate itself. Monodelphis could provide an excellent model species, because the development of its thalamocortical connections is entirely an extrauterine process: The period P0–P15 corresponds to that of E12-P0 in the rat. J. Comp. Neurol. 398:491–514, 1998. © 1998 Wiley-Liss, Inc.     

Connections between cells of the internal capsule,thalamus, and cerebral cortex in embryonic rat

The aim of our study is to understand the development of the earliest connections in the mammalian pallium by documenting the distribution of cells and fibres labelled from the dorsal and ventral thalamus, internal capsule, perirhinal, and dorsal cortex during the period between embryonic day (E) 14 and 17 by using carbocyanine dye tracing in fixed embryonic rat brains. Dye placed in the thalamus of E14 brains backlabels cells in the thalamic reticular nucleus and within the primitive internal capsule. Both anterograde and retrograde tracing confirmed that the first corticofugal projections reach the internal capsule by E14. At E15–E16, after the first cortical plate cells have migrated into the lateral cortex, some cells of the cortical plate and subplate and marginal zone, are backlabelled from the internal capsule, but still not from the dorsal thalamus, even with very long incubation periods. Crystal placement into the perirhinal cortex at E14–E15 labels numerous cells within the internal capsule, whereas no such cells are revealed from dorsal cerebral cortex until E17, suggesting that internal capsule cells establish early connections with the perirhinal and ventral but not dorsal cortex. We propose that the growth of axons from cortex to dorsal thalamus is delayed in two regions: first from E14–E15 at the lateral entrance of the internal capsule and then, from E16, closer to the thalamus, probably within the thalamic reticular nucleus. Subplate projections reach the proximity of the diencephalon at an early stage, but they might never enter the dorsal thalamus. J. Comp. Neurol. 413:1–25, 1999. © 1999 Wiley-Liss, Inc.     

Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.     

Role of semaphorin III in the developing rodent trigeminal system.

Semaphorins are a large family of secreted and transmembrane glycoproteins. Sema III, a member of the Class III semaphorins is a potent chemorepulsive signal for subsets of sensory axons and steers them away from tissue regions with high levels of expression. Previous studies in mutant mice lacking sema III gene showed various neural and nonneural abnormalities. In this study, we focused on the developing trigeminal pathway of sema III knockout mice. We show that the peripheral and central trigeminal projections are impaired during initial pathway formation when they develop into distinct nerves or tracts. These axons defasciculate and compromise the normal bundling of nerves and restricted alignment of the central tract. In contrast to trigeminal projections, thalamocortical projections to the barrel cortex appear normal. Furthermore, sema III receptor, neuropilin, is expressed during a short period of development when the tract is laid down, but not in the developing thalamocortical pathway. Peripherally, trigeminal axons express neuropilin for longer duration than their central counterparts. In spite of projection errors, whisker follicle innervation appears normal and whisker-related patterns form in the trigeminal nuclei and upstream thalamic and cortical centers. Our observations suggest that sema III plays a limited role during restriction of developing trigeminal axons to proper pathways and tracts. Other molecular and cellular mechanisms must act in concert with semaphorins in ensuring target recognition, topographic order of projections, and patterning of neural connections.