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Two-photon in vivo imaging reveals migration of cells with multipolar morphology. a, Schematic of the experimental setup for in vivo time-lapse imaging. New neurons were labeled by injecting an oncoretroviral vector carrying GFP into the VZ adjacent to HVC. Mature HVC X neurons (red dots) were retrogradely labeled with DiI injections into area X (red circle). We imaged HVC, from 4 to 22 dpi, with a two-photon microscope (2PM). Field of view was 700 1000 200 m (depth). b, Example of a maximum intensity projection of a multipolar GFP cell (green) within HVC imaged in vivo at 7 dpi using two-photon microscopy. HVC X somata are labeled with DiI (red). Scale bar, 50 m. c, Maximum intensity projection of two GFP multipolar cells (labeled in green and marked by a blue asterisk and a blue arrowhead) and DiI somata of HVC X neurons (red) at 8 dpi. Arrowhead indicates the same multipolar cell shown in b. Top shows the relative position of the two GFP cells. Scale bar, 100 m. Bottom shows a reconstruction of the migratory trajectory of the multipolar cell in b (marked with a blue arrowhead) over 120 h (white line); white circles indicate the position of the cell body of the migrating cell recorded at 12 h intervals. Note that the cell moved a short distance over the first 12 h of its recorded trajectory; thus, the white circles for the first and second time points are partially overlapping. d, Reconstructed morphology for the multipolar cell shown in b and c at 12 h intervals. Time in hours is indicated in the top left corner. Red line indicates approximate location of the border of HVC. Grid spacing is 80 m. A, Anterior; L, lateral; V, ventral.
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Most non-mammalian vertebrate species add new neurons to existing brain circuits throughout life, a process thought to be essential for tissue maintenance, repair, and learning. How these new neurons migrate through the mature brain and which cues trigger their integration within a functioning circuit is not known. To address these questions, we us...
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... cell over multiple weeks (see Materials and Methods). Therefore, we followed GFP cells (n 92) for different durations, at 6 min, 3 h, 12 h, or 48 h intervals (Table 1). To accurately identify the positions of GFP cells across successive imaging sessions, we retrogradely labeled HVC X neurons by injecting the fluorescent marker DiI into area X ( Fig. 3a; see Materials and Methods). The positions of DiI- labeled HVC X neurons relative to each other did not change over time and were used to register imaging fields across successive imaging sessions (see Materials and ...
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... first examined the behavior of GFP cells (n 14) at 3 or 12 h intervals over 1-6 d. The majority of GFP cells (13 of 14) lacked obvious polarity and had multiple processes extending in different directions, closely resembling the multipolar GFP cells observed in fixed tissue sections (Fig. 3b). The remaining cell had a bipolar morphology that, as expected, migrated in a straight line away from the lateral ventricle. Surprisingly, multipolar cells were also migratory, moving as much as 126 m in a single 12 h period (mean SD, 42.6 27.1 m). These cells appeared to migrate for multiple days. The longest trajectory we were able ...
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... straight line away from the lateral ventricle. Surprisingly, multipolar cells were also migratory, moving as much as 126 m in a single 12 h period (mean SD, 42.6 27.1 m). These cells appeared to migrate for multiple days. The longest trajectory we were able to observe before the multipolar cell left the field of view was 350 m long and took 120 h (Fig. 3c). Two features of the migratory behavior of these cells were surprising and deviated from previ- ously described forms of migration. First, multipolar cells did not migrate along straight paths; instead, they frequently changed directions and appeared to take a meandering course through HVC. As a result, the tortuosity () of their ...
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... to take a meandering course through HVC. As a result, the tortuosity () of their trajectories, defined as the ratio between path length and the distance between the start and end points of the path, was high ( 1.69 0.81). Second, cells changed morphology significantly between imaging ses- sions, adding and removing most of their processes (Fig. 3d). The tortuous paths of multipolar cells could be indicative of migration along blood vessels, which serve as a scaffold for mi- grating neurons in other regions of the brain (Bovetti et al., 2007). We therefore compared the migration paths of 11 multipolar GFP cells with the pattern of blood vessels ( Fig. 4; see Materials and ...
Citations
... 10 Retroviral labeling studies have revealed that migratory neurons in juvenile zebra finches follow tortuous paths that resemble a random walk and do not appear to exclusively follow radial fibers or blood vessels. 11 Together, these observations suggest the existence of a non-radial form of migration in the postnatal songbird brain. However, a detailed description of this process and whether it continues into adulthood is lacking. ...
... These dynamics were similar to those of retrovirally labeled migratory neurons in the juvenile songbird HVC. 11 Similar dynamics were observed in the brains of adult birds 86-800+ days post hatch (dph; 3 males, 3 females, n = 594 cells). Both within HVC and in other regions of the nidopallium, we observed a large number of migrating cells that were distributed throughout the tissue and dispersing in all directions while making turns (Videos S2 and S3). ...
... This is consistent with previous studies that have suggested that migrating neurons in HVC may not be dependent on Vim+ fibers for migration. 7,10,11 To evaluate association with vasculature, we labeled the vasculature with SR-101 at the end of in vivo imaging sessions and compared the migratory trajectories with the pattern of blood vessels (n = 115 cells, n = 2 birds) ( Figure S5; Video S4). Over a 3-h imaging session, 21% of cells exhibited trajectories that closely aligned with the path of the vasculature (n = 17/81 cells, 1 bird; STAR Methods). ...
... Postnatal migration of inhibitory interneurons is prominent in the adult rostral migratory stream (RMS) 28 and early postnatal mouse forebrain 29 . In contrast, excitatory neuron migration is more rare in the adult brain, with local migration of dentate granule neurons 30 and longer excitatory neuron migration in the avian brain 31 . Since PL neurons are excitatory, their migration into nearby brain regions would be remarkable, and could contribute to observed increases in neuron number in some amygdala regions 6,7,32 . ...
The human amygdala paralaminar nucleus (PL) contains immature excitatory neurons that exhibit protracted maturation into adolescence; however, whether a similar population exists in mice is unknown. We discovered a previously undescribed region with immature doublecortin (Dcx)+ excitatory neurons adjacent to the mouse basolateral amygdala, and similar to humans, these neurons mature during adolescence and are distinct from adjacent intercalated cells. Despite their immature features, these neurons are born during embryogenesis, populate the mouse PL prior to birth, and remain in an immature stage of development until adolescence. In the postnatal brain, a subpopulation of these excitatory neurons surprisingly migrate into the neighboring endopiriform cortex, peaking between P21-P28. In humans, cells with the molecular identity of mouse PL neurons populate the PL as early as 18 gestational weeks, and also exhibit migratory morphology into adolescence (13 years). The finding of a similar region in both mice and humans suggests a potentially conserved cellular mechanism for neuron recruitment and migration during adolescence, a key time period for amygdala circuit maturation and behavioral changes.
... In the mouse cortex during prenatal and early postnatal development, high expression levels of Sox11 promote neuron migration, but when Sox11 is downregulated, migration stops and dendrite morphogenesis begins (Hoshiba et al., 2016). Similarly, in the forebrain of juvenile zebra finches, migratory neurons only begin to develop dendritic processes after they stop migrating (Scott et al., 2012). These neurons use somal translocation to migrate along an existing process, rather than following a path pre-defined by blood vessels or radial glia, and travel in a short-range "wandering" pattern. ...
The human amygdala is critical for emotional learning, valence coding, and complex social interactions, all of which mature throughout childhood, puberty, and adolescence. Across these ages, the amygdala paralaminar nucleus (PL) undergoes significant structural changes including increased numbers of mature neurons. The PL contains a large population of immature excitatory neurons at birth, some of which may continue to be born from local progenitors. These progenitors disappear rapidly in infancy, but the immature neurons persist throughout childhood and adolescent ages, indicating that they develop on a protracted timeline. Many of these late-maturing neurons settle locally within the PL, though a small subset appear to migrate into neighboring amygdala subnuclei. Despite its prominent growth during postnatal life and possible contributions to multiple amygdala circuits, the function of the PL remains unknown. PL maturation occurs predominately during late childhood and into puberty when sex hormone levels change. Sex hormones can promote developmental processes such as neuron migration, dendritic outgrowth, and synaptic plasticity, which appear to be ongoing in late-maturing PL neurons. Collectively, we describe how the growth of late-maturing neurons occurs in the right time and place to be relevant for amygdala functions and neuropsychiatric conditions.
... The same applies to other described functions of HVC (X) neurons in the HVC nucleus, such as retinoic acid synthesis (65). Here, the mRNA of its synthesizing enzyme is only expressed by HVC (X) neurons, but its proteins are found in neighboring HVC (RA) neurons (66) or the guidance of newly born HVC (RA) neurons by interactions with HVC (X) neurons (67). All of these hypothesized functions of HVC (X) neurons in the HVC microcircuit would be affected by HVC (X) neuron ablation. ...
Significance
We addressed the question, “How do corticobasal ganglia projecting neurons contribute to vocal learning?” We performed specific ablation of the vocal cortical neurons projecting to the basal ganglia, HVC (X) neurons in a songbird, which generate temporally precise firing during singing. Specific ablation of HVC (X) neurons in juveniles caused deficits in learning the tutor song’s acoustics and less consistency of song sequence. In contrast, adult HVC (X) neuron ablation did not affect the degree of vocal fluctuations or cause alteration in song structure by auditory feedback inhibition. These results support the hypothesis that HVC (X) neurons are a neural substrate for transferring temporal signals, but not for regulating vocal fluctuations or conveying auditory feedback, to the basal ganglia for vocal learning and maintenance.
... Studies in mutant mice with radial glial defects have revealed corresponding defects in neuronal migration suggesting that these cells appear to provide an adequate framework to guide neurons toward their targets (Götz et al., 1998;Pinto and Götz, 2007). In addition, RGL α cells are putative stem cells involved in astrogenesis and neurogenesis, and radial neuronal migration along radial glia fibers has been detected in both immature and adult brain (Scott et al., 2012;Lever et al., 2014;Sun et al., 2015;Bonaguidi et al., 2016;Gebara et al., 2016;Falk and Götz, 2017;Berg et al., 2018). Furthermore, RGL α cells share common molecular signatures with GFAP-immunolabeled astrocytes (Scott et al., 2012;Renzel et al., 2013;Matsue et al., 2018). ...
... In addition, RGL α cells are putative stem cells involved in astrogenesis and neurogenesis, and radial neuronal migration along radial glia fibers has been detected in both immature and adult brain (Scott et al., 2012;Lever et al., 2014;Sun et al., 2015;Bonaguidi et al., 2016;Gebara et al., 2016;Falk and Götz, 2017;Berg et al., 2018). Furthermore, RGL α cells share common molecular signatures with GFAP-immunolabeled astrocytes (Scott et al., 2012;Renzel et al., 2013;Matsue et al., 2018). ...
Little is known about environmental influences on radial glia-like (RGL) α cells (radial astrocytes) and their relation to neurogenesis. Because radial glia is involved in adult neurogenesis and astrogenesis, we investigated this association in two migratory shorebird species that complete their autumnal migration using contrasting strategies. Before their flights to South America, the birds stop over at the Bay of Fundy in Canada. From there, the semipalmated sandpiper (Calidris pusilla) crosses the Atlantic Ocean in a non-stop 5-day flight, whereas the semipalmated plover (Charadrius semipalmatus) flies primarily overland with stopovers for rest and feeding. From the hierarchical cluster analysis of multimodal morphometric features, followed by the discriminant analysis, the radial astrocytes were classified into two main morphotypes, Type I and Type II. After migration, we detected differential changes in the morphology of these cells that were more intense in Type I than in Type II in both species. We also compared the number of doublecortin (DCX)-immunolabeled neurons with morphometric features of radial glial–like α cells in the hippocampal V region between C. pusilla and C. semipalmatus before and after autumn migration. Compared to migrating birds, the convex hull surface area of radial astrocytes increased significantly in wintering individuals in both C. semipalmatus and C. pusilla. Although to a different extent we found a strong correlation between the increase in the convex hull surface area and the increase in the total number of DCX immunostained neurons in both species. Despite phylogenetic differences, it is of interest to note that the increased morphological complexity of radial astrocytes in C. semipalmatus coincides with the fact that during the migratory process over the continent, the visuospatial environment changes more intensely than that associated with migration over Atlantic. The migratory flight of the semipalmated plover, with stopovers for feeding and rest, vs. the non-stop flight of the semipalmated sandpiper may differentially affect radial astrocyte morphology and neurogenesis.
... It is possible that some of these cells retain short-range migratory behavior for years to adjust the final location of their soma. In songbirds, young neurons continue to adjust the location of their cell body in the last stages of neuronal migration in a wandering behavior that maybe key to their ultimate integration 53 . ...
The human amygdala grows during childhood, and its abnormal development is linked to mood disorders. The primate amygdala contains a large population of immature neurons in the paralaminar nuclei (PL), suggesting protracted development and possibly neurogenesis. Here we studied human PL development from embryonic stages to adulthood. The PL develops next to the caudal ganglionic eminence, which generates inhibitory interneurons, yet most PL neurons express excitatory markers. In children, most PL cells are immature (DCX+PSA-NCAM+), and during adolescence many transition into mature (TBR1+VGLUT2+) neurons. Immature PL neurons persist into old age, yet local progenitor proliferation sharply decreases in infants. Using single nuclei RNA sequencing, we identify the transcriptional profile of immature excitatory neurons in the human amygdala between 4-15 years. We conclude that the human PL contains excitatory neurons that remain immature for decades, a possible substrate for persistent plasticity at the interface of the hippocampus and amygdala.
... These findings were confirmed posteriorly by ultrastructural analysis [88]. In other areas such as the high vocal center (HVC), approximately 30% of new neurons are associated with radial glia [89]. ...
Adult neurogenesis occurs in many species, from fish to mammals, with an apparent reduction in the number of both neurogenic zones and new neurons inserted into established circuits with increasing brain complexity. Although the absolute number of new neurons is high in some species, the ratio of these cells to those already existing in the circuit is low. Continuous replacement/addition plays a role in spatial navigation (migration) and other cognitive processes in birds and rodents, but none of the literature relates adult neurogenesis to spatial navigation and memory in primates and humans. Some models developed by computational neuroscience attribute a high weight to hippocampal adult neurogenesis in learning and memory processes, with greater relevance to pattern separation. In contrast to theories involving neurogenesis in cognitive processes, absence/rarity of neurogenesis in the hippocampus of primates and adult humans was recently suggested and is under intense debate. Although the learning process is supported by plasticity, the retention of memories requires a certain degree of consolidated circuitry structures, otherwise the consolidation process would be hampered. Here, we compare and discuss hippocampal adult neurogenesis in different species and the inherent paradoxical aspects.
... If true, given that intra-system correlations are pervasive and nuclei can change in size quickly, perhaps one or a few genes coordinate system-wide effects, or mutations affecting one nucleus propagate along a pathway by affecting neuroblast recruitment and/or survival elsewhere [56]. System-specific correlations seem unlikely to result from changes in proliferative zones, because mature nuclei contain neurons originating from distinct progenitor pools and migrating neuroblasts do not appear committed to join specific networks [57,58]. Future studies on nucleus specification and circuit integration will help identify the mechanisms underlying this type of mosaic evolution. ...
Vertebrate brains differ in overall size, composition and functional capacities, but the evolutionary processes linking these traits are unclear. Two leading models offer opposing views: the concerted model ascribes major dimensions of covariation in brain structures to developmental events, whereas the mosaic model relates divergent structures to functional capabilities. The models are often cast as incompatible, but they must be unified to explain how adaptive changes in brain structure arise from pre-existing architectures and developmental mechanisms. Here we show that variation in the sizes of discrete neural systems in songbirds, a species-rich group exhibiting diverse behavioural and ecological specializations, supports major elements of both models. In accordance with the concerted model, most variation in nucleus volumes is shared across functional domains and allometry is related to developmental sequence. Per the mosaic model, residual variation in nucleus volumes is correlated within functional systems and predicts specific behavioural capabilities. These comparisons indicate that oscine brains evolved primarily as a coordinated whole but also experienced significant, independent modifications to dedicated systems from specific selection pressures. Finally, patterns of covariation between species and brain areas hint at underlying developmental mechanisms.
... Of note, in contrast to the mammalian SVZ that generates GABAergic interneurons, the VZ of the zebra finch generates mainly projection neurons . Furthermore, whereas mammalian neuroblasts migrate in chains (Lois et al., 1996), postnatally generated neuroblasts in zebra finch brain migrate individually Scott et al., 2012). Strikingly, in spite of these species differences, we found that serotonergic axons project along all streams of postnatally migrating neuroblasts in zebra finch brain ( Figure 8K; Figures S8A-S8C2). ...
... Interestingly, serotonergic projections were present not only in regions of migratory streams rich in glial fibers, but also in regions devoid thereof ( Figure S8A1). Thus, since in bird brain the majority of postnatally migrating neuroblasts are not guided by glial cells and blood vessels (Scott et al., 2012), serotonergic projections might be involved in the regulation of neuroblast migration. ...
In many vertebrates, postnatally generated neurons often migrate long distances to reach their final destination, where they help shape local circuit activity. Concerted action of extrinsic stimuli is required to regulate long-distance migration. Some migratory principles are evolutionarily conserved, whereas others are species and cell type specific. Here we identified a serotonergic mechanism that governs migration of postnatally generated neurons in the mouse brain. Serotonergic axons originating from the raphe nuclei exhibit a conspicuous alignment with subventricular zone-derived neuroblasts. Optogenetic axonal activation provides functional evidence for serotonergic modulation of neuroblast migration. Furthermore, we show that the underlying mechanism involves serotonin receptor 3A (5HT3A)-mediated calcium influx. Thus, 5HT3A receptor deletion in neuroblasts impaired speed and directionality of migration and abolished calcium spikes. We speculate that serotonergic modulation of postnatally generated neuroblast migration is evolutionarily conserved as indicated by the presence of serotonergic axons in migratory paths in other vertebrates.
... To quantify the number of HVC-projecting RA neurons, we injected a retrograde tracer into HVC (DiI, Invitrogen D3911; 46 nL total injection volume) that labels neurons with high efficiency in zebra finches (Scott et al., 2012). Following a two-day incubation period, animals were perfused with 4% paraformaldehyde, and 100 mm sagittal sections were cut across the entirety of RA, Nucleus Interfacialis (NIf), and nucleus Uvaeformis (Uva). ...
The sequential activation of neurons has been observed in various areas of the brain,
but in no case is the underlying network structure well understood. Here we examined the circuit anatomy of zebra finch HVC, a cortical region that generates sequences underlying the temporal progression of the song. We combined serial block-face electron microscopy with light microscopy to determine the cell types targeted by HVC (RA) neurons, which control song timing. Close to their soma, axons almost exclusively
targeted inhibitory interneurons, consistent with what had been found with electrical recordings from pairs of cells. Conversely, far from the soma the targets were mostly other excitatory neurons, about half of these being other HVC (RA) cells. Both observations are consistent with the notion that the neural sequences that pace the song are generated by global synaptic chains in HVC embedded within local inhibitory networks.
