Differentiation Of Projection Neurons Research Articles

Spatial dynamics of mammalian brain development and neuroinflammation by multimodal tri-omics mapping.

The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag-RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.

Spatial dynamics of mammalian brain development and neuroinflammation by multimodal tri-omics mapping.

The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag-RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.

Neurogenesis and neuronal differentiation in the postnatal frontal cortex in Down syndrome.

Although Down syndrome (DS), the most common developmental genetic cause of intellectual disability, displays proliferation and migration deficits in the prenatal frontal cortex (FC), a knowledge gap exists on the effects of trisomy 21 upon postnatal cortical development. Here, we examined cortical neurogenesis and differentiation in the FC supragranular (SG, II/III) and infragranular (IG, V/VI) layers applying antibodies to doublecortin (DCX), non-phosphorylated heavy-molecular neurofilament protein (NHF, SMI-32), calbindin D-28K (Calb), calretinin (Calr), and parvalbumin (Parv), as well as β-amyloid (APP/Aβ and Aβ1–42) and phospho-tau (CP13 and PHF-1) in autopsy tissue from age-matched DS and neurotypical (NTD) subjects ranging from 28-weeks (wk)-gestation to 3 years of age. Thionin, which stains Nissl substance, revealed disorganized cortical cellular lamination including a delayed appearance of pyramidal cells until 44 wk of age in DS compared to 28 wk in NTD. SG and IG DCX-immunoreactive (-ir) cells were only visualized in the youngest cases until 83 wk in NTD and 57 wk DS. Strong SMI-32 immunoreactivity was observed in layers III and V pyramidal cells in the oldest NTD and DS cases with few appearing as early as 28 wk of age in layer V in NTD. Small Calb-ir interneurons were seen in younger NTD and DS cases compared to Calb-ir pyramidal cells in older subjects. Overall, a greater number of Calb-ir cells were detected in NTD, however, the number of Calr-ir cells were comparable between groups. Diffuse APP/Aβ immunoreactivity was found at all ages in both groups. Few young cases from both groups presented non-neuronal granular CP13 immunoreactivity in layer I. Stronger correlations between brain weight, age, thionin, DCX, and SMI-32 counts were found in NTD. These findings suggest that trisomy 21 affects postnatal FC lamination, neuronal migration/neurogenesis and differentiation of projection neurons and interneurons that likely contribute to cognitive impairment in DS.

Prefrontal Cortical Projection Neurons Targeting Dorsomedial Striatum Control Behavioral Inhibition

A neural pathway from prefrontal cortex (PFC) to dorsal striatum (DS) has been suggested to mediate cognitive control of behavior, including proactive inhibitory control and attention. However, a direct causal demonstration thereof is lacking. Here, we show that selective chemogenetic silencing of corticostriatal PFC neurons in rats increases premature responses. Wireless single-unit electrophysiological recordings of optogenetically identified corticostriatal PFC neurons revealed that the majority of these neurons encode behavioral trial outcome with persistent changes in firing rate. Attentional parameters were not affected by silencing corticostriatal PFC neurons, suggesting that these projection neurons encode a specific subset of cognitive behaviors. Compared to the general non-identified neuronal population in the PFC, frontostriatal neurons showed selective engagement during periods of inhibitory control. Our results demonstrate a role for corticostriatal neurons in inhibitory control and possibly suggest that distinct domains of cognitive control over behavior are encoded by specific projection neuron populations.

Adult Neurogenesis in Injury-Induced Self-Repair: Use It or Lose It.

One of the most hotly pursued topics in neuroscience and therapeutic medicine is the use of stem cells in the adult brain. Growing in parallel to this emerging field is the recognition that the adult brain is indeed capable of generating new cells. While neurogenesis was understood to be restricted to a few areas, recent studies suggest that damage to the adult brain can trigger neurogenesis even in regions outside of these specific areas. This finding raises the possibility that neurons born in response to perturbation in the brain may be involved in the recovery of function in the damaged adult brain. The key is understanding how to cultivate these newborn cells, because they do not remain viable if they are not accepted into the damaged network of interconnected neurons which support specific functions. From a birth site, undifferentiated precursor cells or neurons undergo migration and differentiation. Many factors influence the safe journey of migrating cells and their survival after maturation at their destination.This review will present evidence from ring dove studies that an activity-dependent mechanism underlies the survival of adult newborn neurons and establishment of their functionality. This evidence includes: [1] unique electrophysiological properties or specific connectivity associated with various type of neurons involved in ring dove coo behavior and reproductive function, [2] emergence of electrophysiological properties and specific projection neurons emanating from newborn neurons after hypothalamic lesion, and finally [3] collective behavioral analyses of social stimulations suggesting that sensorimotor events contribute to the integration of new neurons and reinstatement of function.

Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity.

Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

Expression of transcription factor Coup-TF1 (NR2F1) in developing occipital cortex in humans

PURPOSES: We aimed to investigate the presence, distribution and abundance of transcription factor (TF) coup-tf1 (also known as NR2F1) in the germinative zones of human telencephalon during the fetal period. This transcription factor is of significance for the normal neuronal migration and differentiation of projection neurons in the mouse forebrain. MATERIALS AND METHODS: Brain tissue samples from spontaneously aborted human fetuses aged between 12 and 28 gestational weeks (g.w.) were fixed in paraformaldehyde and histologically investigated. Sections immunostained for coup-tf1 were scanned and its expression in different zones of the occipital lobe of developing pallium was evaluated. COUP-TF1 positive cells were counted and their percentage of all DAPI-stained nuclei was calculated in order to establish the abundance of the COUP-TF1-expressing cells among the total cell population. RESULTS: COUP-TF1 expression was prominent in all investigated zones of the developing human occipital lobe at stage 17 g.w. In all zones studied, COUP-TF1 positive cells presented a relatively high fraction (~71-95 %) of the cells counted with the highest value of 95.24% in the ventricular zone. CONCLUSIONS: The present data on the location, abundance and distribution pattern of coup-tf1-expressing cells in the human occipital lobe provide information on the possibility that this TF might participate to human to corticogenesis, similarly to the mouse.

Simultaneous visualization of extrinsic and intrinsic axon collaterals in Golgi-like detail for mouse corticothalamic and corticocortical cells: a double viral infection method

Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive “TET-ON/TET-OFF system” in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral S1 for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both S1BF and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1–6), bilateral S1 and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.

Correlated Gene Expression and Target Specificity Demonstrate Excitatory Projection Neuron Diversity

The neocortex contains diverse populations of excitatory neurons segregated by layer and further definable by their specific cortical and subcortical projection targets. The current study describes a systematic approach to identify molecular correlates of specific projection neuron classes in mouse primary somatosensory cortex (S1), using a combination of in situ hybridization (ISH) data mining, marker gene colocalization, and combined retrograde labeling with ISH for layer-specific marker genes. First, we identified a large set of genes with specificity for each cortical layer, and that display heterogeneous patterns within those layers. Using these genes as markers, we find extensive evidence for the covariation of gene expression and projection target specificity in layer 2/3, 5, and 6, with individual genes labeling neurons projecting to specific subsets of target structures. The combination of gene expression and target specificity imply a great diversity of projection neuron classes that is similar to or greater than that of GABAergic interneurons. The covariance of these 2 phenotypic modalities suggests that these classes are both discrete and genetically specified.

Reduction in inhibitory control is sufficient to generate hyperalgesia in a spiking model of nociceptive integration in the superficial dorsal horn

The spinal cord’s dorsal horn is a major termination site for primary afferents carrying sensory information from the periphery. The superfic ial dorsal horn (SDH, laminae I and II), in particular, receives inputs from nociceptive C and Aδ fibers, and have an important role in relaying and processing nociceptive information. This work focuses on the pain condition named hyperalgesia, characterized by an increased response to a stimulus that is normally painful. Much is still unknown regarding the central mechanisms giving rise to this condition. Here we present a large network spiking model for hyperalgesia which includes significant functional components of the nociceptive circuit in the SDH. The network architecture (Figure 1A) was defined according to key experimental results, namely: the somatotopic mapping between body surface and the superficial dorsal horn [1]; the presence of an inhibitory control which, under normal conditions, leads to a proper perception of pain [2]; the existence of divergent/convergent fiber inputs to lamina I (LI) neurons [3]; and the presence of inhibitory interneurons, i.e. islet cells, with inputs/outputs within lamina II (LII) [4]. In the model, the connectivity profile between LII interneurons (excitatory, Exc and inhibitory, Inh) and LI projection neurons creates a mexican-hat input profile with a strong centered excitation flanked by a surrounding inhibition. Activity level control was provided by feed-forward inhibition from the inhibitory interneurons in LII. The network model was created and simulated using the simulation environment NeuralSyns [http://sourceforge. net/projects/neuralsyns/]. Neurons were represented as integrate-and-fire units with conductance based synapses. The full model was comprised of 2.600 neurons and 320.000 synapses. The analysis was performed by assessing the changes produced in the receptive fields of LI nociceptive specific projection neurons (NS), both in normal (control) conditions and after reduction in inhibitory control

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry.

Synaptic inhibition controls transient oscillatory synchronization in a model of the insect olfactory system

In a variety of neuronal systems it has been hypothesized that inhibitory interneurons corral principal neurons into synchronously firing groups that encode sensory information and sub-serve behavior (Buzsáki and Chrobak, 1995; Buzsáki, 2008). This mechanism is particularly relevant to the olfactory system where spatiotemporal patterns of projection neuron (PN) activity act as robust markers of odor attributes (Laurent et al., 1996; Wehr and Laurent, 1996). In the insect antennal lobe (AL), a network of local inhibitory interneurons arborizes extensively throughout the AL (Leitch and Laurent, 1996) providing inhibitory input to the cholinergic PNs. Our theoretical work has attempted to elaborate the exact role of inhibition in the generation of odor specific PN responses (Bazhenov et al., 2001a,b; Assisi et al., 2011). In large-scale AL network models we characterized the inhibitory sub-network by its coloring (Assisi et al., 2011) and showed that it can entrain excitatory PNs to the odor specific patterns of transient synchronization. In this focused review, we further examine the dynamics of entrainment in more detail by simulating simple model networks in various parameter regimes. Our simulations in conjunction with earlier studies point to the key role played by lateral (between inhibitory interneurons) and feedback (from inhibitory interneurons to principal cells) inhibition in the generation of experimentally observed patterns of transient synchrony.

Visualization of the morphology and activity of specific projection neurons using rabies virus vector and voltage-sensitive probe

Visualization of the morphology and activity of specific projection neurons using rabies virus vector and voltage-sensitive probe

The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting

BackgroundThe Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program.ResultsWe find that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (TIP60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b.ConclusionsOur results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions.

Targeting expression to projection neurons that innervate specific mushroom body calyx and antennal lobe glomeruli in larval Drosophila

The first and secondary olfactory centers in the olfactory pathway in Drosophila are organized into neuropil structures called glomeruli. The antennal lobe (AL), the first olfactory center in larval Drosophila, is organized in 21 glomeruli. Each AL glomerulus receives innervation from a specific olfactory sensory neuron (OSN), and is therefore identifiable anatomically by the position of the OSN terminal. Olfactory projection neurons (PNs) send a dendrite to a single AL glomerulus and an axon that usually terminates in a single glomerulus in the mushroom body (MB) calyx, a secondary olfactory center, and in the lateral horn. By random labeling of single PNs that express GH146–GAL4, it was previously shown that PNs stereotypically innervate specific AL and calyx glomeruli, and most of these connections have been mapped. Here we report the pattern of innervation of GAL4 lines that drive expression of reporter genes in single or a few PNs, including PNs not identified by the widely used GH146–GAL4 driver. We have mapped the AL and calyx glomeruli innervated by these labeled PNs. This study provides a collection of GAL4 lines to molecularly mark the connections between specific AL and calyx glomeruli. It thus confirms and extends the previous map of AL–calyx connectivity that was based only on randomly labeled single PNs, and provides tools for targeted manipulation of specific PNs for developmental and functional studies.

MicroRNA Processing Pathway Regulates Olfactory Neuron Morphogenesis

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs [1, 2]. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes [3, 4]. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition (Figure 1A). Here, we identify two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections [5, 6]. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers [7-9]. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. Our findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system.

Homotopic glial regulation of striatal projection neuron differentiation.

We have studied the differentiation of striatal projection neurons in co-culture with expanded glia from different regions of the embryonic mouse telencephalon. Our results show that when striatal progenitors are cultured on glia derived from the same region as they originate (i.e. the lateral ganglionic eminence), the neurons formed exhibit long processes. This is not the case when the cells are co-cultured with glia derived from the adjacent telencephalic region, the medial ganglionic eminence. Moreover, expression of the striatal projection neuron marker, dopamine- and cAMP-regulated phosphoprotein (DARPP-32) was significantly enhanced in neurons cultured on the homotopic glia. Thus, glial cultures derived from the lateral ganglionic eminence positively regulate the differentiation of striatal projection neurons in vitro.

Projections to higher olfactory centers from subdivisions of the antennal lobe macroglomerular complex of the male silkmoth.

The macroglomerular complex (MGC) is the first-order center for synaptic processing of olfactory information about the female sex pheromone in the male moth brain. We have investigated the MGC subdivisions of the male silkmoth Bombyx mori by use of three-dimensional reconstruction of the MGC from sequential series of confocal slice images. The B. mori MGC consists of three subdivisions similar to those of Manduca sexta: the cumulus, toroid and horseshoe. Intracellular recording and staining revealed that responses of MGC projection neurons to pheromonal stimulation correlate with their dendritic arborizations in the subdivisions of the MGC (the cumulus, toroid and horseshoe) and each subdivision specific projection neuron transmits information to different regions in the calyces of the mushroom body and the inferior lateral protocerebrum. We revealed that major pheromone component information is transferred to the medial part of the inferior lateral protocerebrum through three different antennocerebral pathways. Although it is generally accepted that the calyces of the mushroom body and the inferior lateral protocerebrum are the target sites for pheromone information from the MGC in moths, our results suggest that the medial part of the inferior lateral protocerebrum may be a more important processing site for major pheromonal information in B. mori.

Targeted Retrograde Gene Delivery for Neuronal Protection

The cellular heterogeneity and complex circuitry of the central nervous system make it difficult to achieve precise delivery of experimental and therapeutic agents. We report here an in vivo retrograde gene delivery strategy to target mature projection neurons using adeno-associated virus, a vector with low toxicity and the capacity for long-term gene expression. Viral delivery to axon terminal fields in the hippocampus and striatum resulted in viral internalization, retrograde transport, and transgene expression in specific projection neurons in entorhinal cortex and substantia nigra. Retrograde delivery of the anti-apoptotic gene Bcl2l (also known as Bcl-xL) protected entorhinal projection neurons from subsequent damage-induced cell death. Given the broad distribution of neurons affected by neurodegenerative diseases, gene delivery to both the terminal fields and the projection neurons through retrograde infection provides for strategic therapeutic intervention at both levels of the neural circuit. This approach may also facilitate experimental studies of defined neural circuits.

Development of cholinergic and GABAergic neurons in the rat medial septum: Effect of target removal in early postnatal development

During normal development of the nervous system, the target fields influence the survival and differentiation of projection neurons, but the factors regulating this interaction remain obscure. In the present study, we have raised the question whether the target region is essential for the postnatal development and maintenance of two different types of central projection neurons, cholinergic and GABAergic septohippocampal cells. In early postnatal rats (P5, P10), the hippocampus was eliminated by unilateral intrahippocampal injections of the excitotoxin N-methyl-D-aspartate. After a long survival time (at P70), we have immunostained serial sections of the septal region with antibodies against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, or the calcium-binding protein parvalbumin (PARV) which is known to be contained in GABAergic septohippocampal neurons. In the medial septum ipsilateral to the lesioned side, about 60% of ChAT-immunoreactive neurons and 62% of PARV-immunoreactive neurons were found in adulthood even after complete elimination of the hippocampus. Some immunoreactive cells appeared heavily shrunken, but electron microscopic analysis revealed ultrastructural characteristics typical for medial septal neurons obtained from controls. Our results indicate that target elimination during development affected both types of projection cells, although only the cholinergic cells are known to be responsive to target-derived factors.