The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn.

The deep dorsal horn is a poorly characterized spinal cord region implicated in processing low-threshold mechanoreceptor (LTMR) information. We report an array of mouse genetic tools for defining neuronal components and functions of the dorsal horn LTMR-recipient zone (LTMR-RZ), a role for LTMR-RZ processing in tactile perception, and the basic logic of LTMR-RZ organization. We found an unexpectedly high degree of neuronal diversity in the LTMR-RZ: seven excitatory and four inhibitory subtypes of interneurons exhibiting unique morphological, physiological, and synaptic properties. Remarkably, LTMRs form synapses on between four and 11 LTMR-RZ interneuron subtypes, while each LTMR-RZ interneuron subtype samples inputs from at least one to three LTMR classes, as well as spinal cord interneurons and corticospinal neurons. Thus, the LTMR-RZ is a somatosensory processing region endowed with a neuronal complexity that rivals the retina and functions to pattern the activity of ascending touch pathways that underlie tactile perception.

The cellular and molecular basis of direction selectivity of Aδ-LTMRs.

The perception of touch, including the direction of stimulus movement across the skin, begins with activation of low-threshold mechanosensory neurons (LTMRs) that innervate the skin. Here, we show that murine Aδ-LTMRs are preferentially tuned to deflection of body hairs in the caudal-to-rostral direction. This tuning property is explained by the finding that Aδ-LTMR lanceolate endings around hair follicles are polarized; they are concentrated on the caudal (downward) side of each hair follicle. The neurotrophic factor BDNF is synthesized in epithelial cells on the caudal, but not rostral, side of hair follicles, in close proximity to Aδ-LTMR lanceolate endings, which express TrkB. Moreover, ablation of BDNF in hair follicle epithelial cells disrupts polarization of Aδ-LTMR lanceolate endings and results in randomization of Aδ-LTMR responses to hair deflection. Thus, BDNF-TrkB signaling directs polarization of Aδ-LTMR lanceolate endings, which underlies direction-selective responsiveness of Aδ-LTMRs to hair deflection.

The functional organization of cutaneous low-threshold mechanosensory neurons.

Innocuous touch of the skin is detected by distinct populations of neurons, the low-threshold mechanoreceptors (LTMRs), which are classified as Aß-, Aδ-, and C-LTMRs. Here, we report genetic labeling of LTMR subtypes and visualization of their relative patterns of axonal endings in hairy skin and the spinal cord. We found that each of the three major hair follicle types of trunk hairy skin (guard, awl/auchene, and zigzag hairs) is innervated by a unique and invariant combination of LTMRs; thus, each hair follicle type is a functionally distinct mechanosensory end organ. Moreover, the central projections of Aß-, Aδ-, and C-LTMRs that innervate the same or adjacent hair follicles form narrow LTMR columns in the dorsal horn. These findings support a model of mechanosensation in which the activities of Aß-, Aδ-, and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.

Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.

In the enteric nervous system (ENS), glia outnumber neurons by 4-fold and form an extensive network throughout the gastrointestinal tract. Growing evidence for the essential role of enteric glia in bowel function makes it imperative to understand better their molecular marker expression and how they relate to glia in the rest of the nervous system. We analyzed expression of markers of astrocytes and oligodendrocytes in the ENS and found, unexpectedly, that proteolipid protein 1 (PLP1) is specifically expressed by glia in adult mouse intestine. PLP1 and S100ß are the markers most widely expressed by enteric glia, while glial fibrillary acidic protein expression is more restricted. Marker expression in addition to cellular location and morphology distinguishes a specific subpopulation of intramuscular enteric glia, suggesting that a combinatorial code of molecular markers can be used to identify distinct subtypes. To assess the similarity between enteric and extraenteric glia, we performed RNA sequencing analysis on PLP1-expressing cells in the mouse intestine and compared their gene expression pattern to that of other types of glia. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes, although there is no evidence of myelination in the murine ENS. GLIA 2015;63:2040-2057.

Cortical RORß is required for layer 4 transcriptional identity and barrel integrity.

Retinoic acid-related orphan receptor beta (RORß) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORß is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORß delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORß, Thsd7a, is down-regulated without RORß, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches.

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals formed significantly fewer neurospheres. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon.

Cas Adaptor Proteins Coordinate Sensory Axon Fasciculation.

Development of complex neural circuits like the peripheral somatosensory system requires intricate mechanisms to ensure axons make proper connections. While much is known about ligand-receptor pairs required for dorsal root ganglion (DRG) axon guidance, very little is known about the cytoplasmic effectors that mediate cellular responses triggered by these guidance cues. Here we show that members of the Cas family of cytoplasmic signaling adaptors are highly phosphorylated in central projections of the DRG as they enter the spinal cord. Furthermore, we provide genetic evidence that Cas proteins regulate fasciculation of DRG sensory projections. These data establish an evolutionarily conserved requirement for Cas adaptor proteins during peripheral nervous system axon pathfinding. They also provide insight into the interplay between axonal fasciculation and adhesion to the substrate.

Prenatal thalamic waves regulate cortical area size prior to sensory processing.

The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorß upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing.

Coding and Plasticity in the Mammalian Thermosensory System.

Perception of the thermal environment begins with the activation of peripheral thermosensory neurons innervating the body surface. To understand how temperature is represented in vivo, we used genetically encoded calcium indicators to measure temperature-evoked responses in hundreds of neurons across the trigeminal ganglion. Our results show how warm, hot, and cold stimuli are represented by distinct population responses, uncover unique functional classes of thermosensory neurons mediating heat and cold sensing, and reveal the molecular logic for peripheral warmth sensing. Next, we examined how the peripheral somatosensory system is functionally reorganized to produce altered perception of the thermal environment after injury. We identify fundamental transformations in sensory coding, including the silencing and recruitment of large ensembles of neurons, providing a cellular basis for perceptual changes in temperature sensing, including heat hypersensitivity, persistence of heat perception, cold hyperalgesia, and cold analgesia.

Upregulation of µ3A Drives Homeostatic Plasticity by Rerouting AMPAR into the Recycling Endosomal Pathway.

Synaptic scaling is a form of homeostatic plasticity driven by transcription-dependent changes in AMPA-type glutamate receptor (AMPAR) trafficking. To uncover the pathways involved, we performed a cell-type-specific screen for transcripts persistently altered during scaling, which identified the µ subunit (µ3A) of the adaptor protein complex AP-3A. Synaptic scaling increased µ3A (but not other AP-3 subunits) in pyramidal neurons and redistributed dendritic µ3A and AMPAR to recycling endosomes (REs). Knockdown of µ3A prevented synaptic scaling and this redistribution, while overexpression (OE) of full-length µ3A or a truncated µ3A that cannot interact with the AP-3A complex was sufficient to drive AMPAR to REs. Finally, OE of µ3A acted synergistically with GRIP1 to recruit AMPAR to the dendritic membrane. These data suggest that excess µ3A acts independently of the AP-3A complex to reroute AMPAR to RE, generating a reservoir of receptors essential for the regulated recruitment to the synaptic membrane during scaling up.

Fibroblast growth factor receptor signaling promotes radial glial identity and interacts with Notch1 signaling in telencephalic progenitors.

The Notch and fibroblast growth factor (FGF) pathways both regulate cell fate specification during mammalian neural development. We have shown previously that Notch1 activation in the murine forebrain promotes radial glial identity. This result, together with recent evidence that radial glia can be progenitors, suggested that Notch1 signaling might promote progenitor and radial glial character simultaneously. Consistent with this idea, we found that in addition to promoting radial glial character in vivo, activated Notch1 (ActN1) increased the frequency of embryonic day 14.5 (E14.5) ganglionic eminence (GE) progenitors that grew into neurospheres in FGF2. Constitutive activation of C-promoter binding factor (CBF1), a Notch pathway effector, also increased neurosphere frequency in FGF2, suggesting that the effect of Notch1 on FGF responsiveness is mediated by CBF1. The observation that ActN1 promoted FGF responsiveness in telencephalic progenitors prompted us to examine the effect of FGF pathway activation in vivo. We focused on FGFR2 because it is expressed in radial glia in the GEs where ActN1 increases FGF2 neurosphere frequency, but not in the septum where it does not. Like ActN1, activated FGFR2 (ActFGFR2) promoted radial glial character in vivo. However, unlike ActN1, ActFGFR2 did not enhance neurosphere frequency at E14.5. Additional analysis demonstrated that, unexpectedly, neither ActFGFR2 nor ActFGFR1 could replace the need for ligand in promoting neurosphere proliferation. This study suggests that telencephalic progenitors with radial glial morphology are maintained by interactions between the Notch and FGF pathways, and that the mechanisms by which FGF signaling promotes radial glial character in vivo and progenitor proliferation in vitro can be uncoupled.

Brain-derived neurotrophic factor is required for axonal growth of selective groups of neurons in the arcuate nucleus.

OBJECTIVE: Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal development, and the Bdnf gene produces two populations of transcripts with either a short or long 3' untranslated region (3' UTR). Deficiencies in BDNF signaling have been shown to cause severe obesity in humans; however, it remains unknown how BDNF signaling impacts the organization of neuronal circuits that control energy balance. METHODS: We examined the role of BDNF on survival, axonal projections, and synaptic inputs of neurons in the arcuate nucleus (ARH), a structure critical for the control of energy balance, using Bdnf (klox/klox) mice, which lack long 3' UTR Bdnf mRNA and develop severe hyperphagic obesity. RESULTS: We found that a small fraction of neurons that express the receptor for BDNF, TrkB, also expressed proopiomelanocortin (POMC) or neuropeptide Y (NPY)/agouti-related protein (AgRP) in the ARH. Bdnf(klox/klox) mice had normal numbers of POMC, NPY, and TrkB neurons in the ARH; however, retrograde labeling revealed a drastic reduction in the number of ARH axons that project to the paraventricular hypothalamus (PVH) in these mice. In addition, fewer POMC and AgRP axons were found in the dorsomedial hypothalamic nucleus (DMH) and the lateral part of PVH, respectively, in Bdnf (klox/klox) mice. Using immunohistochemistry, we examined the impact of BDNF deficiency on inputs to ARH neurons. We found that excitatory inputs onto POMC and NPY neurons were increased and decreased, respectively, in Bdnf (klox/klox) mice, likely due to a compensatory response to marked hyperphagia displayed by the mutant mice. CONCLUSION: This study shows that the majority of TrkB neurons in the ARH are distinct from known neuronal populations and that BDNF plays a critical role in directing projections from these neurons to the DMH and PVH. We propose that hyperphagic obesity due to BDNF deficiency is in part attributable to impaired axonal growth of TrkB-expressing ARH neurons.

Sexually dimorphic BDNF signaling directs sensory innervation of the mammary gland.

How neural circuits associated with sexually dimorphic organs are differentially assembled during development is unclear. Here, we report a sexually dimorphic pattern of mouse mammary gland sensory innervation and the mechanism of its formation. Brain-derived neurotrophic factor (BDNF), emanating from mammary mesenchyme and signaling through its receptor TrkB on sensory axons, is required for establishing mammary gland sensory innervation of both sexes at early developmental stages. Subsequently, in males, androgens promote mammary mesenchymal expression of a truncated form of TrkB, which prevents BDNF-TrkB signaling in sensory axons and leads to a rapid loss of mammary gland innervation independent of neuronal apoptosis. Thus, sex hormone regulation of a neurotrophic factor signal directs sexually dimorphic axonal growth and maintenance, resulting in generation of a sex-specific neural circuit.

MeCP2: phosphorylated locally, acting globally.

In this issue of Neuron, Greenberg and colleagues revise our understanding of how activity-dependent MeCP2 phosphorylation regulates distinct aspects of brain development and circuit function. The study also suggests a prominent role for MeCP2 in the regulation of global chromatin state in vivo.

Removal of Pax6 partially rescues the loss of ventral structures in Shh null mice.

Pax6 and Gli3 are dorsally expressed genes that are known to antagonize sonic hedgehog (Shh) activity. We have previously shown that dorsoventral patterning defects seen in Shh(-/-) mutants are rescued in Shh(-/-);Gli3(-/-) compound mutants. Here we investigate if the loss of Pax6 can also ameliorate defects seen in Shh(-/-) mutants. In support of this notion, we observe that the fusion of the cerebral vesicles seen in Shh(-/-) mutants is partially corrected in E12.5 Shh(-/-);Pax6(-/-) compound mutants. Investigation of pan-ventral markers such as Dlx2 also shows that, unlike Shh(-/-), a broad domain of expression of this gene is observed in Shh(-/-);Pax6(-/-) mice. Interestingly, we observe that while the expression of ER81 in the ventral telencephalon is expanded, the expression of Ebf1 is lost. This suggests that the rescued ventral domain observed in Shh(-/-);Pax6(-/-) mice is the dorsal lateral ganglionic eminence region. With regard to dorsal telencephalic patterning, we also observe rescue of the pallial-subpallial boundary, as well as a partial rescue of the dorsal midline. Together, our findings are consistent with Pax6 function being required for aspects of Gli3-mediated telencephalic patterning.

Combinatorial function of the homeodomain proteins Nkx2.1 and Gsh2 in ventral telencephalic patterning.

Regional patterning of the mammalian telencephalon requires the function of three homeodomain-containing transcription factors, Pax6, Gsh2 and Nkx2.1. These factors are required for the development of the dorsal, lateral and medial domains of the telencephalon, respectively. Previous work has indicated that two of the genes encoding these factors, Pax6 and Gsh2, cross-repress one another in the formation of the border between dorsal and lateral region of the telencephalon. Here, we examine whether similar interactions are responsible for the establishment of other boundaries of telencephalic gene expression. Surprisingly, despite the fact that, at specific times in development, both Pax6 and Gsh2 maintain a complementary pattern of expression with Nkx2.1, in neither case are these boundaries maintained through a similar cross-repressive mechanism. Rather, as revealed by analysis of double-mutant mice, Nkx2.1 and Gsh2 act cooperatively in many aspects to pattern the ventral telencephalon. By contrast, as indicated by both loss- and gain-of-function analysis, Gsh2 expression in the medial ganglionic eminence after E10.5 may negatively regulate Nkx2.1 dependent specification of oligodendrocytes. Therefore, both integrative and antagonistic interactions between homeodomain-containing transcription factors contribute to the patterning of the telencephalon.