Red nucleus interleukin-1ß evokes tactile allodynia through activation of JAK/STAT3 and JNK signaling pathways.

We previously reported that interleukin-1ß (IL-1ß) in the red nucleus (RN) is involved in pain modulation and exerts a facilitatory effect in the development of neuropathic pain. Here, we explored the actions of signaling pathways, including the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB (NF-κB) pathways, on RN IL-1ß-mediated pain modulation. After a single dose of recombinant rat IL-1ß (rrIL-1ß, 10 ng) injected into the RN in normal rats, a tactile allodynia was evoked in the contralateral but not ipsilateral hindpaw, commencing 75 min and peaking 120 min postinjection. Up-regulated protein levels of phospho-STAT3 (p-STAT3) and p-JNK were observed in the RN 120 min after rrIL-1ß injection, the increases of p-STAT3 and p-JNK were blocked by anti-IL-1ß antibody. However, the expression levels of p-ERK, p-p38 MAPK, and NF-κB in the RN were not affected by rrIL-1ß injection. RN neurons and astrocytes contributed to IL-1ß-evoked up-regulation of p-STAT3 and p-JNK. Further studies demonstrated that injection of the JAK2 antagonist AG490 or JNK antagonist SP600125 into the RN 30 min prior to the administration of rrIL-1ß could completely prevent IL-1ß-evoked tactile allodynia, while injection of the ERK antagonist PD98059, p38 MAPK antagonist SB203580, or NF-κB antagonist PDTC did not affect IL-1ß-evoked tactile allodynia. In conclusion, our data provide additional evidence that RN IL-1ß is involved in pain modulation, and that it exerts a facilitatory effect by activating the JAK/STAT3 and JNK signaling pathways.

Dopamine D2 receptors are involved in the regulation of Fyn and metabotropic glutamate receptor 5 phosphorylation in the rat striatum in vivo.

Fyn, a major Src family kinase (SFK) member that is densely expressed in striatal neurons, is actively involved in the regulation of cellular and synaptic activities in local neurons. This SFK member is likely regulated by dopamine signaling through a receptor mechanism involving dopamine D2 receptors (D2Rs). This study characterizes the D2R-dependent regulation of Fyn in the rat striatum in vivo. Moreover, we explore whether D2Rs regulate metabotropic glutamate receptor 5 (mGluR5) in its tyrosine phosphorylation and whether the D2R-SFK pathway modulates trafficking of mGluR5. We found that blockade of D2Rs by systemic administration of a D2R antagonist, eticlopride, substantially increased SFK phosphorylation in the striatum. This increase was a transient and reversible event. The eticlopride-induced SFK phosphorylation occurred predominantly in immunopurified Fyn but not in another SFK member, Src. Eticlopride also elevated tyrosine phosphorylation of mGluR5. In parallel, eticlopride enhanced synaptic delivery of active Fyn and mGluR5. Pretreatment with an SFK inhibitor blocked the eticlopride-induced tyrosine phosphorylation and synaptic trafficking of mGluR5. These results indicate that D2Rs inhibit SFK (mainly Fyn) phosphorylation in the striatum. D2Rs also inhibit tyrosine phosphorylation and synaptic recruitment of mGluR5 through a signaling mechanism likely involving Fyn.

Red nucleus glutamate facilitates neuropathic allodynia induced by spared nerve injury through non-NMDA and metabotropic glutamate receptors.

Previous studies have demonstrated that glutamate plays an important role in the development of pathological pain. This study investigates the expression changes of glutamate and the roles of different types of glutamate receptors in the red nucleus (RN) in the development of neuropathic allodynia induced by spared nerve injury (SNI). Immunohistochemistry indicated that glutamate was constitutively expressed in the RN of normal rats. After SNI, the expression levels of glutamate were significantly increased in the RN at 1 week and reached the highest level at 2 weeks postinjury compared with sham-operated and normal rats. The RN glutamate was colocalized with neurons, oligodendrocytes, and astrocytes but not microglia under physiological and neuropathic pain conditions. To elucidate further the roles of the RN glutamate and different types of glutamate receptors in the development of neuropathic allodynia, antagonists to N-methyl-D-aspartate (NMDA), non-NMDA, or metabotropic glutamate receptors (mGluRs) were microinjected into the RN contralateral to the nerve-injury side of rats with SNI, and the paw withdrawal threshold (PWT) was dynamically assessed with von Frey filaments. Microinjection of the NMDA receptor antagonist MK-801 into the RN did not show any effect on SNI-induced mechanical allodynia. However, microinjection of the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3(1H,4H)-dione or the mGluR antagonist (±)-α-methyl-(4-carboxyphenyl) glycine into the RN significantly increased the PWT and alleviated SNI-induced mechanical allodynia. These findings suggest that RN glutamate is involved in regulating neuropathic pain and facilitates the development of SNI-induced neuropathic allodynia. The algesic effect of glutamate is transmitted by the non-NMDA glutamate receptor and mGluRs.

Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release.

Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.

Regulation of synaptic MAPK/ERK phosphorylation in the rat striatum and medial prefrontal cortex by dopamine and muscarinic acetylcholine receptors.

Dopamine and acetylcholine are two principal transmitters in the striatum and are usually balanced to modulate local neural activity and to maintain striatal homeostasis. This study investigates the role of dopamine and muscarinic acetylcholine receptors in the regulation of a central signaling protein, i.e., the mitogen-activated protein kinase (MAPK). We focus on the synaptic pool of MAPKs because of the fact that these kinases reside in peripheral synaptic structures in addition to their somatic locations. We show that a systemic injection of dopamine D1 receptor (D1R) agonist SKF81297 enhances phosphorylation of extracellular signal-regulated kinases (ERKs), a prototypic subclass of MAPKs, in the adult rat striatum. Similar results were observed in another dopamine-responsive region, the medial prefrontal cortex (mPFC). The dopamine D2 receptor agonist quinpirole had no such effects. Pretreatment with a positive allosteric modulator (PAM) of muscarinic acetylcholine M4 receptors (M4Rs), VU0152100, attenuated the D1R agonist-stimulated ERK phosphorylation in the two regions, whereas the PAM itself did not alter basal ERK phosphorylation. All drug treatments had no effect on phosphorylation of c-Jun N-terminal kinases (JNKs), another MAPK subclass, in the striatum and mPFC. These results demonstrate that dopamine and acetylcholine are integrated to control synaptic ERK but not JNK activation in striatal and mPFC neurons in vivo. Activation of M4Rs exerts an inhibitory effect on the D1R-mediated upregulation of synaptic ERK phosphorylation.

Upregulation of AMPA receptor GluA1 phosphorylation by blocking adenosine A1 receptors in the male rat forebrain.

OBJECTIVE: The adenosine A1 receptor is a Gαi/o protein-coupled receptor and inhibits upon activation cAMP formation and protein kinase A (PKA) activity. As a widely expressed receptor in the mammalian brain, A1 receptors are implicated in the modulation of a variety of neuronal and synaptic activities. In this study, we investigated the role of A1 receptors in the regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the adult rat brain in vivo. METHODS: Adult male Wistar rats were used in this study. After a systemic injection of the A1 antagonist DPCPX, rats were sacrificed and several forebrain regions were collected for assessing changes in phosphorylation of AMPA receptors using Western blots. RESULTS: A systemic injection of the A1 antagonist DPCPX induced an increase in phosphorylation of AMPA receptor GluA1 subunits at a PKA-dependent site, serine 845 (S845), in the two subdivisions of the striatum, the caudate putamen, and nucleus accumbens. DPCPX also increased S845 phosphorylation in the medial prefrontal cortex (mPFC) and hippocampus. The DPCPX-stimulated S845 phosphorylation was a transient and reversible event. Blockade of Gαs/olf -coupled dopamine D1 receptors with a D1 antagonist SCH23390 abolished the responses of S845 phosphorylation to DPCPX in the striatum, mPFC, and hippocampus. DPCPX had no significant impact on phosphorylation of GluA1 at serine 831 and on expression of total GluA1 proteins in all forebrain regions surveyed. CONCLUSION: These data demonstrate that adenosine A1 receptors maintain an inhibitory tone on GluA1 S845 phosphorylation under normal conditions. Blocking this inhibitory tone leads to the upregulation of GluA1 S845 phosphorylation in the striatum, mPFC, and hippocampus via a D1 -dependent manner.

Post-crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord.

The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post-crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post-crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post-crossing dI1 axons in mice, using the Atoh1 enhancer-based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post-crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post-crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.

Quantitative ultrastructural analysis of fibers expressing parvalbumin, calretinin, calbindin D-28k, stage specific embryonic antigen-4, and phosphorylated neurofilament 200 in the peripheral sensory root of the rat trigeminal ganglion.

Parvalbumin (PV), calretinin (CR), calbindin D-28k (CB), stage specific embryonic antigen-4 (SSEA4), and phosphorylated neurofilament 200 (pNF200) have been commonly used as markers for primary afferent neurons with large myelinated (A) fibers but detailed information on the expression of these markers in specific primary afferent fiber types is still lacking. We here examined the fibers that express PV, CR, CB, SSEA4, and pNF200 in the trigeminal ganglion and its peripheral sensory root by light- and electron-microscopic immunohistochemistry and quantitative analysis. We found that all CR-immunopositive (+), CB+, and SSEA4+ fibers and virtually all (98.8%) PV+ fibers were myelinated, most CR+ fibers were large myelinated, whereas most CB+ and SSEA4+ fibers were small myelinated. One half of the PV+ fibers were small myelinated and the other half were large myelinated. Of all pNF200+ fibers, about a third each were small myelinated, large myelinated, and unmyelinated. These findings suggest that PV, CR, CB, and SSEA4 can be used as specific markers for primary afferent neurons with myelinated fibers, but that pNF200 is not suitable as a specific marker for primary afferent neurons with myelinated fibers, and also raise the possibility that PV, CR, CB, and SSEA4 may be expressed in both mechanoreceptive and nociceptive neurons.

Ultrastructural comparison of the Drosophila larval and adult ventral abdominal neuromuscular junction.

Drosophila melanogaster has recently emerged as model system for studying synaptic transmission and plasticity during adulthood, aging and neurodegeneration. However, still little is known about the basic neuronal mechanisms of synaptic function in the adult fly. Per se, adult Drosophila neuromuscular junctions should be highly suited for studying these aspects as they allow for genetic manipulations in combination with ultrastructural and electrophysiological analyses. Although different neuromuscular junctions of the adult fly have been described during the last years, a direct ultrastructural comparison with their larval counterpart is lacking. The present study was designed to close this gap by providing a detailed ultrastructural comparison of the larval and the adult neuromuscular junction of the ventrolongitudinal muscle. Assessment of several parameters revealed similarities but also major differences in the ultrastructural organisation of the two model neuromuscular junctions. While basic morphological parameters are retained from the larval into the adult stage, the analysis discovered major differences of potential functional relevance in the adult: The electron-dense membrane apposition of the presynaptic and postsynaptic membrane is shorter, the subsynaptic reticulum is less elaborated and the number of synaptic vesicles at a certain distance of the presynaptic membrane is higher.

Structural basis for cholinergic regulation of neural circuits in the mouse olfactory bulb.

Odor information is regulated by olfactory inputs, bulbar interneurons, and centrifugal inputs in the olfactory bulb (OB). Cholinergic neurons projecting from the nucleus of the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus are one of the primary centrifugal inputs to the OB. In this study, we focused on cholinergic regulation of the OB and analyzed neural morphology with a particular emphasis on the projection pathways of cholinergic neurons. Single-cell imaging of a specific neuron within dense fibers is critical to evaluate the structure and function of the neural circuits. We labeled cholinergic neurons by infection with virus vector and then reconstructed them three-dimensionally. We also examined the ultramicrostructure of synapses by electron microscopy tomography. To further clarify the function of cholinergic neurons, we performed confocal laser scanning microscopy to investigate whether other neurotransmitters are present within cholinergic axons in the OB. Our results showed the first visualization of complete cholinergic neurons, including axons projecting to the OB, and also revealed frequent axonal branching within the OB where it innervated multiple glomeruli in different areas. Furthermore, electron tomography demonstrated that cholinergic axons formed asymmetrical synapses with a morphological variety of thicknesses of the postsynaptic density. Although we have not yet detected the presence of other neurotransmitters, the range of synaptic morphology suggests multiple modes of transmission. The present study elucidates the ways that cholinergic neurons could contribute to the elaborate mechanisms involved in olfactory processing in the OB. J. Comp. Neurol. 525:574-591, 2017. © 2016 Wiley Periodicals, Inc.

Spatial patterning of excitatory and inhibitory neuropil territories during spinal circuit development.

To generate rhythmic motor behaviors, both single neurons and neural circuits require a balance between excitatory inputs that trigger action potentials and inhibitory inputs that promote a stable resting potential (E/I balance). Previous studies have focused on individual neurons and have shown that, over a short spatial scale, excitatory and inhibitory (E/I) synapses tend to form structured territories with inhibitory inputs enriched on cell bodies and proximal dendrites and excitatory inputs on distal dendrites. However, systems-level E/I patterns, at spatial scales larger than single neurons, are largely uncharted. We used immunostaining for PSD-95 and gephyrin postsynaptic scaffolding proteins as proxies for excitatory and inhibitory synapses, respectively, to quantify the numbers and map the distributions of E/I synapses in zebrafish spinal cord at both an embryonic stage and a larval stage. At the embryonic stage, we found that PSD-95 puncta outnumber gephyrin puncta, with the number of gephyrin puncta increasing to match that of PSD-95 puncta at the larval stage. At both stages, PSD-95 puncta are enriched in the most lateral neuropil corresponding to distal dendrites while gephyrin puncta are enriched on neuronal somata and in the medial neuropil. Significantly, similar to synaptic puncta, neuronal processes also exhibit medial-lateral territories at both developmental stages with enrichment of glutamatergic (excitatory) processes laterally and glycinergic (inhibitory) processes medially. This establishment of neuropil excitatory-inhibitory structure largely precedes dendritic arborization of primary motor neurons, suggesting that the structured neuropil could provide a framework for the development of E/I balance at the cellular level. J. Comp. Neurol. 525:1649-1667, 2017. © 2016 Wiley Periodicals, Inc.

Subcellular organization of UBE3A in neurons.

Ubiquitination regulates a broad array of cellular processes, and defective ubiquitination is implicated in several neurological disorders. Loss of the E3 ubiquitin-protein ligase UBE3A causes Angelman syndrome. Despite its clinical importance, the normal role of UBE3A in neurons is still unclear. As a step toward deciphering its possible functions, we performed high-resolution light and electron microscopic immunocytochemistry. We report a broad distribution of UBE3A in neurons, highlighted by concentrations in axon terminals and euchromatin-rich nuclear domains. Our findings suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. J. Comp. Neurol. 525:233-251, 2017. © 2016 Wiley Periodicals, Inc.

Somatosensory organ topography across the star of the star-nosed mole (Condylura cristata).

Quantifying somatosensory receptor distribution in glabrous skin is usually difficult because of the diversity of skin receptor subtypes and their location within the dermis and epidermis. However, the glabrous noses of moles are an exception. In most species of moles, the skin on the nose is covered with domed mechanosensory units known as an Eimer's organs. Eimer's organs contain a stereotyped array of different mechanosensory neurons, meaning that the distribution of mechanosensitive nerve endings can be inferred by visual inspection of the skin surface. Here we detail the distribution of Eimer's organs on the highly derived somatosensory star on the rostrum of the star-nosed mole (Condylura cristata). The star consists of 22 fleshy appendages, or rays, that are covered in Eimer's organs. We find that the density of Eimer's organs increases from proximal to distal locations along the length of the star's rays with a ratio of 1:2.3:3.1 from the surface nearest to the nostril, to the middle part of ray, to the ray tip, respectively. This ratio is comparable to the increase in receptor unit density reported for the human hand, from the palm, to the middle of the digits, to the distal fingertips. We also note that the tactile fovea of the star-nosed mole, located on the medial ventral ray, does not have increased sensory organ density, and we describe these findings in comparison with other sensory fovea.

Neomycin damage and regeneration of hair cells in both mechanoreceptor and electroreceptor lateral line organs of the larval Siberian sturgeon (Acipenser baerii).

The lateral line found in some amphibians and fishes has two distinctive classes of sensory organs: mechanoreceptors (neuromasts) and electroreceptors (ampullary organs). Hair cells in neuromasts can be damaged by aminoglycoside antibiotics and they will regenerate rapidly afterward. Aminoglycoside sensitivity and the capacity for regeneration have not been investigated in ampullary organs. We treated Siberian sturgeon (Acipenser baerii) larvae with neomycin and observed loss and regeneration of sensory hair cells in both organs by labeling with DASPEI and scanning electron microscopy (SEM). The numbers of sensory hair cells in both organs were reduced to the lowest levels at 6 hours posttreatment (hpt). New sensory hair cells began to appear at 12 hpt and were regenerated completely in 7 days. To reveal the possible mechanism for ampullary hair cell regeneration, we analyzed cell proliferation and the expression of neural placodal gene eya1 during regeneration. Both cell proliferation and eya1 expression were concentrated in peripheral mantle cells and both increased to the highest level at 12 hpt, which is consistent with the time course for regeneration of the ampullary hair cells. Furthermore, we used Texas Red-conjugated gentamicin in an uptake assay following pretreatment with a cation channel blocker (amiloride) and found that entry of the antibiotic was suppressed in both organs. Together, our results indicate that ampullary hair cells in Siberian sturgeon larvae can be damaged by neomycin exposure and they can regenerate rapidly. We suggest that the mechanisms for aminoglycoside uptake and hair cell regeneration are conserved for mechanoreceptors and electroreceptors. J. Comp. Neurol. 524:1443-1456, 2016. © 2015 Wiley Periodicals, Inc.

EphA4 has distinct functionality from EphA7 in the corticothalamic system during mouse brain development.

Deciphering the molecular basis for guiding specific aspects of neocortical development remains a challenge because of the complexity of histogenic events and the vast array of protein interactions mediating these events. The Eph family of receptor tyrosine kinases is implicated in a number of neurodevelopmental activities. Eph receptors have been known to be capable of responding to several ephrin ligands within their subgroups, often eliciting similar downstream effects. However, several recent studies have indicated specificity between receptor-ligand pairs within each subfamily, the functional relevance of which is not defined. Here we show that a receptor of the EphA subfamily, EphA4, has effects distinct from those of its close relative, EphA7, in the developing brain. Both EphA4 and EphA7 interact similarly with corresponding ligands expressed in the developing neocortex. However, only EphA7 shows strong interaction with ligands in the somatosensory thalamic nuclei; EphA4 affects only cortical neuronal migration, with no visible effects on the guidance of corticothalamic (CT) axons, whereas EphA7 affects both cortical neuronal migration and CT axon guidance. Our data provide new evidence that Eph receptors in the same subfamily are not simply interchangeable but are functionally specified through selective interactions with distinct ligands in vivo. J. Comp. Neurol. 524:2080-2092, 2016. © 2015 Wiley Periodicals, Inc.

S-phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex.

The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal.

Fezf2 expression in layer 5 projection neurons of mature mouse motor cortex.

The mature cerebral cortex contains a wide diversity of neuron phenotypes. This diversity is specified during development by neuron-specific expression of key transcription factors, some of which are retained for the life of the animal. One of these key developmental transcription factors that is also retained in the adult is Fezf2, but the neuron types expressing it in the mature cortex are unknown. With a validated Fezf2-Gfp reporter mouse, whole-cell electrophysiology with morphology reconstruction, cluster analysis, in vivo retrograde labeling, and immunohistochemistry, we identify a heterogeneous population of Fezf2(+) neurons in both layer 5A and layer 5B of the mature motor cortex. Functional electrophysiology identified two distinct subtypes of Fezf2(+) neurons that resembled pyramidal tract projection neurons (PT-PNs) and intratelencephalic projection neurons (IT-PNs). Retrograde labeling confirmed the former type to include corticospinal projection neurons (CSpPNs) and corticothalamic projection neurons (CThPNs), whereas the latter type included crossed corticostriatal projection neurons (cCStrPNs) and crossed-corticocortical projection neurons (cCCPNs). The two Fezf2(+) subtypes expressed either CTIP2 or SATB2 to distinguish their physiological identity and confirmed that specific expression combinations of key transcription factors persist in the mature motor cortex. Our findings indicate a wider role for Fezf2 within gene expression networks that underpin the diversity of layer 5 cortical projection neurons.

Synaptic ultrastructure changes in trigeminocervical complex posttrigeminal nerve injury.

Trigeminal nerves collecting sensory information from the orofacial area synapse on second-order neurons in the dorsal horn of subnucleus caudalis and cervical C1/C2 spinal cord (Vc/C2, or trigeminocervical complex), which is critical for sensory information processing. Injury to the trigeminal nerves may cause maladaptive changes in synaptic connectivity that plays an important role in chronic pain development. Here we examined whether injury to the infraorbital nerve, a branch of the trigeminal nerves, led to synaptic ultrastructural changes when the injured animals have developed neuropathic pain states. Transmission electron microscopy was used to examine synaptic profiles in Vc/C2 at 3 weeks postinjury, corresponding to the time of peak behavioral hypersensitivity following chronic constriction injury to the infraorbital nerve (CCI-ION). Using established criteria, synaptic profiles were classified as associated with excitatory (R-), inhibitory (F-), and primary afferent (C-) terminals. Each type was counted within the superficial dorsal horn of the Vc/C2 and the means from each rat were compared between sham and injured animals; synaptic contact length was also measured. The overall analysis indicates that rats with orofacial pain states had increased numbers and decreased mean synaptic length of R-profiles within the Vc/C2 superficial dorsal horn (lamina I) 3 weeks post-CCI-ION. Increases in the number of excitatory synapses in the superficial dorsal horn of Vc/C2 could lead to enhanced activation of nociceptive pathways, contributing to the development of orofacial pain states.

Interstitial branch formation within the red nucleus by deep cerebellar nuclei-derived commissural axons during target recognition.

Target recognition by developing axons is one of the fundamental steps for establishing the proper pattern of neuronal connectivity during development. However, knowledge of the mechanisms that underlie this critical event is still limited. In this study, to examine how commissural axons in vertebrates recognize their targets after crossing the midline, we analyzed in detail the behavior of postcrossing commissural axons derived from the deep cerebellar nuclei (DCN) in the developing mouse cerebellum. For this, we employed a cell-type-specific genetic labeling approach to selectively visualize DCN axons during the time when these axons project to the red nucleus (RN), one of the well-characterized targets of DCN axons. We found that, when DCN axons initially entered the RN at its caudal end, these axons continued to grow rostrally through the RN without showing noticeable morphological signs of axon branching. Interestingly, after a delay, DCN axons started forming interstitial branches from the portion of the axon shaft selectively within the RN. Because commissural axons acquire responsiveness to several guidance cues when they cross the midline, we further addressed whether midline crossing is a prerequisite for subsequent targeting by using a Robo3 knockdown strategy. We found that DCN axons were still capable of forming interstitial branches within the RN even in the absence of midline crossing. These results therefore suggest that the mechanism of RN recognition by DCN axons involves a delayed interstitial branching, and that these axons possess an intrinsic ability to respond to the target-derived cues irrespective of midline crossing.

Molecular features distinguish ten neuronal types in the mouse superficial superior colliculus.

The superior colliculus (SC) is a midbrain center involved in controlling head and eye movements in response to inputs from multiple sensory modalities. Visual inputs arise from both the retina and visual cortex and converge onto the superficial layer of the SC (sSC). Neurons in the sSC send information to deeper layers of the SC and to thalamic nuclei that modulate visually guided behaviors. Presently, our understanding of sSC neurons is impeded by a lack of molecular markers that define specific cell types. To better understand the identity and organization of sSC neurons, we took a systematic approach to investigate gene expression within four molecular families: transcription factors, cell adhesion molecules, neuropeptides, and calcium binding proteins. Our analysis revealed 12 molecules with distinct expression patterns in mouse sSC: cadherin 7, contactin 3, netrin G2, cadherin 6, protocadherin 20, retinoid-related orphan receptor ß, brain-specific homeobox/POU domain protein 3b, Ets variant gene 1, substance P, somatostatin, vasoactive intestinal polypeptide, and parvalbumin. Double labeling experiments, by either in situ hybridization or immunostaining, demonstrated that the 12 molecular markers collectively define 10 different sSC neuronal types. The characteristic positions of these cell types divide the sSC into four distinct layers. The 12 markers identified here will serve as valuable tools to examine molecular mechanisms that regulate development of sSC neuronal types. These markers could also be used to examine the connections between specific cell types that form retinocollicular, corticocollicular, or colliculothalamic pathways. J. Comp. Neurol. 524:2300-2321, 2016. © 2016 Wiley Periodicals, Inc.