Shared Autonomic Pathways Connect Bone Marrow and Peripheral Adipose Tissues Across the Central Neuraxis.

Bone marrow adipose tissue (BMAT) is increased in both obesity and anorexia. This is unique relative to white adipose tissue (WAT), which is generally more attuned to metabolic demand. It suggests that there may be regulatory pathways that are common to both BMAT and WAT and also those that are specific to BMAT alone. The central nervous system (CNS) is a key mediator of adipose tissue function through sympathetic adrenergic neurons. Thus, we hypothesized that central autonomic pathways may be involved in BMAT regulation. To test this, we first quantified the innervation of BMAT by tyrosine hydroxylase (TH) positive nerves within the metaphysis and diaphysis of the tibia of B6 and C3H mice. We found that many of the TH+ axons were concentrated around central blood vessels in the bone marrow. However, there were also areas of free nerve endings which terminated in regions of BMAT adipocytes. Overall, the proportion of nerve-associated BMAT adipocytes increased from proximal to distal along the length of the tibia (from ~3-5 to ~14-24%), regardless of mouse strain. To identify the central pathways involved in BMAT innervation and compare to peripheral WAT, we then performed retrograde viral tract tracing with an attenuated pseudorabies virus (PRV) to infect efferent nerves from the tibial metaphysis (inclusive of BMAT) and inguinal WAT (iWAT) of C3H mice. PRV positive neurons were identified consistently from both injection sites in the intermediolateral horn of the spinal cord, reticular formation, rostroventral medulla, solitary tract, periaqueductal gray, locus coeruleus, subcoeruleus, Barrington's nucleus, and hypothalamus. We also observed dual-PRV infected neurons within the majority of these regions. Similar tracings were observed in pons, midbrain, and hypothalamic regions from B6 femur and tibia, demonstrating that these results persist across mouse strains and between skeletal sites. Altogether, this is the first quantitative report of BMAT autonomic innervation and reveals common central neuroanatomic pathways, including putative "command" neurons, involved in coordinating multiple aspects of sympathetic output and facilitation of parallel processing between bone marrow/BMAT and peripheral adipose tissue.

Corrigendum: Shared Autonomic Pathways Connect Bone Marrow and Peripheral Adipose Tissues Across the Central Neuraxis.

[This corrects the article DOI: 10.3389/fendo.2019.00668.].

Whisker-related circuitry in the trigeminal nucleus principalis: Topographic precision.

Single whiskers are topographically represented in the trigeminal (V) nucleus principalis (PrV) by a set of cylindrical aggregates of primary afferent terminals and somata (barrelettes). This isomorphic pattern is transmitted to the thalamus and barrel cortex. However, it is not known if terminals in PrV from neighboring whiskers interdigitate so as to violate rules of spatial parcellation predicted by barrelette borders; nor is it known the extent to which higher order inputs are topographic. The existence of inter-whisker arbor overlap or diffuse higher order inputs would demand additional theoretical principles to account for single whisker dominance in PrV cell responses. In adult rats, first, primary afferent pairs responding to the same or neighboring whiskers and injected with Neurobiotin or horseradish peroxidase were rendered brown or black to color-code their terminal boutons. When collaterals from both fibers appeared in the same topographic plane through PrV, the percentage of the summed area of the two arbor envelopes that overlapped was computed. For same-whisker pairs, overlap was 5 ± 6% (mean ± SD). For within-row neighbors, overlap was 2 ± 5%. For between-row neighbors, overlap was 1 ± 4%. Second, the areas of whisker primary afferent arbors and their corresponding barrelettes in the PrV were compared. In the transverse plane, arbor envelopes significantly exceeded the areas of cytochrome oxidase-stained barrelettes; arbors often extended into neighboring barrelettes. Third, bulk tracing of the projections from the spinal V subnucleus interpolaris (SpVi) to the PrV revealed strict topography such that they connect same-whisker barrelettes in the SpVi and PrV. Thus, whisker primary afferents do not exclusively project to their corresponding PrV barrelette, whereas higher order SpVi inputs to the PrV are precisely topographic.

Whisker-related circuitry in the trigeminal nucleus principalis: ultrastructure.

Trigeminal (V) nucleus principalis (PrV) is the requisite brainstem nucleus in the whisker-to-barrel cortex model system that is widely used to reveal mechanisms of map formation and information processing. Yet, little is known of the actual PrV circuitry. In the ventral "barrelette" portion of the adult mouse PrV, relationships between V primary afferent terminals, thalamic-projecting PrV neurons, and gamma-aminobutyric acid (GABA)-ergic terminals were analyzed in the electron microscope. Primary afferents, thalamic-projecting cells, and GABAergic terminals were labeled, respectively, by Neurobiotin injections in the V ganglion, horseradish peroxidase injections in the thalamus, and postembedding immunogold histochemistry. Primary afferent terminals (Neurobiotin- and glutamate-immunoreactive) display asymmetric and multiple synapses predominantly upon the distal dendrites and spines of PrV cells that project to the thalamus. Primary afferents also synapse upon GABAergic terminals. GABAergic terminals display symmetric synapses onto primary afferent terminals, the somata and dendrites (distal, mostly) of thalamic-projecting neurons, and GABAergic dendrites. Thus, primary afferent inputs through the PrV are subject to pre- and postsynaptic GABAergic influences. As such, circuitry exists in PrV "barrelettes" for primary afferents to directly activate thalamic-projecting and inhibitory local circuit cells. The latter are synaptically associated with themselves, the primary afferents, and with the thalamic-projecting neurons. Thus, whisker-related primary afferent inputs through PrV projection neurons are pre- and postsynaptically modulated by local circuits.

Null mutations of NT-3 and Bax affect trigeminal ganglion cell number but not brainstem barrelette pattern formation.

Trigeminal ganglion (TG) neurons innervate the grid-like array of whisker follicles on the face of the mouse. Central TG axons project to the trigeminal (V) brainstem nuclear complex, including the nucleus principalis (PrV) and the spinal subnucleus interpolaris (SpVi), where they innervate barrelettes that are organized in a pattern that recapitulates the whisker pattern on the face. Neurotrophin-3 (NT-3) supports a population of TG cells that supply slowly adapting mechanoreceptors in the whisker pad. We examined mice at embryonic day 17 (E17) and on the day of birth (P0) with null mutations of NT-3, Bax, a proapoptotic gene associated with naturally occurring cell death, and Bax/NT-3 double knockout (KO) mutants to determine if: (1) the number of TG cells would be reduced; (2) eliminating the Bax gene would rescue the NT-3-dependent neurons; and (3) the central projections of the rescued axons in the Bax/NT-3 double KO mice would fail to develop the barrelette patterns in the PrV and SpVi subnuclei. In mice at E17, NT-3(-/-) mutants had 65% fewer TG neurons than found in age-matched wild-type (WT) mice, and at P0, the number was reduced by 55% (p < 0.001 for both). Bax null mutant mice at E17 had 132% of the WT number of TG cells (p < 0.001), although the numbers returned to WT levels by P0. Bax/NT-3 double KO mice at E17 had TG cell numbers equal to those seen in WT, but the double KO failed to retain WT TG neuron numbers in P0 mice (39% fewer cells; p < 0.001). In all cases of reduced experimental neuron numbers, and in the E17 Bax(-/-) mice with supernumerary cells, the barrelette patterns in the PrV and SpVi were normal. Only a slight qualitative reduction in overall barrelette field area and clarity of barrelettes were seen. These results suggest that NT-3 is not necessary for barrelette pattern formation in the brainstem.

The transcription factor, Lmx1b, promotes a neuronal glutamate phenotype and suppresses a GABA one in the embryonic trigeminal brainstem complex.

Achieving an appropriate balance between inhibitory and excitatory neuronal fate is critical for development of effective synaptic transmission. However, the molecular mechanisms dictating such phenotypic outcomes are not well understood, especially in the whisker-to-barrel cortex neuraxis, an oft-used model system for revealing developmental mechanisms. In trigeminal nucleus principalis (PrV), the brainstem link in the whisker-barrel pathway, the transcription factor Lmx1b marks glutamatergic cells. In PrV of Lmx1b knockout mice (-/-), initial specification of glutamatergic vs. GABAergic cell fate is normal until embryonic day 14.5. Subsequently, until the day of birth, glutamatergic markers (e.g., VGLUT2) stain significantly fewer PrV neurons, whereas, GABAergic markers (Pax2 and Gad1) stain significantly more PrV cells, notably in Lmx1b null PrV cells. These changes also occurred in Lmx1b/Bax double-/- mice, where PrV cells are rescued from Lmx1b-/- induced apoptosis; thus, effects upon excitatory/inhibitory cell ratios do not reflect a cell death confound. Electroporation-induced ectopic expression of Lmx1b in an array of sites decreases numbers of neurons that express GABAergic markers, but increases VGLUT2+ cell numbers or stain intensity. Thus, Lmx1b is not involved in the initial specification of glutamatergic cell fate, but is essential for maintaining a glutamatergic phenotype. Other experiments suggest that Lmx1b acts to suppress Pax2, a promoter of GABAergic cell fate, in a cell-autonomous manner, which may be a mechanism for maintaining a functional balance of glutamatergic and GABAergic cell types in development.

Passive vs. active touch-induced activity in the developing whisker pathway.

The mouse trigeminal (V) system undergoes significant postnatal structural and functional developmental changes. Histological modules (barrelettes, barreloids and barrels) in the brainstem, thalamus and cortex related to actively moved (whisking) tactile hairs (vibrissae) on the face allow detailed studies of development. High-resolution [(3) H]2-deoxyglucose (2DG) emulsion autoradiography with cytochrome oxidase histochemistry was used to analyze neuronal activity changes related to specific whisker modules in the developing and mature mouse V system provoked by passive (experimenter-induced) and active (animal-induced) displacements of a single whisker (D4). We tested the hypothesis that neuronal activity patterns change in relation to the onset of active touch (whisking) on postnatal day (P)14. Quantitative image analyses revealed: (i) on P7, when whisker-like patterns of modules are clear, heightened 2DG activity in all appropriate modules in the brainstem, thalamus and cortex; (ii) on P14, a transitory activity pattern coincident with the emergence of whisking behavior that presages (iii) strong labeling of the spinal V subnucleus interpolaris and barrel cortex produced by single-whisker-mediated active touch in adults and (iv) at all above-listed ages and structures, significant suppression of baseline activity in some modules surrounding those representing the stimulated whisker. Differences in activity patterns before and after the onset of whisking behavior may be caused by neuronal activity induced by whisking, and by strengthening of modulatory projections that alter the activity of subcortical inputs produced by whisking behavior during active touch.

The transcription factor, Lmx1b, is necessary for the development of the principal trigeminal nucleus-based lemniscal pathway.

Little is known of transcriptional mechanisms underlying the development of the trigeminal (V) principal sensory nucleus (PrV), the brainstem nucleus responsible for the development of the whisker-to-barrel cortex pathway. Lmx1b, a LIM homeodomain transcription factor, is expressed in embryonic PrV. In Lmx1b knockout ((-)(/)(-)) mice, V primary afferent projections to PrV are normal, albeit reduced in number, whereas the PrV-thalamic lemniscal pathway is sparse and develops late. Excess cell death occurs in the embryonic Lmx1b(-)(/)(-) PrV, but not in Lmx1b/Bax double null mutants. Expression of Drg11, a downstream transcription factor essential for PrV development and pattern formation, is abolished in PrV, but not in the V ganglion. Consequently, whisker patterns fail to develop in PrV by birth. Rescued PrV cells in Lmx1b/Bax double (-)(/)(-)s failed to rescue whisker-related PrV pattern formation. Thus, Lmx1b and Drg11 may act in the same genetic signaling pathway that is essential for PrV pattern formation.

In DRG11 knock-out mice, trigeminal cell death is extensive and does not account for failed brainstem patterning.

A previous study (Ding et al., 2003) showed that the homeodomain transcription factor DRG11 is necessary for pattern formation in the trigeminal nucleus principalis (PrV), the requisite brainstem nucleus for development of the whisker-to-barrel cortex pathway. However, it is not known how DRG11 contributes to pattern formation. Anatomical studies were performed in DRG11 knock-out (-/-) and DRG11/Bax double -/- mice to test the hypotheses that DRG11 is required for neuronal survival in the V pathway and that PrV cell death is sufficient to explain pattern alterations. At birth, DRG11(-/-) mice had equivalent cell loss in the V ganglion, PrV, and spinal V subnucleus interpolaris (SpVi). Because whisker-related patterns were normal in the SpVi, cell death would not appear to explain failed pattern formation in the mutant PrV. Electron microscopy revealed exuberant apoptosis and necrosis as the mechanisms of PrV cell death occurring in the late prenatal and newborn DRG11(-/-), when such cell death was up to six times more prevalent than normal. DRG11 heterozygote and Bax(-/-) mice were crossed in an attempt to dissociate PrV patterning anomalies from exuberant apoptosis in DRG11(-/-) mice. Both DRG11(-/-) and DRG11/Bax double -/- mutants lacked whisker-related patterning in their PrV, despite Bax(-/-)-induced rescue of V ganglion and PrV cells. Thus, apoptotic cell death is not a sufficient cause of failed pattern formation in the PrV of the DRG11(-/-). A signaling pathway involving DRG11 may, therefore, be the elusive PrV pattern maker.

Development of the mesencephalic trigeminal nucleus requires a paired homeodomain transcription factor, Drg11.

The mesencephalic trigeminal nucleus (Me5) innervates muscle spindles and is responsible for receiving and transmitting proprioception from the oro-facial region. Molecular mechanisms underlying the development of the Me5 are poorly understood. Evidence is provided here that transcription factor Drg11 is required for Me5 development. Drg11 was expressed in the Me5 cells of the embryonic and early postnatal mouse brains, and the Me5 cells were absent in Drg11-/- mice at birth. The absence of the Me5 cells in Drg11-/- mice appeared to be caused by increased cell death in the Me5 during embryonic development. In postnatal Drg11-/- mice, Me5 cell innervation of masseter muscle spindles was undetectable, while robust trigeminal motoneuron innervation of masseter muscle fibers was detected. The postnatal body weight of Drg11-/- mice was notably less than that of wild-type mice, and this might result, in part, from disruption of the oro-facial proprioceptive afferent pathway. Taken together, our results demonstrate an essential role for Drg11 in the development of the Me5.

Molecular determinants of the face map development in the trigeminal brainstem.

The perception of external sensory information by the brain requires highly ordered synaptic connectivity between peripheral sensory neurons and their targets in the central nervous system. Since the discovery of the whisker-related barrel patterns in the mouse cortex, the trigeminal system has become a favorite model for study of how its connectivity and somatotopic maps are established during development. The trigeminal brainstem nuclei are the first CNS regions where whisker-specific neural patterns are set up by the trigeminal afferents that innervate the whiskers. In particular, barrelette patterns in the principal sensory nucleus of the trigeminal nerve provide the template for similar patterns in the face representation areas of the thalamus and subsequently in the primary somatosensory cortex. Here, we describe and review studies of neurotrophins, multiple axon guidance molecules, transcription factors, and glutamate receptors during early development of trigeminal connections between the whiskers and the brainstem that lead to emergence of patterned face maps. Studies from our laboratories and others' showed that developing trigeminal ganglion cells and their axons depend on a variety of molecular signals that cooperatively direct them to proper peripheral and central targets and sculpt their synaptic terminal fields into patterns that replicate the organization of the whiskers on the muzzle. Similar mechanisms may also be used by trigeminothalamic and thalamocortical projections in establishing patterned neural modules upstream from the trigeminal brainstem.

Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord.

Murine embryonic stem cells were induced to differentiate into neural lineage cells by exposure to retinoic acid. Approximately one million cells were transplanted into the lesion site in the spinal cords of adult rats which had received moderate contusion injuries 9 days previously. One group received transplants of cells genetically modified to over-express bcl-2, which codes for an anti-apoptotic protein. A second group received transplants of the wild-type ES cells from which the bcl-2 line was developed. In the untransplanted control group, only medium was injected. Locomotor abilities were assessed using the Basso, Beattie and Bresnahan (BBB) rating scale for 6 weeks. There was no incremental locomotor improvement in either transplant group when compared to control over the survival period. Morbidity and mortality were significantly more prevalent in the transplant groups than in controls. At the conclusion of the 6-week survival period, the spinal cords were examined. Two of six cords from the bcl-2 group and one of 12 cords from the wild-type group showed gross evidence of abnormal growths at the site of transplantation. No similar growth was seen in the control. Pathological examination of the abnormal cords showed very large numbers of undifferentiated cells proliferating at the injection site and extending up to 1.5 cm rostrally and caudally. These results suggest that transplanting KD3 ES cells, or apoptosis-resistant cells derived from the KD3 line, into the injured spinal cord does not improve locomotor recovery and can lead to tumor-like growth of cells, accompanied by increased debilitation, morbidity and mortality.

The development of myelinated nociceptors is dependent upon trks in the trigeminal ganglion.

Cell size of primary sensory neurons and distribution patterns of neurons that are immunopositive (ip) for VRL-1, a newly cloned capsaicin-receptor homologue, were examined in trigeminal ganglia (TGs) of knockout mice for trkA, trkB or trkC to determine the developmental dependency of myelinated nociceptors on expression of the genes. The number of TG neurons was strongly decreased in the knockout mice as compared to wildtype and heterozygous mice (82%, 39%, and 48% reduction for trkA, trkB and trkC, respectively). The absence of trkA and trkC reduced the number of TG neurons in all cell-size ranges. The number of medium-sized and large TG neurons was decreased in trkB-knockout mice, whereas that of small TG neurons was barely affected by trkB deficiency. TG contained abundant VRL-1-ip neurons in wildtype and heterozygous mice; 9% of TG neurons exhibited immunopositivity. In trkA-knockout mice, VRL-1-ip neurons almost disappeared (1% of TG neurons were VRL-1-ip). However, 13% and 9% of TG neurons in trkB- and trkC-knockout mice, respectively, were immunostained for the ion channel protein. In trkC-knockout mice, the proportion of large VRL-1-ip neurons decreased whereas that of small and medium-sized VRL-1-ip neurons increased. In addition, immunohistochemistry of the protein gene product 9.5 (PGP 9.5) demonstrated that trkA deficiency caused a marked reduction of varicose endings in the epithelium of the palatal mucosa. Loss of trkC diminished the number of PGP 9.5-ip varicose fibers in the deep layer of mucosal connective tissue of the palate. In tooth pulp, PGP 9.5-ip nerve fibers were absent in trkA-knockout mice but abundant in trkB- and trkC-knockout mice. The present study suggests that the development of myelinated nociceptors is dependent on trkA and trkC but not on trkB.

Mechanical and cold allodynia in a rat spinal cord contusion model.

This study examined the time course of mechanical and cold allodynia in rat hindpaw after spinal cord contusion. Hindpaw withdrawal threshold to graded von Frey hair stimulation and withdrawal frequency to acetone application were measured in rats subjected to contusions of varying severity, produced by a MASCIS impactor device with a 10 g weight dropped from 6.25, 12.5, or 25 mm. Mechanical and cold allodynia developed following the injury, and differences in the incidence of allodynia and in withdrawal threshold were significant among these groups. The least severe injury (6.25 mm) most consistently caused a decreased hindpaw threshold to mechanical stimulation and an increased withdrawal frequency to cold.

Enhanced oligodendrocyte survival after spinal cord injury in Bax-deficient mice and mice with delayed Wallerian degeneration.

Mechanisms of oligodendrocyte death after spinal cord injury (SCI) were evaluated by T9 cord level hemisection in wild-type mice (C57BL/6J and Bax+/+ mice), Wlds mice in which severed axons remain viable for 2 weeks, and mice deficient in the proapoptotic protein Bax (Bax-/-). In the lateral white-matter tracts, substantial oligodendrocyte death was evident in the ipsilateral white matter 3-7 mm rostral and caudal to the hemisection site 8 d after injury. Ultrastructural analysis and expression of anti-activated caspase-3 characterized the ongoing oligodendrocyte death at 8 d as primarily apoptotic. Oligodendrocytes were selectively preserved in Wlds mice compared with C57BL/6J mice at 8 d after injury, when severed axons remained viable as verified by antereograde labeling of the lateral vestibular spinal tract. However, 30 d after injury when the severed axons in Wlds animals were already degenerated, the oligodendrocytes preserved at 8 d were lost, and numbers were then equivalent to control C57BL/6J mice. In contrast, oligodendrocyte death was prevented at both time points in Bax-/- mice. When cultured oligodendrocytes were exposed to staurosporine or cyclosporin A, drugs known to stimulate apoptosis in oligodendrocytes, those from Bax-/- mice but not from Bax+/+ or Bax+/- mice were resistant to the apoptotic death. In contrast, the three groups were equally vulnerable to excitotoxic necrosis death induced by kainate. On the basis of these data, we hypothesize that the Wallerian degeneration of white matter axons that follows SCI removes axonal support and induces apoptotic death in oligodendrocytes by triggering Bax expression.

Formation of whisker-related principal sensory nucleus-based lemniscal pathway requires a paired homeodomain transcription factor, Drg11.

Little is known about the molecular mechanisms underlying the formation of the principal sensory nucleus (PrV) of the trigeminal nerve, a major relay station for somatotopic pattern formation in the trigeminal system. Here, we show that mice lacking Drg11, a homeodomain transcription factor, exhibit defects within the PrV, which include an aberrant distribution of Drg11-/- cells, altered expression of a molecular marker, unusual projections of primary afferents from trigeminal ganglion cells, and, subsequently, increased cell death. In addition, surviving PrV cells exhibit delayed and more spatially restricted ascending projections to the ventral posterior medial nucleus of the thalamus (VPm). These early embryonic abnormalities in the PrV lead to the failure to develop whisker-related patterns in the PrV, VPm, and somatosensory cortex. By contrast, somatotopic patterns exist in the spinal trigeminal subnuclei interpolaris (SpVi) and subnuclei caudalis (SpVc) and the dorsal column nucleus-based lemniscal and cortical pathway. Thus, the deficits in the trigeminal system of Drg11-/- mice are specific to the PrV. Our results demonstrate that Drg11 is essential for proper cellular differentiation and, subsequently, for the formation of the whisker-related lemniscal and cortical structures.

Ruffini endings are absent from the periodontal ligament of trkB knockout mice.

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.