Selective activation and down-regulation of Trk receptors by neurotrophins in human neurons co-expressing TrkB and TrkC.

In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.

Anomalous branching of the aortic arch in a dog.

Anomalies in the subclavian and common carotid arteries can be of interest in cases of cranial mediastinal surgeries, as well as to diagnose the cause of oesophageal constrictions leading to clinical signs of dysphagia (dysphagia lusoria). The development and regression of the aortic arches are of key importance in understanding the origin of these type of vascular anomalies. This report describes the congenital anomalous aortic origin of the common carotid and the subclavian arteries in a 14-year-old dog and the plausible developmental pattern failure. Academic dissection revealed a common bicarotid trunk and bisubclavian trunk arising from the most cranial aspect of the aortic arch. Despite the abnormal origin, these vessels displayed a predominantly standard anatomical course. All the anticipated branches were identified and described. Cardiac abnormalities were also noted including right atrial dilation, coronary sinus enlargement, right and left valvular endocardiosis, a patent foramen ovale and marked concentric left ventricular hypertrophy with compensatory left atrial dilation. Additionally, the right recurrent laryngeal nerve demonstrated an aberrant course consistent with a 'non-recurrent laryngeal nerve' (non-RLN). Awareness of the anatomical variations of the aortic arch is important for surgical interventions of the cranial mediastinum as well as radiological interpretation. Although infrequent, the variants similar to the one described here have been reported in different species.

LMK235, a small molecule inhibitor of HDAC4/5, protects dopaminergic neurons against neurotoxin- and α-synuclein-induced degeneration in cellular models of Parkinson's disease.

Epigenetic modifications in neurodegenerative disease are under investigation for their roles in disease progression. Alterations in acetylation rates of certain Parkinson's disease (PD)-linked genes have been associated with the pathological progression of this disorder. In light of this, and given the lack of disease-modifying therapies for PD, HDAC inhibitors (HDIs) are under consideration as potential pharmacological agents. The neuroprotective effects of pan-HDACs and some class-specific inhibitors have been tested in in vivo and in vitro models of PD, with varying outcomes. Here we used gene co-expression analysis to identify HDACs that are associated with human dopaminergic (DA) neuron development. We identified HDAC3, HDAC5, HDAC6 and HDAC9 as being highly correlated with the DA markers, SLC6A3 and NR4A2. RT-qPCR revealed that mRNA expression of these HDACs exhibited similar temporal profiles during embryonic mouse midbrain DA (mDA) neuron development. We tested the neuroprotective potential of a number of class-specific small molecule HDIs on human SH-SY5Y cells, using neurite growth as a phenotypic readout of neurotrophic action. Neither the class I-specific HDIs, RGFP109 and RGFP966, nor the HDAC6 inhibitor ACY1215, had significant effects on neurite outgrowth. However, the class IIa HDI, LMK235 (a HDAC4/5 inhibitor), significantly increased histone acetylation and neurite outgrowth. We found that LMK235 increased BMP-Smad-dependent transcription in SH-SY5Y cells and that this was required for its neurite growth-promoting effects on SH-SY5Y cells and on DA neurons in primary cultures of embryonic day (E) 14 rat ventral mesencephalon (VM). These effects were also seen in SH-SY5Y cells transfected with HDAC5 siRNA. Furthermore, LMK235 treatment exerted neuroprotective effects against degeneration induced by the DA neurotoxin 1-methyl-4-phenylpyridinium (MPP+), in both SH-SY5Y cells and cultured DA neurons. Treatment with LMK235 was also neuroprotective against axonal degeneration induced by overexpression of wild-type (WT) or A53T mutant α-synuclein in both SH-SY5Y cells and primary cultures of DA neurons. In summary, these data show the neuroprotective potential of the class IIa HDI, LMK235, in cell models of relevance to PD.

Neuregulin-4 contributes to the establishment of cutaneous sensory innervation.

Recent work has shown that neuregulin-4 (NRG4) is a physiological regulator of the growth of sympathetic axons and CNS dendrites in the developing nervous system. Here, we have investigated whether NRG4 plays a role in sensory axon growth and the establishment of cutaneous sensory innervation. Imaging early nerve fibers in the well-characterized cutaneous trigeminal territory, the brachial plexus, and thorax revealed very marked and highly significant decreases in nerve fiber length and branching density in Nrg4-/- embryos compared with Nrg4+/+ littermates. NRG4 promoted neurotrophin-independent sensory axon growth from correspondingly early trigeminal ganglion and DRG neurons in culture but not from enteroceptive nodose ganglion neurons. High levels of Nrg4 mRNA were detected in cutaneous tissues but not in sensory ganglia. Our findings suggest that NRG4 is an important target-derived factor that participates in the establishment of early cutaneous sensory innervation.

STRAP and NME1 Mediate the Neurite Growth-Promoting Effects of the Neurotrophic Factor GDF5.

Loss of midbrain dopaminergic (mDA) neurons and their axons is central to Parkinson's disease (PD). Growth differentiation factor (GDF)5 is a potential neurotrophic factor for PD therapy. However, the molecular mediators of its neurotrophic action are unknown. Our proteomics analysis shows that GDF5 increases the expression of serine threonine receptor-associated protein kinase (STRAP) and nucleoside diphosphate kinase (NME)1 in the SH-SY5Y neuronal cell line. GDF5 overexpression increased NME1 expression in adult rat brain in vivo. NME and STRAP mRNAs are expressed in developing and adult rodent midbrain. Expression of both STRAP and NME1 is necessary and sufficient for the promotion of neurite growth in SH-SY5Y cells by GDF5. NME1 treatment increased neurite growth in both SH-SY5Y cells and cultured mDA neurons. Expression patterns of NME and STRAP are altered in PD midbrain. NME1 and STRAP are thus key mediators of GDF5's neurotrophic effects, rationalizing their future study as therapeutic targets for PD.

Association of distinct type 1 bone morphogenetic protein receptors with different molecular pathways and survival outcomes in neuroblastoma.

Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan-Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increase BMPR1B expression. In agreement, nerve growth factor treatment of cultured sympathetic neurons decreased Bmpr1a expression and increased Bmpr1b expression. Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB.

CD40L Reverse Signaling Influences Dendrite Spine Morphology and Expression of PSD-95 and Rho Small GTPases.

CD40-activated CD40L reverse signaling is a major physiological regulator of neural process growth from many kinds of developing neurons. Here we have investigated whether CD40L-reverse signaling also influences dendrite spine number and morphology in striatal medium spiny neurons (MSNs). Golgi preparations revealed no differences in the spine density, but because the dendrite arbors of MSNs were larger and branched in Cd40 -/- mice, the total number of spines was greater in Cd40 -/- mice. We also detected more mature spines compared with wild-type littermates. Western blot revealed that MSN cultures from Cd40 -/- mice had significantly less PSD-95 and there were changes in RhoA/B/C and Cdc42. Immunocytochemistry revealed that PSD-95 was clustered in spines in Cd40 -/- neurons compared with more diffuse labeling in Cd40 +/+ neurons. Activation of CD40L-reverse signaling with CD40-Fc prevented the changes observed in Cd40 -/- cultures. Our findings suggest that CD40L-reverse signaling influences dendrite spine morphology and related protein expression and distribution.

Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling.

Parkinson's disease is characterized by the intracellular accumulation of α-synuclein which has been linked to early dopaminergic axonal degeneration. Identifying druggable targets that can promote axonal growth in cells overexpressing α-synuclein is important in order to develop strategies for early intervention. Class-IIa histone deacetylases (HDACs) have previously emerged as druggable targets, however, it is not known which specific class-IIa HDACs should be targeted to promote neurite growth in dopaminergic neurons. To provide insight into this, we used gene co-expression analysis to identify which, if any, of the class-IIa HDACs had a positive correlation with markers of dopaminergic neurons in the human substantia nigra. This revealed that two histone deacetylases, HDAC5 and HDAC9, are co-expressed with TH, GIRK2 and ALDH1A1 in the human SN. We further found that HDAC5 and HDAC9 are expressed in dopaminergic neurons in the adult mouse substantia nigra. We show that siRNAs targeting HDAC5 or HDAC9 can promote neurite growth in SH-SY5Y cells, and that their pharmacological inhibition, using the drug MC1568, promoted neurite growth in cultured rat dopaminergic neurons. Moreover, MC1568 treatment upregulated the expression of the neurotrophic factor, BMP2, and its downstream transcription factor, SMAD1. In addition, MC1568 or siRNAs targeting HDAC5 or HDAC9 led to an increase in Smad-dependent GFP expression in a reporter assay. Furthermore, MC1568 treatment of cultured rat dopaminergic neurons increased cellular levels of phosphorylated Smad1, which was prevented by the BMP receptor inhibitor, dorsomorphin. Dorsomorphin treatment prevented the neurite growth-promoting effects of siRNAs targeting HDAC5, as did overexpression of dominant-negative Smad4 or of the inhibitory Smad7, demonstrating a functional link to BMP signaling. Supplementation with BMP2 prevented the neurite growth-inhibitory effects of nuclear-restricted HDAC5. Finally, we report that siRNAs targeting HDAC5 or HDAC9 promoted neurite growth in cells overexpressing wild-type or A53T-α-synuclein and that MC1568 protected cultured rat dopaminergic neurons against the neurotoxin, MPP+. These findings establish HDAC5 and HDAC9 as novel regulators of BMP-Smad signaling, that additionally may be therapeutic targets worthy of further exploration in iPSC-derived human DA neurons and in vivo models of Parkinson's disease.

CD40 forward signalling is a physiological regulator of early sensory axon growth.

Multiple members of the tumour necrosis factor superfamily (TNFSF) regulate the growth and branching of neural processes late in development, when neurons are establishing and refining connections. Here, we present the first evidence that a TNFSF member acts much earlier in development, when axons are growing to their targets. CD40L transiently enhanced axon growth from embryonic mouse DRG neurons cultured at this early stage. Early spinal nerves of embryos lacking the CD40L receptor (Cd40-/- mice) were significantly shorter in vivo than those of Cd40+/+ littermates. CD40L was synthesized in early DRG targets and was co-expressed with CD40 in early DRG neurons. Whereas CD40L enhanced early axon growth independently of neurotrophins, disruption of a CD40L/CD40 autocrine loop impaired early neurotrophin-promoted axon growth. In marked contrast to the widespread regulation of axon and dendrite growth by CD40L reverse signalling later in development, CD40-Fc, which activates reverse signalling, had no effect on early sensory axon growth. These results suggest that CD40 forward signalling is a novel physiological regulator of early axon growth that acts by target-derived and autocrine mechanisms.

Neuregulin-4 Is Required for the Growth and Elaboration of Striatal Medium Spiny Neuron Dendrites.

Medium spiny neurons (MSNs) comprise the vast majority of neurons in the striatum. Changes in the exuberant dendrites of these widely connected neurons are associated with a multitude of neurological conditions and are caused by a variety of recreational and medicinal drugs. However, we have a poor understanding of the physiological regulators of dendrite growth and elaboration of this clinically important population of neurons. Here, we show that MSN dendrites are markedly smaller and less branched in neonatal mice that possess a homozygous null mutation in the neuregulin-4 gene (Nrg4-/-) compared with wild type (Nrg4+/+) littermates. Nrg4-/- mice also had a highly significant reduction in MSN dendrite spine number in neonates and adults. The striking stunted dendrite arbor phenotype of MSNs observed in Nrg4-/- neonates was replicated in MSNs cultured from Nrg4-/- embryos and was completely rescued by soluble recombinant neuregulin-4. MSNs cultured from wild type mice coexpressed NRG4 and its receptor ErbB4. Our findings show that NRG4 is a major novel regulator of dendritic growth and arborization and spine formation in the striatum and suggest that it exerts its effects by an autocrine/paracrine mechanism.

Regional Differences in the Contributions of TNF Reverse and Forward Signaling to the Establishment of Sympathetic Innervation.

Members of the TNF and TNF receptor superfamilies acting by both forward and reverse signaling are increasingly recognized as major physiological regulators of axon growth and tissue innervation in development. Studies of the experimentally tractable superior cervical ganglion (SCG) neurons and their targets have shown that only TNF reverse signaling, not forward signaling, is a physiological regulator of sympathetic innervation. Here, we compared SCG neurons and their targets with prevertebral ganglion (PVG) neurons and their targets. Whereas all SCG targets were markedly hypoinnervated in both TNF-deficient and TNFR1-deficient mice, PVG targets were not hypoinnervated in these mice and one PVG target, the spleen, was significantly hyperinnervated. These in vivo regional differences in innervation density were related to in vitro differences in the responses of SCG and PVG neurons to TNF reverse and forward signaling. Though TNF reverse signaling enhanced SCG axon growth, it did not affect PVG axon growth. Whereas activation of TNF forward signaling in PVG axons inhibited growth, TNF forward signaling could not be activated in SCG axons. These latter differences in the response of SCG and PVG axons to TNF forward signaling were related to TNFR1 expression, whereas PVG axons expressed TNFR1, SCG axons did not. These results show that both TNF reverse and forward signaling are physiological regulators of sympathetic innervation in different tissues.

CD40L reverse signaling suppresses prevertebral sympathetic axon growth and tissue innervation.

CD40-activated CD40L reverse signaling is a major physiological regulator of the growth of neural processes in the developing nervous system. Previous work on superior cervical ganglion (SCG) neurons of the paravertebral sympathetic chain has shown that CD40L reverse signaling enhances NGF-promoted axon growth and tissue innervation. Here we show that CD40L reverse signaling has the opposite function in prevertebral ganglion (PVG) sympathetic neurons. During a circumscribed perinatal window of development, PVG neurons cultured from Cd40-/- mice had substantially larger, more exuberant axon arbors in the presence of NGF than PVG neurons cultured from wild-type mice. Tissues that receive their sympathetic innervation from PVG neurons were markedly hyperinnervated in Cd40-/- mice compared with wild-type mice. The exuberant axonal growth phenotype of cultured CD40-deficient perinatal PVG neurons was pared back to wild-type levels by activating CD40L reverse signaling with a CD40-Fc chimeric protein, but not by activating CD40 forward signaling with CD40L. The co-expression of CD40 and CD40L in PVG neurons suggests that these proteins engage in an autocrine signaling loop in these neurons. Our work shows that CD40L reverse signaling is a physiological regulator of NGF-promoted sympathetic axon growth and tissue innervation with opposite effects in paravertebral and prevertebral neurons.

TWE-PRIL reverse signalling suppresses sympathetic axon growth and tissue innervation.

TWE-PRIL is a naturally occurring fusion protein of components of two TNF superfamily members: the extracellular domain of APRIL; and the intracellular and transmembrane domains of TWEAK with no known function. Here, we show that April-/- mice (which lack APRIL and TWE-PRIL) exhibited overgrowth of sympathetic fibres in vivo, and sympathetic neurons cultured from these mice had significantly longer axons than neurons cultured from wild-type littermates. Enhanced axon growth from sympathetic neurons cultured from April-/- mice was prevented by expressing full-length TWE-PRIL in these neurons but not by treating them with soluble APRIL. Soluble APRIL, however, enhanced axon growth from the sympathetic neurons of wild-type mice. siRNA knockdown of TWE-PRIL but not siRNA knockdown of APRIL alone also enhanced axon growth from wild-type sympathetic neurons. Our work reveals the first and physiologically relevant role for TWE-PRIL and suggests that it mediates reverse signalling.

An essential role for neuregulin-4 in the growth and elaboration of developing neocortical pyramidal dendrites.

Neuregulins, with the exception of neuregulin-4 (NRG4), have been shown to be extensively involved in many aspects of neural development and function and are implicated in several neurological disorders, including schizophrenia, depression and bipolar disorder. Here we provide the first evidence that NRG4 has a crucial function in the developing brain. We show that both the apical and basal dendrites of neocortical pyramidal neurons are markedly stunted in Nrg4-/- neonates in vivo compared with Nrg4+/+ littermates. Neocortical pyramidal neurons cultured from Nrg4-/- embryos had significantly shorter and less branched neurites than those cultured from Nrg4+/+ littermates. Recombinant NRG4 rescued the stunted phenotype of embryonic neocortical pyramidal neurons cultured from Nrg4-/- mice. The majority of cultured wild type embryonic cortical pyramidal neurons co-expressed NRG4 and its receptor ErbB4. The difference between neocortical pyramidal dendrites of Nrg4-/- and Nrg4+/+ mice was less pronounced, though still significant, in juvenile mice. However, by adult stages, the pyramidal dendrite arbors of Nrg4-/- and Nrg4+/+ mice were similar, suggesting that compensatory changes in Nrg4-/- mice occur with age. Our findings show that NRG4 is a major novel regulator of dendritic arborisation in the developing cerebral cortex and suggest that it exerts its effects by an autocrine/paracrine mechanism.

TNF superfamily member APRIL enhances midbrain dopaminergic axon growth and contributes to the nigrostriatal projection in vivo.

We have studied the role of the tumor necrosis factor superfamily member APRIL in the development of embryonic mouse midbrain dopaminergic neurons in vitro and in vivo. In culture, soluble APRIL enhanced axon growth during a window of development between E12 and E14 when nigrostriatal axons are growing to their targets in the striatum in vivo. April transcripts were detected in both the striatum and midbrain during this period and at later stages. The axon growth-enhancing effect of APRIL was similar to that of glial cell-derived neurotrophic factor (GDNF), but in contrast to GDNF, APRIL did not promote the survival of midbrain dopaminergic neurons. The effect of APRIL on axon growth was prevented by function-blocking antibodies to one of its receptors, BCMA (TNFRSF13A), but not by function-blocking antibodies to the other APRIL receptor, TACI (TNFRSF13B), suggesting that the effects of APRIL on axon growth are mediated by BCMA. In vivo, there was a significant reduction in the density of midbrain dopaminergic projections to the striatum in April-/- embryos compared with wild type littermates at E14. These findings demonstrate that APRIL is a physiologically relevant factor for the nigrostriatal projection. Given the importance of the degeneration of dopaminergic nigrostriatal connections in the pathogenesis and progression of Parkinson's disease, our findings contribute to our understanding of the factors that establish nigrostriatal integrity.

Zeb2 is a negative regulator of midbrain dopaminergic axon growth and target innervation.

Neural connectivity requires neuronal differentiation, axon growth, and precise target innervation. Midbrain dopaminergic neurons project via the nigrostriatal pathway to the striatum to regulate voluntary movement. While the specification and differentiation of these neurons have been extensively studied, the molecular mechanisms that regulate midbrain dopaminergic axon growth and target innervation are less clear. Here we show that the transcription factor Zeb2 cell-autonomously represses Smad signalling to limit midbrain dopaminergic axon growth and target innervation. Zeb2 levels are downregulated in the embryonic rodent midbrain during the period of dopaminergic axon growth, when BMP pathway components are upregulated. Experimental knockdown of Zeb2 leads to an increase in BMP-Smad-dependent axon growth. Consequently there is dopaminergic hyperinnervation of the striatum, without an increase in the numbers of midbrain dopaminergic neurons, in conditional Zeb2 (Nestin-Cre based) knockout mice. Therefore, these findings reveal a new mechanism for the regulation of midbrain dopaminergic axon growth during central nervous system development.

T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling.

Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca2+ influx, and highly selective T-type Ca2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca2+ channels, prevented enhanced axon growth. T-type Ca2+ channel-specific inhibitors eliminated Ca2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca2+ channels along sympathetic axons, which is required for enhanced axon growth.

Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation.

BACKGROUND: Nerve growth factor (NGF) is the prototypical target-derived neurotrophic factor required for sympathetic neuron survival and for the growth and ramification of sympathetic axons within most but not all sympathetic targets. This implies the operation of additional target-derived factors for regulating terminal sympathetic axon growth and branching. RESULTS: Here report that growth differentiation factor 5 (GDF5), a widely expressed member of the transforming growth factor beta (TGFß) superfamily required for limb development, promoted axon growth from mouse superior cervical ganglion (SCG) neurons independently of NGF and enhanced axon growth in combination with NGF. GDF5 had no effect on neuronal survival and influenced axon growth during a narrow window of postnatal development when sympathetic axons are ramifying extensively in their targets in vivo. SCG neurons expressed all receptors capable of participating in GDF5 signaling at this stage of development. Using compartment cultures, we demonstrated that GDF5 exerted its growth promoting effect by acting directly on axons and by initiating retrograde canonical Smad signalling to the nucleus. GDF5 is synthesized in sympathetic targets, and examination of several anatomically circumscribed tissues in Gdf5 null mice revealed regional deficits in sympathetic innervation. There was a marked, highly significant reduction in the sympathetic innervation density of the iris, a smaller though significant reduction in the trachea, but no reduction in the submandibular salivary gland. There was no reduction in the number of neurons in the SCG. CONCLUSIONS: These findings show that GDF5 is a novel target-derived factor that promotes sympathetic axon growth and branching and makes a distinctive regional contribution to the establishment of sympathetic innervation, but unlike NGF, plays no role in regulating sympathetic neuron survival.

Novel Application of Behavioral Assays Allows Dissociation of Joint Pathology from Systemic Extra-Articular Alterations Induced by Inflammatory Arthritis.

Introduction: Although rheumatoid arthritis (RA) is a disease of articular joints, patients often suffer from co-morbid neuropsychiatric changes, such as anxiety, that may reflect links between heightened systemic inflammation and abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we apply behavioral neuroscience methods to assess the impact of antigen-induced arthritis (AIA) on behavioral performance in wild type (WT) and interleukin-10 deficient (Il10-/-) mice. Our aim was to identify limb-specific motor impairments, as well as neuropsychological responses to inflammatory arthritis. Methods: Behavioral testing was performed longitudinally in WT and Il10-/- mice before and after the induction of arthritic joint pathology. Footprint analysis, beam walking and open field assessment determined a range of motor, exploratory and anxiety-related parameters. Specific gene changes in HPA axis tissues were analyzed using qPCR. Results: Behavioral assessment revealed transient motor and exploratory impairments in mice receiving AIA, coinciding with joint swelling. Hind limb coordination deficits were independent of joint pathology. Behavioral impairments returned to baseline by 10 days post-AIA in WT mice. Il10-/- mice demonstrated comparable levels of swelling and joint pathology as WT mice up to 15 days post-AIA, but systemic differences were evident in mRNA expression in HPA axis tissues from Il10-/- mice post-AIA. Interestingly, the behavioral profile of Il10-/- mice revealed a significantly longer time post-AIA for activity and anxiety-related behaviors to recover. Conclusions: The novel application of sensitive behavioral tasks has enabled dissociation between behaviors that occur due to transient joint-specific pathology and those generated by more subtle systemic alterations that manifest post-AIA.