The emerging literature implicates a role for glia/cytokines in persistent pain. However, the mechanisms by which these non-neural elements contribute to CNS activity-dependent plasticity and pain are unclear. Using a trigeminal model of inflammatory hyperalgesia, here we provide evidence that demonstrates a mechanism by which glia interact with neurons, leading to activity-dependent plasticity and hyperalgesia. In response to masseter inflammation, there was an upregulation of glial fibrillary acidic proteins (GFAPs), a marker of astroglia, and interleukin-1␤ (IL-1␤), a prototype proinflammatory cytokine, in the region of the trigeminal nucleus specifically related to the processing of deep orofacial input. The activated astroglia exhibited hypertrophy and an increased level of connexin 43, an astroglial gap junction protein. The upregulated IL-1␤ was selectively localized to astrocytes but not to microglia and neurons. Local anesthesia of the masseter nerve prevented the increase in GFAP and IL-1␤ after inflammation, and substance P, a prototype neurotransmitter of primary afferents, induced similar increases in GFAP and IL-1␤, which was blocked by a nitric oxide synthase inhibitor

SUMMARYNeural stem cells in the subventricular zone (SVZ) of the adult mammalian forebrain are a potential source of neurons for neural tissue repair after brain insults such as ischemic stroke and traumatic brain injury (TBI). Recent studies show that neurogenesis in the ventricular zone (VZ) of the adult zebrafish telencephalon has features in common with neurogenesis in the adult mammalian SVZ. Here, we established a zebrafish model to study injury-induced neurogenesis in the adult brain. We show that the adult zebrafish brain possesses a remarkable capacity for neuronal regeneration. Telencephalon injury prompted the proliferation of neuronal precursor cells (NPCs) in the VZ of the injured hemisphere, compared with in the contralateral hemisphere. The distribution of NPCs, viewed by BrdU labeling and ngn1-promoter-driven GFP, suggested that they migrated laterally and reached the injury site via the subpallium and pallium. The number of NPCs reaching the injury site significantly decreased when the fish were treated with an inhibitor of γ-secretase, a component of the Notch signaling pathway, suggesting that injury-induced neurogenesis mechanisms are at least partly conserved between fish and mammals. The injury-induced NPCs differentiated into mature neurons in the regions surrounding the injury site within a week after the injury. Most of these cells expressed T-box brain protein (Tbr1), suggesting they had adopted the normal neuronal fate in this region. These results suggest that the telencephalic VZ contributes to neural tissue recovery following telencephalic injury in the adult zebrafish, and that the adult zebrafish is a useful model for regenerative medicine.

SUMMARYAmplification of genomic DNA by endoreduplication often marks the initiation of cell differentiation in animals and plants. The transition from mitotic cycles to endocycles should be developmentally programmed but how this process is regulated remains largely unknown. We show that the plant growth regulator auxin modulates the switch from mitotic cycles to endocycles in Arabidopsis; high levels of TIR1-AUX/IAA-ARF-dependent auxin signalling are required to repress endocycles, thus maintaining cells in mitotic cycles. By contrast, lower levels of TIR1-AUX/IAA-ARF-dependent auxin signalling trigger an exit from mitotic cycles and an entry into endocycles. Our data further demonstrate that this auxin-mediated modulation of the mitotic-to-endocycle switch is tightly coupled with the developmental transition from cell proliferation to cell differentiation in the Arabidopsis root meristem. The transient reduction of auxin signalling by an auxin antagonist PEO-IAA rapidly downregulates the expression of several core cell cycle genes, and we show that overexpressing one of the genes, CYCLIN A2;3 (CYCA2;3), partially suppresses an early initiation of cell differentiation induced by PEO-IAA. Taken together, these results suggest that auxin-mediated mitotic-to-endocycle transition might be part of the developmental programmes that balance cell proliferation and cell differentiation in the Arabidopsis root meristem.

In the brain of adult mammals, neuronal precursors are generated in the subventricular zone in the lateral wall of the lateral ventricles and migrate into the olfactory bulbs (OBs) through a well-studied route called the rostral migratory stream (RMS). Recent studies have revealed that a comparable neural stem cell niche is widely conserved at the ventricular wall of adult vertebrates. However, little is known about the migration route of neuronal precursors in nonmammalian adult brains. Here, we show that, in the adult zebrafish, a cluster of neuronal precursors generated in the telencephalic ventricular zone migrates into the OB via a route equivalent to the mammalian RMS. Unlike the mammalian RMS, these neuronal precursors are not surrounded by glial tubes, although radial glial cells with a single cilium lined the telencephalic ventricular wall, much as in embryonic and neonatal mammals. To observe the migrating neuronal precursors in living brain tissue, we established a brain hemisphere culture using a zebrafish line carrying a GFP transgene driven by the neurogenin1 (ngn1) promoter. In these fish, GFP was observed in the neuronal precursors migrating in the RMS, some of which were aligned with blood vessels. Numerous ngn1:gfp-positive cells were observed migrating tangentially in the RMS-like route medial to the OB. Taken together, our results suggest that the RMS in the adult zebrafish telencephalon is a functional migratory pathway. This is the first evidence for the tangential migration of neuronal precursors in a nonmammalian adult telencephalon.

BackgroundIn addition to caudal subnucleus caudalis (Vc) of the spinal trigeminal complex, recent studies indicate that the subnuclei interpolaris/caudalis (Vi/Vc) transition zone plays a unique role in processing deep orofacial nociceptive input. Studies also suggest that glia and inflammatory cytokines contribute to the development of persistent pain. By systematically comparing the effects of microinjection of the antiinflammatory cytokine interleukin (IL)-10 and two glial inhibitors, fluorocitrate and minocycline, we tested the hypothesis that there was a differential involvement of Vi/Vc and caudal Vc structures in deep and cutaneous orofacial pain.ResultsDeep or cutaneous inflammatory hyperalgesia, assessed with von Frey filaments, was induced in rats by injecting complete Freund's adjuvant (CFA) into the masseter muscle or skin overlying the masseter, respectively. A unilateral injection of CFA into the masseter or skin induced ipsilateral hyperalgesia that started at 30 min, peaked at 1 d and lasted for 1-2 weeks. Secondary hyperalgesia on the contralateral site also developed in masseter-, but not skin-inflamed rats. Focal microinjection of IL-10 (0.006-1 ng), fluorocitrate (1 μg), and minocycline (0.1-1 μg) into the ventral Vi/Vc significantly attenuated masseter hyperalgesia bilaterally but without an effect on hyperalgesia after cutaneous inflammation. Injection of the same doses of these agents into the caudal Vc attenuated ipsilateral hyperalgesia after masseter and skin inflammation, but had no effect on contralateral hyperalgesia after masseter inflammation. Injection of CFA into the masseter produced significant increases in N-methyl-D-aspartate (NMDA) receptor NR1 serine 896 phosphorylation and glial fibrillary acidic protein (GFAP) levels, a marker of reactive astrocytes, in Vi/Vc and caudal Vc. In contrast, cutaneous inflammation only produced similar increases in the Vc.ConclusionThese results support the hypothesis that the Vi/Vc transition zone is involved in deep orofacial injury and suggest that glial inhibition and interruption of the cytokine cascade after inflammation may provide pain relief.

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BackgroundThe purpose of the present study is to evaluate the mechanisms underlying tongue-referred pain associated with tooth pulp inflammation.MethodUsing mechanical and temperature stimulation following dental surgery, we have demonstrated that dental inflammation and hyperalgesia correlates with increased immunohistochemical staining of neurons for TLR4 and HSP70.ResultsMechanical or heat hyperalgesia significantly enhanced in the ipsilateral tongue at 1 to 9 days after complete Freund’s adjuvant (CFA) application to the left lower molar tooth pulp compared with that of sham-treated or vehicle-applied rats. The number of fluorogold (FG)-labeled TLR4-immunoreactive (IR) cells was significantly larger in CFA-applied rats compared with sham-treated or vehicle-applied rats to the molar tooth. The number of heat shock protein (Hsp) 70-IR neurons in trigeminal ganglion (TG) was significantly increased on day 3 after CFA application compared with sham-treated or vehicle-applied rats to the molar tooth. About 9.2% of TG neurons were labeled with DiI applied to the molar tooth and FG injected into the tongue, and 15.4% of TG neurons were labeled with FG injected into the tongue and Alexa-labeled Hsp70-IR applied to the tooth. Three days after Hsp70 or lipopolysaccharide (LPS) application to the tooth in naive rats, mechanical or heat hyperalgesia was significantly enhanced compared with that of saline-applied rats. Following successive LPS-RS, an antagonist of TLR4, administration to the TG for 3 days, the enhanced mechanical or heat hyperalgesia was significantly reversed compared with that of saline-injected rats. Noxious mechanical responses of TG neurons innervating the tongue were significantly higher in CFA-applied rats compare with sham rats to the tooth. Hsp70 mRNA levels of the tooth pulp and TG were not different between CFA-applied rats and sham rats.ConclusionsThe present findings indicate that Hsp70 transported from the tooth pulp to TG neurons or expressed in TG neurons is released from TG neurons innervating inflamed tooth pulp, and is taken by TG neurons innervating the tongue, suggesting that the Hsp70-TLR4 signaling in TG plays a pivotal role in tongue-referred pain associated with tooth pulp inflammation.

In order to clarify the peripheral mechanisms of ectopic persistent pain in a tooth pulp following pulpal inflammation of an adjacent tooth, masseter muscle activity, phosphorylated extracellular signal-regulated protein kinase (pERK) and TRPV1 immunohistochemistries and satellite cell activation using glial fibrillary acidic protein (GFAP) immunohistochemistry in the trigeminal ganglion (TG) were studied in the rats with molar tooth-pulp inflammation. And, Fluorogold (FG) and DiI were also used in a neuronal tracing study to analyze if some TG neurons innervate more than one tooth pulp. Complete Freund’s adjuvant (CFA) or saline was applied into the upper first molar tooth pulp (M1) in pentobarbital-anesthetized rats, and capsaicin was applied into the upper second molar tooth pulp (M2) on day 3 after the CFA or saline application. Mean EMG activity elicited in the masseter muscle by capsaicin application to M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. The mean number of pERK-immunoreactive (IR) TG cells was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. Application of the satellite cell inhibitor fluorocitrate (FC) into TG caused a significant depression of capsaicin-induced masseter muscle activity and a significant reduction of satellite cell activation. The number of TRPV1-IR TG cells innervating M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats, and that was decreased following FC injection into TG. Furthermore, 6% of TG neurons innervating M1 and/or M2 innervated both M1 and M2. These findings suggest that satellite cell activation following tooth pulp inflammation and innervation of multiple tooth pulps by single TG neurons may be involved in the enhancement of the activity of TG neurons innervating adjacent non-inflamed teeth that also show enhancement of TRPV1 expression in TG neurons, resulting in the ectopic persistent tooth-pulp pain following pulpal inflammation of adjacent teeth.