Ultrastructure of dorsal root ganglia.

Dorsal root ganglia (DRG) contains thousands of sensory neurons that transmit information about our external and internal environment to the central nervous system. This includes signals related to proprioception, temperature, and nociception. Our understanding of DRG has increased tremendously over the last 50 years and has established the DRG as an active participant in peripheral processes. This includes interactions between neurons and non-neuronal cells such as satellite glia cells and macrophages that contribute to an increasingly complex cellular environment that modulates neuronal function. Early ultrastructural investigations of the DRG have described subtypes of sensory neurons based on differences in the arrangement of organelles such as the Golgi apparatus and the endoplasmic reticulum. The neuron-satellite cell complex and the composition of the axon hillock in DRG have also been investigated, but, apart from basic descriptions of Schwann cells, ultrastructural investigations of other cell types in DRG are limited. Furthermore, detailed descriptions of key components of DRG, such as blood vessels and the capsule that sits at the intersection of the meninges and the connective tissue covering the peripheral nervous system, are lacking to date. With rising interest in DRG as potential therapeutic targets for aberrant signalling associated with chronic pain conditions, gaining further insights into DRG ultrastructure will be fundamental to understanding cell-cell interactions that modulate DRG function. In this review, we aim to provide a synopsis of the current state of knowledge on the ultrastructure of the DRG and its components, as well as to identify areas of interest for future studies.

Neuroanatomical relationship between Jingbi and the brachial plexus.

BACKGROUND: This study examined the stratified anatomy of the traditional acupuncture point Jingbi and the neuroanatomical relationship between Jingbi and the brachial plexus, and investigated neural pathways that could be affected by acupuncture stimulation at Jingbi. METHODS: Twelve dissected specimens were used to study the pathway of an acupuncture needle inserted at Jingbi. The stratified anatomy and the neuroanatomical relationship between Jingbi and the brachial plexus were studied. Our samples were grouped by gender and cause of death for comparative analysis. RESULTS: All needles (n = 24, on both sides of a total of 12 cadavers) punctured the anterior scalene muscle medial to the brachial plexus and external jugular vein, lateral to the phrenic nerve and internal jugular vein, and superior to the clavicle and subclavian artery/vein. The depth of needle insertion at Jingbi on the right side of male samples was 28.0 (interquartile range (IQR), 22.5-30.8) mm, which was approximately 8 mm deeper than for female subjects (p < 0.05). The needle was 3.0 (IQR, 2.0-5.0) mm and 7.0 (IQR, 5.5-8.0) mm medial to the brachial plexus on the left and right sides, respectively. CONCLUSION: Deep needle insertion at Jingbi can puncture the anterior scalene muscle. The mechanism of action of acupuncture stimulation at Jingbi might be related to its close relationship with the brachial plexus. Significant differences in needling depth were observed when our samples were grouped by gender. More studies are needed.

Immortalized Dorsal Root Ganglion Neuron Cell Lines.

Pain is one of the most significant causes of suffering and disability world-wide, and arguably the most burdensome global health challenge. The growing number of patients suffering from chronic pain conditions such as fibromyalgia, complex regional pain syndrome, migraine and irritable bowel syndrome, not only reflect the complexity and heterogeneity of pain types, but also our lack of understanding of the underlying mechanisms. Sensory neurons within the dorsal root ganglia (DRG) have emerged as viable targets for effective chronic pain therapy. However, DRG's contain different classes of primary sensory neurons including pain-associated nociceptive neurons, non-nociceptive temperature sensing, mechanosensory and chemoreceptive neurons, as well as multiple types of immune and endothelial cells. This cell-population heterogeneity makes investigations of individual subgroups of DRG neurons, such as nociceptors, difficult. In attempts to overcome some of these difficulties, a limited number of immortalized DRG-derived cell lines have been generated over the past few decades. In vitro experiments using DRG-derived cell lines have been useful in understanding sensory neuron function. In addition to retaining phenotypic similarities to primary cultured DRG neurons, these cells offer greater suitability for high throughput assays due to ease of culture, maintenance, growth efficiency and cost-effectiveness. For accurate interpretation and translation of results it is critical, however, that phenotypic similarities and differences of DRG-derived cells lines are methodically compared to native neurons. Published reports to date show notable variability in how these DRG-derived cells are maintained and differentiated. Understanding the cellular and molecular differences stemming from different culture methods, is essential to validate past and future experiments, and enable these cells to be used to their full potential. This review describes currently available DRG-derived cell lines, their known sensory and nociceptor specific molecular profiles, and summarize their morphological features related to differentiation and neurite outgrowth.

Emerging Evidence of Macrophage Contribution to Hyperinnervation and Nociceptor Sensitization in Vulvodynia.

Vulvodynia is an idiopathic chronic pain disorder and a leading cause of dyspareunia, or pain associated with sexual intercourse, for women. The key pathophysiological features of vulvodynia are vaginal hyperinnervation and nociceptor sensitization. These features have been described consistently by research groups over the past 30 years, but currently there is no first-line recommended treatment that targets this pathophysiology. Instead, psychological interventions, pelvic floor physiotherapy and surgery to remove painful tissue are recommended, as these are the few interventions that have shown some benefit in clinical trials. Recurrence of vulvodynia is frequent, even after vestibulectomy and questions regarding etiology remain. Vestibular biopsies from women with vulvodynia contain increased abundance of immune cells including macrophages as well as increased numbers of nerve fibers. Macrophages have multiple roles in the induction and resolution of inflammation and their function can be broadly described as pro-inflammatory or anti-inflammatory depending on their polarization state. This state is not fixed and can alter rapidly in response to the microenvironment. Essentially, M1, or classically activated macrophages, produce pro-inflammatory cytokines and promote nociceptor sensitization and mechanical allodynia, whereas M2, or alternatively activated macrophages produce anti-inflammatory cytokines and promote functions such as wound healing. Signaling between macrophages and neurons has been shown to promote axonal sprouting and nociceptor sensitization. This mini review considers emerging evidence that macrophages may play a role in nociceptor sensitization and hyperinnervation relevant to vulvodynia and considers the implications for development of new therapeutic strategies.

Human Dorsal Root Ganglia.

Sensory neurons with cell bodies situated in dorsal root ganglia convey information from external or internal sites of the body such as actual or potential harm, temperature or muscle length to the central nervous system. In recent years, large investigative efforts have worked toward an understanding of different types of DRG neurons at transcriptional, translational, and functional levels. These studies most commonly rely on data obtained from laboratory animals. Human DRG, however, have received far less investigative focus over the last 30 years. Nevertheless, knowledge about human sensory neurons is critical for a translational research approach and future therapeutic development. This review aims to summarize both historical and emerging information about the size and location of human DRG, and highlight advances in the understanding of the neurochemical characteristics of human DRG neurons, in particular nociceptive neurons.

A study to investigate needle insertion at Shenshu (BL23) to puncture psoas major muscle.

OBJECTIVE: It is unknown whether the psoas major muscle, thought to be a key muscle for treatment of lower back pain, can be punctured at Shenshu (BL23). METHODS: Twelve dissected specimens were used for studying the needling pathway of BL23 by perpendicularly inserting the depth-measuring blade of a vernier caliper at BL23. Dimensions of psoas muscle were measured. Correlation studies were conducted. In addition, our samples were grouped by gender and underlying medical conditions for analysis. RESULTS: Half (50%) of the needle insertions successfully punctured psoas muscle. The mean depth of needle insertion to puncture psoas muscle (Dmin) in the group with short-term underlying medical conditions was 38.0 mm (interquartile range 29.0-51.8 mm), approximately 6 mm deeper than 32.0 (29.3-42.5) mm in the group with long-term health problems (P = 0.041). The cross-sectional area (CSA) of psoas muscle in the former group was on average approximately 1.5 times that of the latter group (P = 0.04). When the data were analysed by gender, the thickness of psoas muscle in the male group was 19.0 (6.5-24.0) mm compared to 19.5 (5.8-34.8) mm in the female group (P = 0.02). The age in the female group (P = 0.04) and the body length of the total group (P =0.04) negatively correlated to Dmin. CONCLUSION: Needle insertion at BL23 might be able to puncture psoas muscle. Differences in the CSA of psoas muscle and Dmin were observed in groups with short-term and long-term underlying medical conditions.

Differential expression of microRNA-1 in dorsal root ganglion neurons.

Damage to sensory neurons induces neural repair, regrowth and hyperexcitability. The regulation of such responses to injury must be organized in some way by the neurons. Regulation can occur at the post-transcriptional level via microRNAs (miRNAs). miRNAs are small non-coding RNAs that influence the stability or translation of mRNAs and thereby regulate gene expression. Although nociceptive neurons show transcriptional and post-transcriptional regulatory mechanisms at many levels, miRNAs have not yet been systematically investigated in these neurons. Based on our preliminary array data we investigated the presence of miR-1 in dorsal root ganglion (DRG) neurons of mice and humans. We detected miR-1 in total RNA from human and mouse DRG and localised miR-1 in human and murine sensory neurons in situ. In Situ Hybridization detected miR-1 expression by nearly all DRG neurons. In vitro studies of enriched sensory neuron subpopulations from mouse DRG showed higher miR-1 expression levels in I-B4 negative neurons compared with I-B4 positive cells. Culturing of primary sensory neurons reduced the relative miR-1 expression levels independent of the presence or absence of laminin on the culture substrate. Transfection with a miR-1 mimic induced a massive increase in neuronal miR-1 associated with attenuated neurite outgrowth. This first description of miR-1 in sensory neurons including nociceptors suggests that miR-1 has a role in modulating neurite outgrowth.

TRPC channels and diacylglycerol dependent calcium signaling in rat sensory neurons.

Transient receptor potential (TRP) channels of the TRPV, TRPA, and TRPM subfamilies play important roles in somatosensation including nociception. While particularly the Thermo TRPs have been extensively investigated in sensory neurons, the relevance of the subclass of "canonical" TRPC channels in primary afferents is yet elusive. In the present study, we investigated the presence and contribution to Ca(2+) transients of TRPC channels in dorsal root ganglion neurons. We found that six of the seven known TRPC subtypes were expressed in lumbar DRG, with TRPC1, C3, and C6 being the most abundant. Microfluorimetric calcium measurements showed Ca(2+) influx induced by oleylacylglycerol (OAG), an activator of the TRPC3/C6/C7 subgroup. Furthermore, OAG induced rises in [Ca(2+)](i) were inhibited by SKF96365, an inhibitor of receptor and store operated calcium channel. OAG induced calcium transients were also inhibited by blockers of diacylglycerol (DAG) lipase, lipoxygenase or cyclooxygenase and, intriguingly, by inhibitors of the capsaicin receptor TRPV1. Notably, SKF96365 did not affect capsaicin-induced calcium transients. Taken together, our findings suggest that TRPC are functionally expressed in subpopulations of DRG neurons. These channels, along with TRPV1, contribute to calcium homeostasis in rat sensory neurons.

Intermedin: a skin peptide that is downregulated in atopic dermatitis.

Intermedin (IMD), also called adrenomedullin-2, is a peptide that belongs to the calcitonin/calcitonin gene-related peptide/amylin peptide family. IMD exerts many effects on the cardiovascular system, gastrointestinal tract, and central nervous system. Here, we analyzed the expression of the IMD peptide in human skin of healthy controls, in biopsies from lesional and non-lesional areas of atopic dermatitis (AD) skin, in cultured human keratinocytes, and in the HaCaT keratinocyte cell line at the transcriptional (quantitative reverse transcription-PCR) and translational (immunohistochemistry) level. IMD messenger RNA (mRNA) and protein could be detected in keratinocytes and human skin. Keratinocytes, nerve fibers, periglandular cells, arterial/arteriolar smooth muscle cells, and pericytes of dermal microvessels were intensely IMD-immunoreactive. The IMD mRNA was, compared to healthy skin, significantly reduced in lesional and non-lesional areas of AD skin. This was accompanied by a reduction of IMD immunoreactivity in pericytes of the upper dermis indicating that skin from AD patients is generally affected, and downregulation of IMD in AD skin is not a secondary phenomenon caused by acute inflammation but is a general characteristic of AD skin. These data further point to a role of IMD expressed by pericytes in conferring higher susceptibility of the skin of AD patients to inflammatory stimuli.

Coexpression and spatial association of nicotinic acetylcholine receptor subunits alpha7 and alpha10 in rat sympathetic neurons.

Fast excitatory synaptic transmission in sympathetic ganglia is mediated by nicotinic acetylcholine receptors (nAChRs). Although it is known that the nAChR alpha7-subunit occurs in sympathetic ganglia, the expression of the recently cloned subunit alpha10 (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002) has not been analyzed. Until now, functional receptors containing alpha10-subunits have been found only in combination with alpha9-subunits (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002). The alpha9-subunit exhibits a restricted expression pattern, whereas the alpha10-subunit is expressed more widely. This broad distribution resembles more closely that known for subunit alpha7 than for subunit alpha9. On this background, we investigated the distribution of nAChR subunits alpha7, alpha9, and alpha10 in rat sympathetic ganglia and studied a possible interaction between subunit alpha7 and potential partners by double-labeling immunofluorescence and fluorescence resonance energy transfer (FRET) (Kam et al., 1995; Jares-Erijman and Jovin, 2003).

Nicotinic acetylcholine receptors containing subunits alpha3 and alpha5 in rat nociceptive dorsal root ganglion neurons.

Nociceptive primary afferent neurons carry nicotinic acetylcholine receptors (nAChRs). Using RTPCR, mRNAs for all alpha-subunits have been identified in rat dorsal root ganglia (DRG) (Genzen et al., 2001; Lips et al., 2002), but the responses of nociceptive neurons to nicotine are not uniform and the cellular distribution of nAChRs within DRG, in general, and among functionally different subtypes of primary afferent neurons, in particular, are only partially resolved (Rau et al., 2005). These diverse actions might suggest the presence of various nAChR isoforms that are operative under different conditions. The present study was aimed to extend previous studies on nAChRs that contain subunits alpha4, alpha7, and alpha10 in providing data for alpha3- and alpha5-subunit-containing nAChRs (Haberberger et al., 2004; Papadopolou et al., 2004). To this end, calcium-imaging and double-labeling immunofluorescence with nAChR alpha-subunit-specific antibodies, in combination with markers for nociceptive neurons (TRPV1, I-B4), were applied.

Role of acetylcholine and muscarinic receptors in serotonin-induced bronchoconstriction in the mouse.

For the murine trachea, it has been reported that constriction evoked by serotonin (5-HT) is largely dependent on acetylcholine (ACh) released from the epithelium, owing to the sensitivity of the 5-HT response to epithelium removal, sensitivity to atropine, and insensitivity to tetrodotoxin (Moffatt et al., 2003). Consistent with this assumption, the respiratory epithelium contains ACh, its synthesizing enzyme, and the high-affinity choline transporter CHT1 (Reinheimer et al., 1996; Pfeil et al., 2003; Proskocil et al., 2004). Recently, we demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells (Lips et al., 2005). Hence, we proposed that 5-HT evokes release of ACh from epithelial cells via OCTs and that this epithelial-derived ACh induces bronchoconstriction. We tested this hypothesis in a well-established model of videomorphometric analysis of bronchial diameter in precision-cut murine lung slices utilizing epithelium removal to assess the role of the epithelium, OCT mouse knockout (KO) strains to assess the role of OCT isoforms, and muscarinic receptor M2/M3 double-KO mice to assess the cholinergic component of 5-HT induced bronchoconstriction, as bronchi of this strain are entirely unresponsive to cholinergic stimulation(Struckmann et al., 2003).

Activation of the SPHK/S1P signalling pathway is coupled to muscarinic receptor-dependent regulation of peripheral airways.

BACKGROUND: In peripheral airways, acetylcholine induces contraction via activation of muscarinic M2-and M3-receptor subtypes (M2R and M3R). Cholinergic hypersensitivity is associated with chronic obstructive pulmonary disease and asthma, and therefore the identification of muscarinic signaling pathways are of great therapeutic interest. A pathway that has been shown to be activated via MR and to increase [Ca2+]i includes the activation of sphingosine kinases (SPHK) and the generation of the bioactive sphingolipid sphingosine 1-phosphate (S1P). Whether the SPHK/S1P signaling pathway is integrated in the muscarinic control of peripheral airways is not known. METHODS: To address this issue, we studied precision cut lung slices derived from FVB and M2R-KO and M3R-KO mice. RESULTS: In peripheral airways of FVB, wild-type, and MR-deficient mice, SPHK1 was mainly localized to smooth muscle. Muscarine induced a constriction in all investigated mouse strains which was reduced by inhibition of SPHK using D, L-threo-dihydrosphingosine (DHS) and N, N-dimethyl-sphingosine (DMS) but not by N-acetylsphingosine (N-AcS), a structurally related agent that does not affect SPHK function. The initial phase of constriction was nearly absent in peripheral airways of M3R-KO mice when SPHK was inhibited by DHS and DMS but was unaffected in M2R-KO mice. Quantitative RT-PCR revealed that the disruption of the M2R and M3R genes had no significant effect on the expression levels of the SPHK1-isoform in peripheral airways. CONCLUSION: These results demonstrate that the SPHK/S1P signaling pathway contributes to cholinergic constriction of murine peripheral airways. In addition, our data strongly suggest that SPHK is activated via the M2R. Given the important role of muscarinic mechanisms in pulmonary disease, these findings should be of considerable therapeutic relevance.

Nicotinic acetylcholine receptor subtypes in nociceptive dorsal root ganglion neurons of the adult rat.

Stimulation of nicotinic acetylcholine receptors (nAChR) excites peripheral sensory nerve fibres, but also exert antinociceptive effects. The differences in these nAChR-mediated effects could be related to the expression of different nAChR subtypes located on nociceptive neurons. In the present study, we focused on the recently described alpha 10-nAChR subunit, and on alpha 4 and alpha 7 subunits, which are the most abundant subunits in the central nervous system. In nociceptive neurons from thoracic and lumbar dorsal root ganglia (DRG), nAChR subunits were found at transcriptional (RT-PCR), translational (immunohistochemistry) and functional levels. Cultured DRG neurons express mRNA for the subunits alpha 2-7 and alpha 10. The alpha-subunit proteins 4, 7 and 10 were colocalised in virtually all nociceptive neurons that were identified by immunoreactivity for the vanilloid receptor TRPV-1. These findings were corroborated by current recordings and calcium measurements, which revealed excitatory inward currents and calcium responses in capsaicin sensitive neurons.

Nicotinic receptor alpha 7-subunits are coupled to the stimulation of nitric oxide synthase in rat dorsal root ganglion neurons.

In dorsal root ganglia (DRG) intraganglionic communication takes place both among neurons and between neurons and satellite cells. One diffusible substance involved in this signalling is nitric oxide (NO), and acetylcholine (ACh) is a candidate for the stimulation of intraganglionic NO synthesis. DRG neurons react to ACh-receptor stimulation with NO-dependent cGMP production. Here, we investigated the role of the alpha 7-subunit containing Ca(2+)-permeable nicotinic ACh receptors (nAChR) in this process. The alpha 7-nAChR mRNA and the protein were expressed in virtually all lumbar DRG neurons as evidenced by laser-assisted cell picking and oligo cell RT-PCR, in situ hybridisation and immunohistochemistry. Strong alpha 7-nAChR immunoreactivity was present in vanilloid receptor 1-immunoreactive, i.e. nociceptive, neurons. A neuronal production of NO in response to nicotine could be demonstrated in DRG slice preparations utilising the NO-sensitive fluorescent indicator diaminofluorescein diacetate (DAF-2DA). This stimulation of NO production was sensitive to inhibition of alpha 7-nAChR by mecamylamine and alpha-bungarotoxin, to inhibition of nitric oxide synthase (NOS) with L-NAME and L-NMMA, and to the blockade of voltage-operated Ca(2+) channels by verapamil. The results show the presence of the alpha 7-nAChR subunit in nociceptive rat DRG neurons and provide evidence for its coupling to NOS activation, indicating a role of this pathway in the intraganglionic communication in sensory ganglia.

Expression of the high-affinity choline transporter, CHT1, in the rat trachea.

The rate limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline by the high-affinity choline transporter (CHT1). Here, we investigated the distribution of CHT1 in the rat trachea. CHT1-mRNA was detected by reverse transcriptase-polymerase chain reaction in trachea without epithelium, abraded tracheal mucosa, and in epithelial cells obtained by laser-assisted cell-picking. Accordingly, CHT1-mRNA could also be detected in tracheal epithelial cells by in situ hybridization. Recently obtained polyclonal rabbit and guinea-pig antisera against a synthetic peptide corresponding to amino acid residues 29-40 of the rat CHT1 sequence localized CHT1 protein in combination with antisera against the vesicular acetylcholine transporter in cholinergic fibers innervating tracheal glands and the tracheal muscle. In case of the tracheal epithelium, CHT1 was restricted to the apical membrane of the ciliated cells, as demonstrated by confocal laser scanning and electron microscopy using an affinity-purified CHT1 antiserum. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favor of ACh synthesis being fueled by choline uptake via CHT1 after release and breakdown of ACh at the luminal surface. Accordingly, cholinergic regulation of tracheal epithelial function is governed by local release and recycling of ACh by ciliated cells.

Expression of the high-affinity choline transporter, CHT1, in the neuronal and non-neuronal cholinergic system of human and rat skin.

Choline is an essential component in acetylcholine biosynthesis, and is involved in cell signaling. It is unable to permeate the cell membrane and requires a transporter to enter the cell. Neurons that synthesize acetylcholine take up choline by a recently cloned high-affinity choline transporter (choline transporter 1) that is Na+-dependent and can be blocked by hemicholinium-3. The aim of this study was to determine the expression and to analyze the distribution of choline transporter 1 in human and rat skin. The mRNA for choline transporter 1 was detected in rat and human skin and in the human keratinocyte cell line HaCaT. A polyclonal anti-serum was developed against the N-terminal region of the human and rat protein. In rat and human skin, choline transporter 1 immunoreactivity was present in nerve fibers. In addition, keratinocytes, HaCaT cells and cells of the internal root sheath of the hair follicle contained choline transporter 1 immunoreactivity. The labeling patterns of nonconfluent vs confluent cultured cells and the distribution of choline transporter 1 along the epidermal layer suggest an association of choline transporter 1 with keratinocyte differentiation. In conclusion, this study shows the presence of the high-affinity choline transporter choline transporter 1 in nerve fibers and epithelial cells in the human and rat skin supporting the pivotal role of this transporter in both the neuronal and non-neuronal cholinergic system of the skin.