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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Neuropeptides. 2013 Jan 3;47(2):109–115. doi: 10.1016/j.npep.2012.10.006

Spinal Astrocytes Produce and Secrete Dynorphin Neuropeptides

Andrew Wahlert 1, Lydiane Funkelstein 1, Bethany Fitzsimmons 2, Tony Yaksh 2, Vivian Hook 1,3,*
PMCID: PMC3606903  NIHMSID: NIHMS432917  PMID: 23290538

Abstract

Dynorphin peptide neurotransmitters (neuropeptides) have been implicated in spinal pain processing based on the observations that intrathecal delivery of dynorphin results in proalgesic effects and disruption of extracellular dynorphin activity (by antisera) prevents injury evoked hyperalgesia. However, the cellular source of secreted spinal dynorphin has been unknown. For this reason, this study investigated the expression and secretion of dynorphin-related neuropeptides from spinal astrocytes (rat) in primary culture. Dynorphin A(1-17), dynorphin B, and -neoendorphin were found to be present in the astrocytes, illustrated by immunofluorescence confocal microscopy, in a discrete punctate pattern of cellular localization. Measurement of astrocyte cellular levels of these dynorphins by radioimmunoassays confirmed the expression of these three dynorphin-related neuropeptides. Notably, BzATP (3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate) and KLA (di[3-deoxy-D-manno-octulosonyl]-lipid A) activation of purinergic and toll-like receptors, respectively, resulted in stimulated secretion of dynorphins A and B. However, -neoendorphin secretion was not affected by BzATP or KLA. These findings suggest that dynorphins A and B undergo regulated secretion from spinal astrocytes. These findings also suggest that spinal astrocytes may provide secreted dynorphins that participate in spinal pain processing.

Keywords: Astrocyte, Dynorphins, Spinal Cord, Secretion, Pain Processing

Introduction

Work on mechanisms for pain processing has focused on the spinal neural circuits involved in the transmission of information generated by peripheral stimuli to the brain. Several studies have demonstrated increased levels of spinal dynorphin, as well as the prodynorphin mRNA and the translated prodynorphin in pathological pain states (Iadarola et al., 1988; Kajander et al., 1990; Draisci et al., 1991; Dubner and Ruda, 1992; Malan et al., 2000; Abraham et al., 2001; Ji et al., 2002; Shimoyama et al., 2005). Interestingly, although spinal kappa opioid receptor activation with exogenous agonists have shown a reduction in nociceptive processing (Schmauss and Yaksh, 1984; Stevens and Yaksh, 1986; Nagasaka et al., 1996; Pelissie et al., 1990), intrathecal dynorphin peptides (endogenous ligands of kappa opioid receptors) have been shown to produce toxicity and a paradoxical enhancement of the pain state and antagonism of morphine analgesia (Schmauss and Yaksh, 1984; Gaumann et al., 1990; Tan-No et al., 2002; Komatsu et al., 2009; Headrick and Faden, 1995; Rochford et al., 1991). Consistent with these algogenic actions, intrathecal dynorphin will increase spinal release of glutamate and proalgesic lipid mediators such as prostaglandins E2 (Koetzner et al., 2004; Svensson et al., 2005). The role of endogenous dynorphin systems in regulating pain processing is supported by joint observations that intrathecal delivery of antibodies to dynorphin, binding up free extracellular pools of this neuropeptide and disruption of the dynorphin activity prevents injury evoked hyperalgesia (Malan et al., 2000; Wang et al., 2001; Nichols et al., 1997).

The evidence for dynorphins in regulating nociceptive processing raises the issue regarding the cellular origin of the endogenous spinal dynorphins. Therefore, the goal of this study is to evaluate the expression and release of dynorphin-related neuropeptides – composed of dynorphin A (1-17), dynorphin B and α-neoendorphin – from spinal astrocytes in primary cultures from rat. Here we show that the family of dynorphin neuropeptides is expressed in astrocytes and, of particular interest, we show that dynorphins can be released into the extracellular milieu by agents known to act though receptors present on astrocytes, notably the P2X and TLR 4 receptors (Allsopp et al., 2011; Sims et al., 2010). These findings implicate astrocytic dynorphins as factors involved in spinal pain processing.

Materials and Methods

Primary rat spinal cord astrocyte cell culture preparation

Rat pups (48-72 hour-old) were obtained from timed pregnant females (Holtzman Sprague Dawley). All aspects of these studies were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of California, San Diego.

Primary cultures of primary rat spinal cord astrocytes from 48-72 hour-old pups were prepared using a method described previously (Schwartz and Wilson, 1992) with some modifications. Briefly, the spinal cords were removed from the vertebral column by inserting an 18G needle into the spinal column and using steady force to push cold Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) through the spinal column. Once the spinal cords were hydroextruded, the cords were dispersed into pieces by mechanical trituration with a 10 ml pipette in DMEM. The dispersed cord suspension was then passed through a 100 μm cell strainer once, then a 70 μm cell strainer, and collected in a conical tube. After centrifugation at 1400 rpm for 5 min, the cells were resuspended in DMEM containing 10% fetal bovine serum (Gibco) and plated in a flask coated with poly-L-lysine (Sigma, St. Louis, MO). Coating of flasks was achieved by incubation with 3 ml of 20 mg/ml poly-L-lysine in PBS (phosphate buffered saline) at 37o C (in 5% CO2/95% incubator) for one hour, followed by washing three times with PBS (room temperature), and flasks were air dried under the hood. After plating of astrocytes into these flasks, cells were maintained in a humidified atmosphere of 95% air/5% CO2 at 37°C for 10 days, with the media being changed on days 4 and 7. On days 10 and 11, cells (microglia) growing on top of the confluent astrocyte layer were removed by shaking at 200 rpm for 2 h at 37° C and replacing the media; this step provides a pure preparation of astrocytes. On day 12 the cells were trypsinized and cells were replated equally into new flasks coated with poly-L-lysine. These pure astrocyte cultures were used for immunohistochemistry, measurement of intracellular dynorphin, and dynorphin secretion experiments described below.

Immunohistochemistry of dynorphin A (1-17), dynorphin B, and α-neoendorphin, and GFAP in rat spinal cord astrocytes

Rat spinal cord astrocytes in primary culture were plated in 8-chambered well slides at 8,000 cells per well. Astrocytes were fixed 72 hours later in 4% formaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with primary antibodies to dynorphin-related neuropeptides consisting of anti-dynorphin A (rabbit, 1:100, Phoenix Pharmaceuticals, Burlingame, CA), anti-dynorphin B (rabbit, 1:100, Bachem Americas, Torrance, CA), combined with anti-GFAP (glial fibrillary acidic protein) (mouse, 1:1000, Molecular Probes Eugene, OR) in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) for 2 hours at room temperature. After washing with PBS, cells were incubated with secondary goat anti-rabbit Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 (1:200, red and green fluorescent labels, respectively, Molecular Probes, Eugene, OR) in PBS containing 3% BSA for 45 minutes at room temperature. After washing with PBS, the chambers were removed and the glass slides covered with coverlids using ProLong Gold antifade mounting media containing the nuclear stain DAPI (Invitrogen-Life Technologies, Grand Island, NY). Controls with only the secondary fluorescent-labeled antibodies resulted in absence of immunofluorescence, indicating the specificity of immunofluorescent staining by the primary antisera to dynorphins and GFAP. Immunofluorescent images were examined with the Delta Vision Spectris Image Deconvolution System on an Olympus IX70 microscope using the software Softworx Explorer from Applied Precision, conducted as previously described (Funkelstein et al., 2008, 2012).

Treatment of astrocytes with BzATP and KLA and secretion of dynorphin A (1-17), dynorphin B, and α-neoendorphin measured by RIA (radioimmunoassay)

Rat spinal cord astrocytes in primary culture (preparation described above) were cultured on 10 cm2 petri dishes at 2 × 106 cells per dish. 72 hours later, the media was removed and media with treatment agents was added (n = 12). Astrocytes were incubated (37o C) with and without 250 μM BzATP (Sigma, St. Louis, MO) or 100 ng/ml KLA Lipid (Avanti, Alabaster, AL) for 30 minutes and 4 hours. The secretion media was collected after 30 minutes and 4 hours, and stored at −70°C. Secretion of dynorphin peptides into the media was measured by specific radioimmunoassays (RIA) to dynorphin A (1-17), dynorphin B (1-13), and -neoendorphin with RIA reagents (from Phoenix Pharmaceuticals, Burlingame, CA, and Peninsula Laboratories, San Carlos, CA), conducted as previously described (Minokadeh et al., 2010). The specificities of each of these RIAs was tested and each RIA shows lack of cross-reactivity with the other dynorphin-related peptides (Table 1). The specificities of these and our other RIAs have been previously validated and used used to measure dynorphins and other neuropeptides in extracted cellular and secretion samples (Funkelstein et al., 2008a,b, 2012; Beinfeld et al., 2009; Minokadeh al., 2010). Dynorphin neuropeptides in the secretion media are expressed as picograms (pg) per 50 microliter (μl). Differences in group means were tested for statistical significance (p<0.05) with the student’s t-test (n = 12).

Table 1.

Specificities of RIAs for Dynorphin A, Dynorphin B, and -Neoendorphin

Percent Crossreactivity with Indicated Neuropeptide
Radioimmunoassay Dyn. A Dyn. B -Neoend. (Leu)Enk Dyn-8
Dynorphin A 100% 0% 0% 0% 0%
Dynorphin B 0% 100% 0% 0% 0%
 -Neoendorphin 0% 0% 100% 0% 0%
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The percent crossreactivities of the radioimmunoassays (RIA) for dynorphin A (Dyn. A), dynorphin B (Dyn. B), and -neoendorphin ( -Neoend.) were measured for these dynorphin-related neuropeptides, as well as for (Leu)enkephalin ((Leu)Enk) and dynorphin(1-8) (Dyn-8). Results show lack of crossreactivity of each RIA for other related dynorphin-related neuropeptides. Therefore, these RIAs specifically detect and measure dynorphin A, dynorphin B, and -neoendorphin.

Results

Dynorphin-related neuropeptides in rat primary spinal cord astrocytes illustrated by immunofluorescence confocal microscopy

The dynorphin A (1-17), dynorphin B, and - neoendorphin neuropeptides, produced from the common prodynorphin proneuropeptide by proteolysis, are known as related peptide neurotransmitters that include the (Leu)enkephalin motif suggesting opioid activities (Civelli et al., 1985; Evans et al., 1985). To assess the presence of these dynorphin-related neuropeptides in spinal astrocytes, selective antisera to each of these neuropeptides was utilized to assess their cellular presence in astrocytes by immunofluorescence confocal microscopy.

The presence of these dynorphin neuropeptides in GFAP-positive (glial fibrillary acidic protein) spinal astrocytes was evaluated. Dynorphin A (1-17) (red immunofluorescence) is present in astrocytes in a punctate pattern of localization, and is present in GFAP-containing (green immunofluorescence) astrocytes (Figure 1). The majority of astrocytes were GFAP positive, a marker for astrocytes (Baba et al., 1997). However, a small population of astrocytes displayed dynorphin A (1-17) but were not GFAP positive; this observation may be explained by the fact that some astrocytes in culture are immature and do not stain with GFAP (Baba et al., 1997).

Figure 1. Dynorphin A in spinal astrocytes illustrated by immunofluorescence confocal microscopy.

Figure 1

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The presence of dynorphin A in spinal astrocytes in primary culture (from rat) was assessed by anti-dynorphin A in immunofluorescence histochemistry using confocal microscopy (panels a and b). Dynorphin A (red fluorescence) is present in astrocytes with GFAP (green fluorescence), a marker for astrocyte cells. Nuclei are stained with DAPI (blue). In panel b, it is noted that an astrocyte contains dynorphin A but not GFAP (Baba et al., 1997). GFAP labels mature astrocytes, and thus immature astrocytes may be present which are not labeled with GFAP (Baba et al., 1997).

Dynorphin B showed a punctate pattern of immunofluorescence staining in GFAP positive astrocytes (Figure 2). Like dynorphins A and B, -neoendorphin also showed a punctate pattern of localization in GFAP-positive astrocytes (Figure 3). These cellular data demonstrate that the three related dynorphin neuropeptides are produced in astrocytes.

Figure 2. Dynorphin B in astrocytes demonstrated by immunofluorescence confocal microscopy.

Figure 2

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Dynorphin B in spinal astrocytes was observed with anti-dynorphin B by immunofluorescence histochemistry (panels a and b). Dynorphin B (red fluorescence) is present in a discrete, punctate pattern of cellular localization in GFAP-positive (green fluorescence) astrocyte cells. Nuclei are stained with DAPI (blue).

Figure 3. Alpha-neoendorphin in astrocytes illustrated by immunofluorescence confocal microscopy.

Figure 3

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Alpha-neoendorphin was detected by anti- -neoendorphin immunofluorescence confocal microscopy. Alpha-neoendorphin (red fluorescence) is present in a discrete, punctate pattern of cellular localization in GFAP-positive (green fluorescence) astrocytes. Nuclei are stained with DAPI (blue).

Dynorphin A (1-17), dynorphin B, and α-neoendorphin content in primary rat spinal cord astrocytes measured by radioimmunoassay (RIA)

Rat spinal cord astrocytes in primary culture were analyzed for the contents of dynorphin-related neuropeptides by RIA, expressed as pg neuropeptide per one million cells, and as well as fmol neuropeptide per one million cells (Table 2). Similar molar amounts of dynorphins A and B are present in astrocytes. However, - neoendorphin is present at levels more than 3-fold greater than dynorphins A and B. Each of these dynorphin-related peptides are present as one copy within the prodynorphin precursor protein; the different molar ratios of processed dynorphin A/dynorphin B/ -neoendorphin of 1/1.5/4.2 illustrate regulated proteolytic processing of prodynorphin.

Table 2.

Dynorphin A, Dynorphin B, and -Neoendorphin Levels in Astrocytes

Cellular Content of Dynorphin-Related Peptides
Neuropeptide pg peptide/10 million cells pmol peptide/10 million cells (×10−4)
Dynorphin A 0.81 ± 0.03 3.75 ± 0.14
Dynorphin B 0.89 ± 0.13 5.64 ± 0.82
 -Neoendorphin 1.87 ± 0.39 15.2 ± 3.17
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The cellular content of dynorphin A, dynorphin B, and -neoendorphin neuropeptides in astroctyes were measured by RIA, as described in the methods. Cellular neuropeptide levels are expressed as pg peptide/10 million cells and as pmol peptide/10 million cells. Calculation of pmol amounts of dynorphins utilized the sequences and molecular weights (MW) of rat dynorphin A (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln, 2147.5 MW), rat dynorphin B (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr, 1570.8 MW), and rat -neoendorphin (Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys, 1228.4 MW) (Minokadeh et al., 2010). Values are expressed as the mean ± s.e.m. (n = 3).

Secretion of dynorphin A (1-17), dynorphin B, and α-neoendorphin from rat spinal cord astrocytes is stimulated by BzATP and KLA

Based on the presence of the dynorphin-related peptides in astrocytes, the ability of stimulatory agents to induce secretion of these dynorphins was evaluated. Astrocytes were incubated with BzATP (250 μM) to assess its effects for stimulating secretion of dynorphin neuropeptides. After four hours of BzATP treatment, the secretion of dynorphin A and dynorphin B were significantly elevated by approximately 4-fold and 35-fold above basal controls (no BzATP) (Figure 4). The secretion of -neoendorphin was not significantly affected by BzATP.

Figure 4. BzATP stimulates secretion of dynorphin A and dynorphin B from astrocytes.

Figure 4

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Astrocytes in primary culture were incubated with BzATP (250 μM) for 4 hours, and the secretion media was collected for measurement of dynorphin A, dynorphin B, and - neoendorphin by RIAs. BzATP stimulated the secretion of dynorphin A (panel a) and dynorphin B (panel b), but not -neoendorphin (panel c). Results are shown as the mean ± s.e.m. (n = 2), with significant BzATP stimulation of secretion indicated by *p < 0.05 (p = 0.0003 in panel a, p = 0.0001 in panel b).

KLA also significantly stimulated the secretion of dynorphins A and B (Figure 5). KLA (100 ng/ml KLA for 4 hours) stimulated the secretion of dynorphin A by 2.2-fold above basal unstimulated controls. KLA also stimulated the secretion of dynorphin B by 31-fold above basal controls. Secretion of -neoendorphin, however, was not affected by KLA.

Figure 5. KLA stimulates secretion of dynorphins A and B from astrocytes.

Figure 5

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Astrocytes in primary culture were incubated with KLA (100 ng/ml) for 4 hours, and the secretion media was collected for measurement of dynorphin A, dynorphin B, and - neoendorphin by RIAs. KLA stimulated the secretion of dynorphin A (panel a) and dynorphin B (panel b), but not -neoendorphin (panel c). Results are shown as the mean + s.e.m. (n = 2), with significant KLA stimulation of secretion indicated by *p < 0.05 (p = 0.0002 in panel a, and p = 0.0003 in panel b).

Discussion

Results of this study demonstrate the novel findings that spinal astrocytes (rat, in primary culture) express dynorphin A (1-17), dynorphin B (1-13), and -neoendorphin neuropeptides as demonstrated by immunofluorescence confocal microscopy and cellular measurements by radioimmunoassay. The three dynorphin peptides display a punctate pattern of cellular localization. Significantly, increased extracellular concentrations of dynorphins A and B were induced by BzATP and KLA, indicating an evoked transmembrane movement of these molecules. These results indicate that activation of purinergic and toll-like receptor 4 (by BzATP and KLA, respectively) of spinal astrocytes stimulates release of extracellular dynorphins that can potentially participate in the regulation of the excitability of dorsal horn circuits involved in spinal pain processing (Malan et al., 2000; Wang et al., 2001; Nichols et al., 1997). This study demonstrates that astrocyte type glial cells can participate in secreting extracellular dynorphins for chemical communication among glial and neuronal cells and is in accord with the limited data showing that peptide neurotransmitter secretion may occur from astrocytes and not only neurons (Krzan et al., 2003; Paco et al., 2009)

These dynorphin peptides are generated from the common prodynorphin precursor protein that contains one copy each of dynorphin A, dynorphin B, and -neoendorphin (Civelli et al., 1985; Evans et al., 1985). Proteolytic processing of prodynorphin generates the active dynorphin peptides in equimolar ratios. Interestingly, astrocytes display molar ratios of dynA/dynB/ - neoendorphin of approximately 4/6/15, which differs from the equimolar ratios present within the prodynorphin precursor. It is known, too, that turnover of peptides via degradative proteolytic mechanisms (Bateman and Hersh, 1987; Kovac et al., 2009) can result in different cellular levels of the three dynorphin peptides.

The evoked secretion of dynorphins into the extracellular space may contribute to the link between dorsal horn systems, astrocyte activation and dorsal horn excitability. BzATP, a P2X3 receptor agonist, and KLA, a TLR4 receptor agonist, results in prominent activation of astrocytes and serve to increase extracellular concentrations of dynorphins A and B. BzATP and KLA increased the secretion of dynorphin A by approximately 5- and 2-fold, respectively, compared to unstimulated control astrocytes. Secretion of dynorphin B was significantly increased by 10-fold by BzATP and KLA. However, the secretion of -neoendorphin was not affected by BzATP or KLA. These findings suggest that dynorphins A and B may be packaged in discrete subcellular secretory organelles without -neoendorphin, consistent with a co-release of dynorphins A and B without -neoendorphin. This explanation is consistent with the presence of chemical messengers in subpopulations of secretory organelles (Sobota et al., 2006). It will be of interest for future investigations to define the secretory organelles and their expressed enzymes that provide selective formation and secretion of dynorphins from astrocytes and other spinal glial cells, including microglia.

Demonstration of dynorphin secretion from astrocytes opens new mechanisms for modulation of synaptic neurotransmission by glial cells. The ability of astrocytes to release neuropeptides contributes to cell-cell signaling mediated by astrocyte secretion of glutamate and ATP neurotransmitters (Montana et al., 2006; Bergersen and Gundersen, 2009; Stevens, 2008). Little is currently known about the secretory mechanism of astrocyte release of neurotransmitters and its comparison to neuronal secretion. It is noted that spinal astrocytic dynorphin peptide immunofluorescence parallels that in mouse brain neurons (Minokadeh et al., 2010), expressing similar neuropeptide levels. It is also noted that release of glutamate and ATP from astrocytes occurs in a calcium-dependent manner, like neurons (Kreft et al., 2004). Further astrocytes possess many of the vesicle mobilizing proteins necessary for exocytosis (Verderio, et al., 1999; Verderio et al., 2012) It will be of interest in the future to define more precisely the mechanisms of continuous and evoked astrocyte peptide secretion to understand its regulation.

BzATP and KLA are agonists for the purinergic receptor types (P2X and P2Y types) and TLR4 receptors, respectively (Boyer and Harden, 1989; Evans et al., 1995; Conigrave et al., 1998; Carrasquer et al., 2009; Sims et al., 2010; Allsopp et al., 2011). Stimulation dynorphin secretion by these agonists indicate the presence of their specific receptors in astrocytes. Indeed, both receptors are strongly expressed on astrocytes (Bsibsi et al., 2002; Bowman et al., 2003; Jarvis, 2010) and their respective activation is known to result in increased calcium signaling in astrocytes and the secretion of a variety of cytokines (Miller et al., 2009; Saito et al., 2010; Suadicani et al., 2006). The present work suggests that, in addition to these many proalgesic agents, dynorphin must now be included in the nature of the effects produced secondary to the activation of these non-neuronal cells.

The results of this study are intriguing given the known prohyperalgesic effect associated with the intrathecal delivery of TLR4 or P2X7 receptor agonists (Wismer et al., 2003; Christianson et al., 2012), and the hypothesized role of spinal astrocytes in mediating a hyperpathic state after peripheral tissue and nerve injury (Watkins et al., 2003; Jack et al., 2005; De Leo et al., 2006; Gao and Ji, 2010; Xu and Yaksh, 2011). As noted, increased extracellular levels of dynorphin initiate an increased release of prostaglandins and glutamate (Koetzner et al., 2004; Svensson et al., 2005), activating cascades that are believed to initiate a facilitated state in spinal dorsal horn nociceptive processing.

In this regard, following peripheral injury and inflammation, astrocytes in the dorsal horn of the spinal cord have been shown to be activated (Gao and Ji, 2010; Svensson and Brodin, 2010; Gwak et al., 2012). This activation is believed to contribute to the hyperalgesic states that occur with these injury states. Astrocytes are thought to communicate with neurons using a variety receptors and neuropeptides. Release of glutamate, adenosine 5′-triphosphate (ATP) and interleukin-1-beta (IL-1β) by both astrocytes and neurons have been implicated among other substances. In particular, glutamate can stimulate the release ATP, and vice versa, leading to amplification of astrocyte activation in the dorsal horn of the spinal cord (Duan et al., 2003; Werry et al., 2006). ATP activation of astrocytes via the purinergic receptors, P2X and P2Y, increase expression of pro-inflammatory genes (c-fos, c-jun and Tis11), GFAP and calcium waves in astrocyte networks perpetuating signals of noxious stimuli (Neary et al., 1994; Abbracchio et al., 2009; Priller et al., 1998; Anderson et al., 2004; Inagaki et al., 1991; Stevens and Fields, 2000; Hald, 2009). The finding from this study that astrocytes secrete dynorphin neurotransmitters reveals a new role for neuropeptides in the role played by dorsal horn astrocytes in nociceptive processing.

Prior studies have also examined the presence of dynorphin and other neuropeptides in astrocytes of different brain regions (Vilijn et al., 1988; Shinoda et al., 1989; Mika et al., 2011, but not spinal cord astrocytes as examined here. One study analyzed prodynorphin mRNA expression in astrocytes from rat brain regions of striatum, hypothalamus, cortex, and hippocampus (Vilijn et al., 1988), demonstrating expression of prodynorphin in hypothalamic astrocytes. Another study (Shinoda et al., 1989) investigated the presence of neuropeptides (enkephalin and somatostatin) in cerebellar, cortical and striatal astrocytes. A recent review (Mika et al., 2011) summarizes findings in the field that dynorphin is present in spinal cord and is up-regulated in chronic pain. The findings from this study indicate spinal astrocytes to be an important source of extracellular dynorphins .

In summary, this study demonstrates that spinal astrocytes produce and when activated secrete dynorphin peptide neurotransmitters. Given the demonstrated proalgesic actions of actions of spinal dynorphin, this work indicates an important mechanism whereby astrocytes may serve to mediate spinal nociceptive processing.

Acknowledgements

This research was supported by NIH grants (R01 DA04271, R01 MH077305, and T32 DA07315) to VH, and by R01 NS16541 and R01 DA02110 grants to TY. The authors appreciate support of the Neurosciences Microscopy Imaging Core (P30 NS047101 to J. Gleeson, UC San Diego) for the immunofluorescence confocal microscopy experiments of this study.

Abbreviations

BzATP

3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate

GFAP

glial fibrillary acidic protein

KLA

di[3-deoxy-D-manno-octulosonyl]-lipid A and also known as Kdo2-Lipid A

RIA

radioimmunoassay

Footnotes

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