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IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1β and increased transforming growth factor-β1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-β1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1β transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme broadly expressed in noradrenergic and dopaminergic neurons in the central nervous system.57,70 Tyrosine hydroxylase is also expressed by peripheral sympathetic neurons98 and by enteric neurons within the gut.81,84 More than 30 years ago, TH was unexpectedly discovered in developing adult rodent cranial and dorsal root ganglion (DRG) neurons. Today, TH-expressing DRG neurons are being rediscovered as a relevant subpopulation. This review addresses the emerging importance of TH-expressing DRG neurons in sensation and pain mechanisms, focusing specifically on (1) their nature as C-low threshold mechanoreceptors (C-LTMRs); (2) their involvement in nociception/pain; and (3) their catecholaminergic phenotype. Fil: Brumovsky, Pablo Rodolfo. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Investigaciones Biomédicas; Argentina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentina

Afferent and efferent activities in the colorectum are governed by both intrinsic and extrinsic neuronal systems [1–5]. The extrinsic innervation of the colorectum is provided by the pelvic (PN) and lumbar splanchnic nerves [4, 5], containing both sensory (primary afferents) and autonomic (sympathetic and parasympathetic) components. Specifically for the PN, lumbosacral (6th lumbar to the 2nd sacral) dorsal root ganglia (DRG) neurons provide the afferent component [4]. Afferent fibres in the PN are further subdivided into four functional classes, based on responses to mechanical stimulation of their receptive fields [6], including: 1) mucosal (responsive to light mucosal stroking and probing), muscular (responsive to circumferential stretch and probing), muscular-mucosal (responsive to stroking, stretch and probing) or serosal/vascular afferents (only responsive to probing), as described in rat [7], guinea pig [8–14] and mouse [15] colorectum. Responses to application of different chemical compounds further subdivide mechanically sensitive afferents [16–18]. The autonomic component in the PN is supplied by the postganglionic projections of sympathetic neurons in the lumbar sympathetic chain, or sympathetic and parasympathetic neurons present in the ‘mixed’ major pelvic ganglion (MPG) [19, 20]. Most information concerning the origin of the extrinsic innervation of the colorectum has been obtained through the use of retrograde tracers and careful analysis of their accumulation in DRG [21–24] and autonomic [25–27] neurons, several days after injection in the subserosal space. The termination patterns of extrinsic nerves in the colorectum, on the other hand, have been more difficult to elucidate, partly because of the parallel contribution of a rich and complex neuropil produced by local intrinsic neurons [1, 19, 28, 29]. With some limitations, however, various immunohistochemical markers such as the calcitonin gene-related peptide (CGRP), tyrosine hydroxylase and the vesicular acetylcholine transporter (VAChT) or choline acetyl transferase (ChAT) have been used, respectively, for the identification of afferent, sympathetic or parasympathetic nerves innervating the colorectal wall in rodents [3, 4, 30–34]. More recently, the introduction of in vitro anterograde tracing of visceral nerves using biotinamide (BTA)[5, 13, 14, 25, 34–41] and its use in combination with electrophysiology has been fundamental for the detailed analysis of correlation between functional and anatomical characteristics of afferent nerves innervating visceral organs, including the colorectum (see [5]). However, while extremely useful, such characterization has been done mostly by visualization in whole mount preparations after removal of the mucosa and the circular muscle, limiting assessment across all layers of the colorectum. In fact, a detailed description of the anatomy of anterogradely traced extrinsic afferent endings in the different layers of the colorectum has yet to be reported [5]. In this study, we present results in mouse colorectum, analyzing its extrinsic innervation from a different perspective. “Bulk” tracing, followed by transverse or longitudinal tissue sectioning, was used to assess the general distribution of PN axons innervating the organ across layers. In addition, immunohistochemical analysis using CGRP antiserum was employed to attempt identification of sensory extrinsic nerves. Finally, a few observations made on traced MPG are also provided.

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1–VGLUT3 transcripts in lumbar 4–5 (L4–5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4–5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III–IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III–IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury. Fil: Malet, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vieytes, C. A.. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina Fil: Lundgren, K. H.. University of Cincinnati; Estados Unidos Fil: Seal, R. P.. University of Pittsburgh; Estados Unidos Fil: Tomasella, María Eugenia. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Seroogy, K. B.. University of Cincinnati; Estados Unidos Fil: Hökfelt, T.. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia Fil: Gebhart, G. F.. University of Pittsburgh; Estados Unidos Fil: Brumovsky, Pablo Rodolfo. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados Unidos

BACKGROUND: Reinnervation timing after nerve injury is critical for favorable axonal regeneration, remyelination and clinical improvement. Considering bone marrow mononuclear cells (BMMC) are easily obtained and readily available for transplant, this work analyzed the effect of BMMC systemic administration on nerve repair and pain behavior.METHODS: Adult rats with sciatic nerve crush were immediately and systemically injected BMMC through the caudal artery. Nontreated, sham and naïve rats were also included. Histological, immunohistochemical, biochemical, functional and behavioral analyses were performed in nerves harvested from each group at different survival times.RESULTS: Axons in BMMC-treated rats exhibited a more conserved morphological appearance than those in nontreated rats, as observed at different survival times both in semi-thin sections and ultrastructural analysis. BMMC-treated rats also showed a reduction in major myelin protein immunoreactive clusters 7 and 14 days post injury (DPI), as compared to nontreated rats. Electrophysiological analysis showed BMMC treatment to slightly improve the amplitude of compound muscle action potential (CMAP) starting at 14 DPI. Finally, mechanical withdrawal threshold revealed a full preventive action against transient mechanical hypersensitivity in BMMC-treated rats.CONCLUSIONS: These data demonstrate the efficiency of BMMC, systemically and noninvasively transplanted, in correcting morphological, functional and behavioral alterations resulting from peripheral nerve injury. Fil: Usach, Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Malet, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentina Fil: Lopez, Lidia Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Lavalle, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Piñero, Gonzalo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina Fil: Saccoliti, María. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos ; Argentina Fil: Cueto, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital Español. Servicio de Neurología; Argentina Fil: Brumovsky, Pablo Rodolfo. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentina Fil: Brusco, Herminia Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; Argentina Fil: Setton, Clara Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentina

Carbohydrate malabsorption such as in lactose intolerance or enteric infection causes symptoms that include abdominal pain. Because this digestive disorder increases intracolonic osmolarity and acidity by accumulation of undigested carbohydrates and fermented products, we tested whether these two factors (hypertonicity and acidity) would modulate colorectal afferents in association with colorectal nociception and hypersensitivity. In mouse colorectum-pelvic nerve preparations in vitro, afferent activities were monitored after application of acidic hypertonic saline (AHS; pH 6.0, 800 mosM). In other experiments, AHS was instilled intracolonically to mice and behavioral responses to colorectal distension (CRD) measured. Application of AHS in vitro excited 80% of serosal and 42% of mechanically-insensitive colorectal afferents (MIAs), sensitizing a proportion of MIAs to become mechanically sensitive and reversibly inhibiting stretch-sensitive afferents. Acute intracolonic AHS significantly increased expression of the neuronal activation marker pERK in colon sensory neurons and augmented noxious CRD-induced behavioral responses. After three consecutive daily intracolonic AHS treatments, mice were hypersensitive to CRD 4–15 days after the first treatment. In complementary single fiber recordings in vitro, the proportion of serosal class afferents increased at day 4; the proportion of MIAs decreased, and muscular class stretch-sensitive afferents were sensitized at days 11–15 in mice receiving AHS. These results indicate that luminal hypertonicity and acidity, two outcomes of carbohydrate malabsorption, can induce colorectal hypersensitivity to distension by altering the excitability and relative proportions of colorectal afferents, suggesting the potential involvement of these factors in the development of abdominal pain.

Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, ∼14% of colorectal and ∼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons.

Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT(1) and VGLUT(2) , respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naïve mice was included. Previously characterized antibodies against VGLUT(1) , VGLUT(2) , and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT(1) and VGLUT(2) , was also performed. Most colorectal DRG neurons expressed VGLUT(2) and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT(1) . VGLUT(2) -immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT(2) -IR nerves were detected in all layers of the colorectum; VGLUT(1) -IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT(2) , and to a lesser extent, VGLUT(1) ; 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT(2). Altogether, our data suggests a role for VGLUT(2) in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility.

Glutamate is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons. However, to date a glutamatergic phenotype of autonomic neurons has not been described. Therefore, we explored the expression of vesicular glutamate transporter (VGLUT) types 1, 2 and 3 in lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) of naive BALB/C mice, as well as after pelvic nerve axotomy (PNA), using immunohistochemistry and in situ hybridization. Colocalization with activating transcription factor-3 (ATF-3), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT) and calcitonin gene-related peptide was also examined. Sham-PNA, sciatic nerve axotomy (SNA) or naive mice were included. In naive mice, VGLUT(2)-like immunoreactivity (LI) was only detected in fibers and varicosities in LSC and MPG; no ATF-3-immunoreactive (IR) neurons were visible. In contrast, PNA induced upregulation of VGLUT(2) protein and transcript, as well as of ATF-3-LI in subpopulations of LSC neurons. Interestingly, VGLUT(2)-IR LSC neurons coexpressed ATF-3, and often lacked the noradrenergic marker TH. SNA only increased VGLUT(2) protein and transcript in scattered LSC neurons. Neither PNA nor SNA upregulated VGLUT(2) in MPG neurons. We also found perineuronal baskets immunoreactive either for VGLUT(2) or the acetylcholinergic marker VAChT in non-PNA MPGs, usually around TH-IR neurons. VGLUT(1)-LI was restricted to some varicosities in MPGs, was absent in LSCs, and remained largely unaffected by PNA or SNA. This was confirmed by the lack of expression of VGLUT(1) or VGLUT(3) mRNAs in LSCs, even after PNA or SNA. Taken together, axotomy of visceral and non-visceral nerves results in a glutamatergic phenotype of some LSC neurons. In addition, we show previously non-described MPG perineuronal glutamatergic baskets.

Information about colorectal distension (i.e., colorectal dilation by increased intraluminal pressure) is primarily encoded by stretch-sensitive colorectal afferents in the pelvic nerve (PN). Despite anatomic differences between rectum and distal colon, little is known about the functional roles of colonic vs. rectal afferents in the PN pathway or the quantitative nature of mechanosensory encoding. We utilized an in vitro mouse colorectum-PN preparation to investigate pressure-encoding characteristics of colorectal afferents. The colorectum with PN attached was dissected, opened longitudinally, and pinned flat in a Sylgard-lined chamber. Action potentials of afferent fibers evoked by circumferential stretch (servo-controlled force actuator) were recorded from the PN. Stretch-sensitive fibers were categorized into the following four groups: colonic muscular, colonic muscular/mucosal, rectal muscular, and rectal muscular/mucosal. Seventy-nine stretch-sensitive PN afferents evenly distributed into the above four groups were studied. Rectal muscular afferents had significantly greater stretch-responses than the other three groups. Virtually all rectal afferents (98%) had low thresholds for response and encoded stimulus intensity into the noxious range without obvious saturation. Most colonic afferents (72%) also had low thresholds (18 mmHg) for response. These high-threshold colonic afferents were sensitized to stretch by inflammatory soup; response threshold was significantly reduced (from 23 to 12 mmHg), and response magnitude significantly increased. These results suggest that the encoding of mechanosensory information differs between colonic and rectal stretch-sensitive PN afferents. Rectal afferents have a wide response range to stretch, whereas high-threshold colonic afferents likely contribute to visceral nociception.

Viscero-somatic referral and sensitization has been well documented clinically and widely investigated, whereas viscero-visceral referral and sensitization (termed cross-organ sensitization) has only recently received attention as important to visceral disease states. Because second order neurons in the CNS have been extensively shown to receive convergent input from different visceral organs, it has been assumed that cross-organ sensitization arises by the same convergence-projection mechanism as advanced for viscero-somatic referral and sensitization. However, increasing evidence also suggests participation of peripheral mechanisms to explain referral and sensitization. We briefly summarize behavioral, morphological and physiological support of and focus on potential mechanisms underlying cross-organ sensitization.

Studies in humans and rodents suggest that colon inflammation promotes urinary bladder hypersensitivity and, conversely, that cystitis contributes to colon hypersensitivity, events referred to as cross-organ sensitization. To investigate a potential peripheral mechanism, we examined whether cystitis alters the sensitivity of pelvic nerve colorectal afferents. Male C57BL/6 mice were treated with cyclophosphamide (CYP) or saline, and the mechanosensitive properties of single afferent fibers innervating the colorectum were studied with an in vitro preparation. In addition, mechanosensitive receptive endings were exposed to an inflammatory soup (IS) to study sensitization. Urinary bladder mechanosensitive afferents were also tested. We found that baseline responses of stretch-sensitive colorectal afferents did not differ between treatment groups. Whereas IS excited a proportion of colorectal afferents CYP treatment did not alter the magnitude of this response. However, the number of stretch-sensitive fibers excited by IS was increased relative to saline-treated mice. Responses to IS were not altered by CYP treatment, but the proportion of IS-responsive fibers was increased relative to saline-treated mice. In bladder, IS application increased responses of muscular afferents to stretch, although no differences were detected between saline- and CYP-treated mice. In contrast, their chemosensitivity to IS was decreased in the CYP-treated group. Histological examination revealed no changes in colorectum and modest edema and infiltration in the urinary bladder of CYP-treated mice. In conclusion, CYP treatment increased mechanical sensitivity of colorectal muscular afferents and increased the proportion of chemosensitive colorectal afferents. These data support a peripheral contribution to cross-organ sensitization of pelvic organs.

The expression of two vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, was studied with immunohistochemistry in lumbar dorsal root ganglia (DRGs), the lumbar spinal cord and the skin of the adult mouse. About 12% and 65% of the total number of DRG neuron profiles (NPs) expressed VGLUT1 and VGLUT2, respectively. VGLUT1-immunoreactive (IR) NPs were usually medium- to large-sized, in contrast to a majority of small- or medium-sized VGLUT2-IR NPs. Most VGLUT1-IR NPs did not coexpress calcitonin gene-related peptide (CGRP) or bound isolectin B4 (IB4). In contrast, approximately 31% and approximately 42% of the VGLUT2-IR DRG NPs were also CGRP-IR or bound IB4, respectively. Conversely, virtually all CGRP-IR and IB4-binding NPs coexpressed VGLUT2. Moderate colocalization between VGLUT1 and VGLUT2 was also observed. Sciatic nerve transection induced a decrease in the overall number of VGLUT1- and VGLUT2-IR NPs (both ipsi- and contralaterally) and, in addition, a parallel, unilateral increase of VGLUT2-like immunoreactivity (LI) in a subpopulation of mostly small NPs. In the dorsal horn of the spinal cord, strong VGLUT1-LI was detected, particularly in deep dorsal horn layers and in the ventral horns. VGLUT2-LI was abundant throughout the gray spinal matter, 'radiating' into/from the white matter. A unilateral dorsal rhizotomy reduced VGLUT1-LI, while apparently leaving unaffected the VGLUT2-LI. Transport through axons for both VGLUTs was confirmed by their accumulation after compression of the sciatic nerve or dorsal roots. In the hind paw skin, abundant VGLUT2-IR nerve fibers were observed, sometimes associated with Merkel cells. Lower numbers of VGLUT1-IR fibers were also detected in the skin. Some VGLUT1-IR and VGLUT2-IR fibers were associated with hair follicles. Based on these data and those by Morris et al. [Morris JL, Konig P, Shimizu T, Jobling P, Gibbins IL (2005) Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate. J Comp Neurol 483:1-16], we speculate that virtually all DRG neurons in adult mouse express VGLUTs and use glutamate as transmitter.

Research during the past two decades supports a complex role for neuropeptide tyrosine (NPY) and two of its associated receptors, the Y1 receptor and the Y2 receptor, in the modulation of pain, in addition to regeneration and survival mechanisms at the spinal level. Thus, NPY has been shown to both cause and reduce pain, in addition to having biphasic effects. Recent research has focused on the distribution of the spinal NPY-mediated system. Here, we propose various possible scenarios for the role of NPY in pain processing, based on its actions at different sites (axon versus cell body), through different receptors (Y1 receptor versus Y2 receptor) and/or types of neuron (ganglion neurons and intraganglionic cross-excitation versus interneurons versus projection neurons).

The expression of tyrosine hydroxylase (TH) was studied in adult mouse dorsal root ganglia (DRGs) and spinal cord by means of immunohistochemistry and in situ hybridization. TH immunoreactivity and TH mRNA were present in 10-15% of lumbar DRG neurons, in most cases being small/medium-sized. Only very few of these neurons coexpressed calcitonin gene-related peptide (CGRP), and only around 6% bound isolectin B4 (IB4). Dopamine beta-hydroxylase-positive(+) or aromatic amino acid decarboxylase (AADC)+ DRG neurons were rare and did not colocalize TH. No evidence for dopamine transporter expression was obtained. Axotomy of the sciatic nerve only showed a tendency towards reduction in the number of TH+ neurons. In the dorsal horn of the spinal cord, moderately dense and widespread TH+ nerve terminals were observed, mainly in the gray matter and they did not show a typical primary afferent pattern. Also, dorsal rhizotomy or peripheral axotomy had no apparent effect on TH-LI in the dorsal horn. In the skin, along with an abundant TH+ innervation of blood vessels and sweat gland acini, a number of fibers was observed in close relation to the skin surface, some even penetrating into the epithelium. These results demonstrate presence, in normal adult mouse DRGs, of a subpopulation of TH+, essentially CGRP- and IB4-negative small/medium-sized neurons. No evidence for transport of TH into central afferents was obtained, but the enzyme may be present in some sensory fibers in the skin. The fact that neither AADC nor the dopamine transporter could be visualized suggests of non-dopaminergic transmitter phenotype, but the levels of these two dopaminergic markers may be too low to be detected with the present methodology. A further alternative is that L-DOPA after release is extracellularly converted to dopamine.

The expression of the galanin receptor-1 and -2 (Gal(1) and Gal(2)) messenger ribonucleic acids (mRNAs) was studied in the lower spinal cord of rat by means of in situ hybridization, using ribonucleic acid probes (riboprobes). Naïve rats as well as rats with unilateral axotomy of the sciatic nerve or unilateral inflammation of the hindpaw were analyzed. In naïve rats, numerous Gal(1) mRNA-positive (+) neurons were detected in lamina (L) I-III. In addition, several Gal(1) mRNA(+) neurons were seen in deeper layers, including the ventral horns, area X, and the lateral spinal nucleus. In contrast, few and comparatively weakly labeled Gal(2) mRNA(+) neurons were observed, mostly in the ventral horns and in area X, with fewer in the dorsal horn and in the sympathetic and parasympathetic intermediate lateral cell columns. Axotomy induced a strong increase in intensity and number of Gal(2) mRNA(+) motoneurons ipsilateral to the lesion. In contrast, nerve cut or hindpaw inflammation did not alter the expression of Gal(1) or Gal(2) in the dorsal horn. The present (and previous) results suggest that galanin, acting through Gal(1) and Gal(2) receptors, has a modulatory role on spinal excitability, not only via interneurons in superficial dorsal horn, but also on neurons in deep layers and area X, as well as on the sympathetic and parasympathetic outflow. Furthermore, the nerve injury-induced Gal(2) upregulation in motor neurons suggests a role for galanin in survival/regeneration mechanisms.

Neuropeptide Y (NPY), a 36-amino-acid peptide, mediates biological effects by activating Y1, Y2, Y5, and y6 receptors. NPY neurons innervate many brain regions, including the hypothalamus, where NPY is involved in regulation of a broad range of homeostatic functions. We examined, by immunohistochemistry with tyramide signal amplification, the expression of the NPY Y2 receptor (Y2R) in the mouse brain with a newly developed rabbit polyclonal antibody. Y2R immunoreactivity was specific with its absence in Y2R knockout (KO) mice and in adjacent sections following preadsorption with the immunogenic peptide (10−5 M). Y2R-positive processes were located in many brain regions, including the olfactory bulb, some cortical areas, septum, basal forebrain, nucleus accumbens, amygdala, hippocampus, hypothalamus, substantia nigra compacta, locus coeruleus, and solitary tract nucleus. However, colchicine treatment was needed to detect Y2R-like immunoreactivity in cell bodies in many, but not all, areas. The densest distributions of cell bodies were located in the septum basal forebrain, including the bed nucleus, and amygdala, with lower density in the anterior olfactory nucleus, nucleus accumbens, caudal striatum, CA1, CA2, and CA3 hippocampal fields, preoptic nuclei lateral hypothalamus, and A13 DA cells. The widespread distribution of Y2R-positive cell bodies and fibers suggests that NPY signaling through the Y2R is common in the mouse brain. Localization of the Y2R suggests that it is mostly presynaptic, a view supported by its frequent absence in cell bodies in the normal mouse and its dramatic increase in cell bodies of colchicine-treated mice. J. Comp. Neurol. 499:357–390, 2006. © 2006 Wiley-Liss, Inc.

The 29/30 amino acid neuropeptide galanin is present in a small population of DRG neurons under normal condition but is strongly upregulated after nerve injury. There is evidence that this upregulated galanin has trophic actions, for example promoting neurite outgrowth as well as influencing pain processing. In fact, both pro- and antinociceptive effects have been reported, probably relating to activation of different receptors. It has been proposed that presynaptic GalR2 receptors are pro-nociceptive by enhancing release of excitatory transmitters in the dorsal horn, and anti-nociceptive via an action on GalR1-positive interneurons. These neurons have recently been shown to be glutamatergic. Several other peptides and molecules are also regulated by nerve injury. Here we focus on neuropeptide tyrosine (NPY), which is upregulated in parallel with galanin. We review data reporting on coexistence between galanin and NPY and between these two peptides and the two NPY receptors Y1 and Y2. The data show considerable overlap, and it will be an important task to analyse how cross-talk between these neuropeptides can influence pain processing. It is proposed that such cross-talk can occur by release of peptides from DRGs neuron somata within dorsal root ganglia. To what extent these mechanisms shown to exist in rodents also occur in human is important, if one wants to discuss novel strategies for pain treatment on the basis of these findings. So far information is limited, but it has been demonstrated that galanin is expressed in DRGs and possibly also regulated.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Summary: Purpose: Mice carrying a deletion of the GALR1 galanin receptor have recently showed spontaneous seizure phenotype with 25% penetrance. To better understand the role of neuropeptides, which are known to undergo complex plasticity changes with development of epileptic seizures, we characterized their expression in the hippocampal formation in GALR1- knockout (-KO) mice with or without seizures and in wild-type (WT) mice. Methods: Immunohistochemistry and in situ hybridization were used to study expression of galanin, neuropeptide Y (NPY), substance P, enkephalin, dynorphin, and cholecystokinin (CCK). Results: In GALR1-KO mice that had been displaying seizures, a strong upregulation of galanin immunoreactivity (ir) and messenger RNA (mRNA) was found in the polymorph layer of the dentate gyrus; galanin-ir also appeared in a dense fiber network in the supragranular layer. A strong upregulation of enkephalin was found in the granule cells/mossy fibers, whereas dynorphin mRNA levels were modestly decreased. NPY was strongly expressed in the granule cells/mossy fibers, and an increase of NPY mRNA levels in the polymorph cells was paralleled by an increase of NPY-ir in the molecular layer. An upregulation of substance P-ir was confined to the fibers in the granule and molecular layers, whereas substance P mRNA was increased in the cells of the polymorph layer. Both CCK-ir and mRNA were strongly downregulated in the granule cell/mossy fiber system, but CCK-ir appeared increased in the supragranular and molecular layers. No changes in neuropeptide-ir were found in GALR1-KO mice not displaying seizures. Conclusions: Complex changes in neuropeptide expression in some principal hippocampal neurons and interneurons appear as a characteristic feature of the spontaneous-seizure phenotype in GALR1-KO mice. However, to what extent causal relations exist between this “epilepsia peptidergic profile” and development of seizures requires further clarification.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking α-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R. J. Comp. Neurol. 449:390–404, 2002. © 2002 Wiley-Liss, Inc.

Using a sensitive immunohistochemical method, the localization of the neuropeptide Y (NPY) Y1 receptor (Y1R) was studied in contralateral and ipsilateral dorsal root ganglion (DRG) neurons of rats subjected to different unilateral manipulations with focus on their axonal processes and projection areas. Y1R-like immunoreactivity (LI) was observed in the contralateral sciatic nerve and dorsal roots of lesioned rats, and double staining revealed colocalization with calcitonin gene-related peptide (CGRP). Y1R-LI was also seen in fibers close to and even within the epidermis. A fairly small number of nerve endings double-labeled for Y1R and CGRP were present in the dorsal horn. After unilateral crush of the sciatic nerve Y1R- and CGRP-LI accumulated in the same axons proximal to the lesion. After dorsal rhizotomy CGRP-LI was strongly reduced in the ipsilateral dorsal horn. No certain change was observed for Y1R- or NPY-LI, but Y1R/CGRP double-labeled nerve endings disappeared after the lesion. These results strongly suggest centrifugal transport of Y1Rs in DRG neurons, mainly to the peripheral sensory branches. To what extent these Y1Rs are functional has not been analyzed here, but a recent study on Y1R null mice provides evidence for involvement of prejunctional Y1Rs in peripheral sensory functions

Using immunohistochemical techniques, we characterized changes in the expression of several neurochemical markers in lumbar 4-sacral 2 (L4–S2) dorsal root ganglion (DRG) neuron profiles (NPs) and the spinal cord of BALB/c mice after axotomy of the L6 and S1 spinal nerves, major tributaries of the pelvic (targeting pelvic visceral organs) and pudendal (targeting perineum and genitalia) nerves. Sham animals were included. Expression of cyclic AMP-dependent transcription factor 3 (ATF3), calcitonin gene-related peptide (CGRP), transient receptor potential cation channel subfamily V, member 1 (TRPV1), tyrosine hydroxylase (TH) and vesicular glutamate transporters (VGLUT) types 1 and -2 was analysed seven days after injury. L6–S1 axotomy induced dramatic de novo expression of ATF3 in many L6–S1 DRG NPs, and parallel significant downregulations in the percentage of CGRP-, TRPV1-, TH- and VGLUT2-immunoreactive (IR) DRG NPs, as compared to their expression in uninjured DRGs (contralateral L6–S1-AXO; sham mice); VGLUT1 expression remained unaltered. Sham L6–S1 DRGs only showed a small ipsilateral increase in ATF3-IR NPs (other markers were unchanged). L6–S1-AXO induced de novo expression of ATF3 in several lumbosacral spinal cord motoneurons and parasympathetic preganglionic neurons; in sham mice the effect was limited to a few motoneurons. Finally, a moderate decrease in CGRP- and TRPV1-like-immunoreactivities was observed in the ipsilateral superficial dorsal horn neuropil. In conclusion, injury of a mixed visceral/non-visceral nerve leads to considerable neurochemical alterations in DRGs matched, to some extent, in the spinal cord. Changes in these and potentially other nociception-related molecules could contribute to pain due to injury of nerves in the abdominopelvic cavity. Fil: Mccarthy, Carly Jane. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina Fil: Tomasella, María Eugenia. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Malet, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Seroogy, Kim B.. University of Cincinnati; Estados Unidos Fil: Hökfelt, Tomas. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia Fil: Villar, Marcelo Jose. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gebhart, G.F.. University of Pittsburgh; Estados Unidos Fil: Brumovsky, Pablo Rodolfo. University of Pittsburgh; Estados Unidos. Universidad Austral. Facultad de Ciencias Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Galanin is a 29-aa neuropeptide with a complex role in pain processing. Several galanin receptor subtypes are present in dorsal root ganglia and spinal cord with a differential distribution. Here, we describe a generation of a specific galanin R2 (GalR2) agonist, AR-M1896, and its application in studies of a rat neuropathic pain model (Bennett). The results show that in normal rats mechanical and cold allodynia of the hindpaw are induced after intrathecal infusion of low-dose galanin (25 ng per 0.5 μl/h). The same effect is seen with equimolar doses of AR-M1896 or AR-M961, an agonist both at GalR1 and GalR2 receptors. In allodynic Bennett model rats, the mechanical threshold increased dose-dependently after intrathecal injection of a high dose of AR-M961, whereas no effect was observed in the control or AR-M1896 group. No effect of either of the two compounds was observed in nonallodynic Bennett model rats. These data indicate that a low dose of galanin has a nociceptive role at the spinal cord level mediated by GalR2 receptors, whereas the antiallodynic effect of high-dose galanin on neuropathic pain is mediated by the GalR1 receptors. Thus, a selective GalR1 agonist may be used to treat neuropathic pain.

Purpose: Vesicular glutamate transporters (VGLUTs), essential for loading glutamate into synaptic vesicles, are present in various neuronal systems. However, the expression of VGLUTs in neurons innervating the urinary bladder has not yet been analyzed. Here, we study the presence of VGLUTs type-1, -2 and -3 (VGLUT1, VGLUT2 and VGLUT3, respectively) in mouse urinary bladder neurons. Materials and Methods: Expression of VGLUT1, VGLUT2 and calcitonin gene-related peptide (CGRP) was analyzed by immunohistochemistry in retrogradely labeled primary afferent and autonomic neurons of BALB/C mice after injecting Fast Blue in the urinary bladder wall. To study VGLUT3, retrograde tracing of the urinary bladder was performed in transgenic mice where VGLUT3 is identified by detection of enhanced green fluorescent protein (EGFP). Results: Most urinary bladder DRG neurons expressed VGLUT2. A smaller percentage of neurons also expressed VGLUT1 or VGLUT3. Coexpression with CGRP was only observed for VGLUT2. Occasional VGLUT2-immunoreactive (IR) neurons were seen in the major pelvic ganglion (MPG). Abundant VGLUT2-IR nerves were detected in the urinary bladder dome, trigone and also the urethra; VGLUT1-IR nerves were discretely present. Conclusions: We present novel data on the expression of VGLUTs in sensory and autonomic neurons innervating the mouse urinary bladder. The frequent association of VGLUT2 and CGRP in sensory neurons suggests interactions between glutamatergic and peptidergic neurotransmissions, potentially influencing commonly perceived sensations in the urinary bladder, such as discomfort and pain. Fil: Brumovsky, Pablo Rodolfo. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Investigaciones Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh. Department of Anesthesiology. Pittsburgh Center for Pain Research; Estados Unidos Fil: Seal, Rebecca P.. University of Pittsburgh. Department of Anesthesiology. Pittsburgh Center for Pain Research; Estados Unidos Fil: Lundgren, Kerstin H.. University of Cincinnati. Department of Neurology; Estados Unidos Fil: Seroogy, Kim B.. University of Cincinnati. Department of Neurology; Estados Unidos Fil: Watanabe, Masahiko. Hokkaido University School of Medicine. Department of Anatomy; Japón Fil: Gebhart, G. F.. University of Pittsburgh. Department of Anesthesiology. Pittsburgh Center for Pain Research; Estados Unidos

Single ligature nerve constriction (SLNC) of the rat sciatic nerve triggers neuropathic pain-related behaviors and induces changes in neuropeptide expression in primary afferent neurons. Bone marrow stromal cells (MSCs) injected into the lumbar 4 (L4) dorsal root ganglia (DRGs) of animals subjected to a sciatic nerve SLNC selectively migrate to the other ipsilateral lumbar DRGs (L3, L5 and L6) and prevent mechanical and thermal allodynia. In this study, we have evaluated the effect of MSC administration on the expression of the neuropeptides galanin and NPY, as well as the NPY Y(1)-receptor (Y(1)R) in DRG neurons. Animals were subjected to a sciatic nerve SLNC either alone or followed by the administration of MSCs, phosphate-buffered saline (PBS) or bone marrow non-adherent mononuclear cells (BNMCs), directly into the ipsilateral L4 DRG. Seven days after injury, the ipsilateral and contralateral L4-5 DRGs were dissected out and processed for standard immunohistochemistry, using specific antibodies. As previously reported, SLNC induced an ipsilateral increase in the number of galanin and NPY immunoreactive neurons and a decrease in Y(1)R-positive DRG neurons. The intraganglionic injection of PBS or BNMCs did not modify this pattern of expression. In contrast, MSC administration partially prevented the injury-induced changes in galanin, NPY and Y(1)R expression. The large number of Y(1)R-immunoreactive neurons together with high levels of NPY expression in animals injected with MSCs could explain, at least in part, the analgesic effects exerted by these cells. Our results support MSC participation in the modulation of neuropathic pain and give insight into one of the possible mechanisms involved. Fil: Coronel, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad Austral; Argentina Fil: Musolino, P. L.. Universidad Austral; Argentina Fil: Brumovsky, Pablo Rodolfo. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia Fil: Hökfelt, T.. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia Fil: Villar, M. J.. Universidad Austral; Argentina

Afferent nerve firing has been linked to spontaneous bladder contractions in a number of lower urinary tract pathologies and it may lead to urgency and incontinence. Using optical mapping, single unit recording and tension measurements we investigated the correlation between afferent nerve firing and spontaneous bladder contractions in spinal cord transected mice.Bladder-nerve preparations (bladder sheets and the associated L6-S2 pelvic nerves) were dissected from normal and spinal cord transected mice showing overactivity on cystometry and opened along the ventral aspect from base to dome. Bladder sheets were mounted horizontally in a temperature regulated chamber to simultaneously record Ca(2+) transients across the mucosal surface, single unit afferent nerve firing and whole bladder tension.Single unit afferent fibers were identified by probing their receptive fields. Fibers showed a graded response to von Frey stimulation and a frequency of afferent firing that increased as a function of the degree of stretch. Optical maps of Ca(2+) transients in control bladders demonstrated multiple initiation sites that resulted in high frequency, low amplitude spontaneous contractions. Alternatively in maps of the bladders of spinal cord transected mice Ca(2+) transients arose from 1 or 2 focal sites, resulting in low frequency, high amplitude contractions and concomitant afferent firing.Large amplitude, spontaneous bladder contractions evoke afferent nerve activity, which may contribute to incontinence.

The distribution of galanin was studied in the lumbar 5 dorsal root ganglia (DRGs) and spinal cord, superior cervical ganglia (SCGs), and skin of transgenic mice overexpressing galanin under the dopamine beta-hydroxylase (DBH) promoter (GalOE-DBH mice) and in wild type (WT) mice. The DRGs and spinal cord were analysed before and after a unilateral, complete transection (axotomy) of the sciatic nerve and after dorsal rhizotomy. Both galanin protein and transcript were studied by, respectively, immunohistochemistry and in situ hybridization. Increased galanin expression was observed in several small, medium-sized and large DRG neuron profiles (NPs) in the naive transgenic mouse, frequently in neurons lacking calcitonin gene-related peptide (CGRP) and isolectin B4-binding. This lack of coexistence was particularly evident in the medium-sized/large NPs. In the dorsal horn of the spinal cord, no differences were detected between GalOE-DBH and WT mice, both displaying a strong galanin-positive neuropil in the superficial laminae of the dorsal horn, but the transgenic mice showed a more abundant galanin-positive innervation of the ventral horn. A 12-day dorsal rhizotomy, surprisingly, failed to alter the galanin staining patterns in the dorsal (and ventral) dorsal horn. Unilateral axotomy induced upregulation of galanin in DRG NPs of all sizes in both types of mouse. In the hindpaw skin, a profuse galanin-positive fiber plexus was observed in sweat glands and around blood vessels of the transgenic mice, being much more restricted in WT mice. Finally, GalOE mice exhibited a strong galanin-like immunoreactivity in most SCG NPs. The overexpression of the peptide in DRGs and SCGs was paralleled by increased mRNA levels. The present results show that overexpression of galanin under the control of the DBH promoter does not only occur, as expected in these mice, in noradrenline/adrenaline neurons but also in DRG neurons, particularly in large and medium-sized NPs. To what extent and how this overexpression pattern is related to the previously shown elevated pain threshold under normal and lesion conditions is discussed [Grass, S., Crawley, J.N., Xu, X.J., Wiesenfeld-Hallin, Z., 2003a. Reduced spinal cord sensitization to C-fibre stimulation in mice over-expressing galanin. Eur. J. Neurosci. 17, 1829-1832; Hygge-Blakeman, K., Brumovsky, P., Hao, J.X., Xu, X.J., Hokfelt, T., Crawley, J.N., Wiesenfeld-Hallin, Z., 2004. Galanin over-expression decreases the development of neuropathic pain-like behaviour in mice after partial sciatic nerve injury. Brain Res. 1025, 152-158].

The localization of the neuropeptide tyrosine (NPY) Y2 receptor (Y2R) protein was studied in mouse dorsal root ganglia (DRGs) and spinal cord, by using a recently developed rabbit anti-Y2R antibody and a sensitive immunohistochemical method. Y2R-like immunoreactivity (-LI) was observed in about 10% of the small/medium-sized lumbar DRG neurons. Among these, about 44% were calcitonin gene-related peptide-immunoreactive, and about 38% bound isolectin B4. In the dorsal horn of the spinal cord, an intense Y2R-LI was seen in the most superficial layers, mostly restricted to laminae I-II. This immunoreactivity was completely abolished by dorsal rhizotomy. Y2R-L1 was also detected on the skin, more abundantly in hairy than glabrous skin. Specificity experiments showed complete disappearance of the Y2R-LI described above after incubation with antibody preadsorbed with the immunogenic peptide. Furthermore, Y2R-LI was also absent in a Y2R knockout mouse. These results demonstrate that the NPY Y2R is associated mainly with both peptidergic and nonpeptidergic small, presumably nociceptive, neurons projecting to the superficial layers of the dorsal horn. The results also support a role for this receptor and NPY in pain mechanisms.

The neuropeptide galanin may have a role in modulation of nociception, particularly after peripheral nerve injury. Here we assessed the development of neuropathic pain-like behaviors in mice overexpressing galanin under the dopamine beta-hydroxylase promoter. Unoperated galanin over-expressing mice exhibited a moderately reduced sensitivity to noxious heat. Both galanin over-expressing mice and wild-type controls developed mechanical and heat hypersensitivity after photochemically induced partial sciatic nerve ischemic injury. The magnitude and persistence of such pain-like behaviors were significantly less, and recovery was faster in galanin over-expressing mice compared to wild types. However, the recovery from toe-spread deficits did not differ between galanin over-expressing and wild-type mice after a crush injury to the sciatic nerve. Thus, early recovery in pain-like response is unlikely to result from accelerated regeneration in the galanin over-expressing mice. Immunohistochemical analysis showed that galanin is over-expressed both in small and large dorsal root ganglion cells in the transgene mouse, whereas large galanin-positive neurons were never seen in wild-type mice. The present results in general support an inhibitory role of galanin in nociception and indicate that increased availability of galanin in spinal dorsal horn at the time or shortly after nerve injury may reduce the development of pain-like behaviors in mice.

In the present study, the rat sciatic nerve was constricted to varying degrees using only one ligature with a very thin polyethylene sheath placed between nerve and ligature thread. Complete nerve transection was studied for comparison. With a 40-80% constriction of the nerve we observed allodynia to a similar extent as in the so-called Bennett model based on four loose ligatures. We also monitored changes in the expression of neuropeptide Y (NPY) and the NPY Y1 receptor (Y1R) in the lumbar 4-5 dorsal root ganglia (DRG) and dorsal horn and found upregulation of NPY and downregulation of the Y1R in DRG neurons after injury. These results indicate that similar peptide and receptor changes occur in this model as after axotomy and in other nerve injury models, although the immunohistochemical and behavioral changes seem to be dependent on the degree of constriction of the nerve. Thus, it seems relevant to monitor the degree of constriction when evaluating pain and other post-injury events. The possibility that some of the changes in NPY-ergic neurotransmission are related to the generation of allodynia is discussed; as well as the possibility to use this mononeuropathic model based on a single ligature nerve constriction (SLNC) as a complementary approach to other widely used pain models.