Pro-opiomelanocortin neurons in the nucleus of the solitary tract mediate endorphinergic endogenous analgesia in mice.

ABSTRACT: The nucleus of the solitary tract (NTS) contains pro-opiomelanocortin (POMC) neurons that are 1 of the 2 major sources of ß-endorphin in the brain. The functional role of these NTS POMC neurons in nociceptive and cardiorespiratory function is debated. We have shown that NTS POMC optogenetic activation produces bradycardia and transient apnoea in a working heart-brainstem preparation and chemogenetic activation with an engineered ion channel (PSAM) produced opioidergic analgesia in vivo. To better define the role of the NTS POMC neurons in behaving animals, we adopted in vivo optogenetics (ChrimsonR) and excitatory/inhibitory chemogenetic DREADD (hM3Dq/hM4Di) strategies in POMC-Cre mice. We show that optogenetic activation of NTS POMC neurons produces time-locked, graded, transient bradycardia and bradypnoea in anaesthetised mice that is naloxone sensitive (1 mg/kg, i.p.), suggesting a role of ß-endorphin. Both optogenetic and chemogenetic activation of NTS POMC neurons produces sustained thermal analgesia in behaving mice that can be blocked by naloxone. It also produced analgesia in an inflammatory pain model (carrageenan) but not in a neuropathic pain model (tibial nerve transection). Inhibiting NTS POMC neurons does not produce any effect on basal nociception but inhibits stress-induced analgesia (unlike inhibition of arcuate POMC neurons). Activation of NTS POMC neuronal populations in conscious mice did not cause respiratory depression, anxiety, or locomotor deficit (in open field) or affective preference. These findings indicate that NTS POMC neurons play a key role in the generation of endorphinergic endogenous analgesia and can also regulate cardiorespiratory function.

Regulation of food intake by astrocytes in the brainstem dorsal vagal complex.

A role for glial cells in brain circuits controlling feeding has begun to be identified with hypothalamic astrocyte signaling implicated in regulating energy homeostasis. The nucleus of the solitary tract (NTS), within the brainstem dorsal vagal complex (DVC), integrates vagal afferent information from the viscera and plays a role in regulating food intake. We hypothesized that astrocytes in this nucleus respond to, and influence, food intake. Mice fed high-fat chow for 12 hr during the dark phase showed NTS astrocyte activation, reflected in an increase in the number (65%) and morphological complexity of glial-fibrillary acidic protein (GFAP)-immunoreactive cells adjacent to the area postrema (AP), compared to control chow fed mice. To measure the impact of astrocyte activation on food intake, we delivered designer receptors exclusively activated by designer drugs (DREADDs) to DVC astrocytes (encompassing NTS, AP, and dorsal motor nucleus of the vagus) using an adeno-associated viral (AAV) vector (AAV-GFAP-hM3Dq_mCherry). Chemogenetic activation with clozapine-N-oxide (0.3 mg/kg) produced in greater morphological complexity in astrocytes and reduced dark-phase feeding by 84% at 4 hr postinjection compared with vehicle treatment. hM3Dq-activation of DVC astrocytes also reduced refeeding after an overnight fast (71% lower, 4 hr postinjection) when compared to AAV-GFAP-mCherry expressing control mice. DREADD-mediated astrocyte activation did not impact locomotion. hM3Dq activation of DVC astrocytes induced c-FOS in neighboring neuronal feeding circuits (including in the parabrachial nucleus). This indicates that NTS astrocytes respond to acute nutritional excess, are involved in the integration of peripheral satiety signals, and can reduce food intake when activated.

Targeted disruption of the orphan receptor Gpr151 does not alter pain-related behaviour despite a strong induction in dorsal root ganglion expression in a model of neuropathic pain.

BACKGROUND: Gpr151 is an orphan GPCR whose function is unknown. The restricted pattern of neuronal expression in the habenula, dorsal horn of the spinal cord and dorsal root ganglion plus homology with the galanin family of receptors imply a role in nociception. RESULTS: Real-time quantitative RT-PCR demonstrated a 49.9±2.9 fold highly significant (P<0.001) increase in Gpr151 mRNA expression in the dorsal root ganglion 7days after the spared nerve injury model of neuropathic pain. Measures of acute, inflammatory and neuropathic pain behaviours were not significantly different using separate groups of Gpr151 loss-of-function mutant mice and wild-type controls. Galanin at concentrations between 100nM and 10µM did not induce calcium signalling responses in ND7/23 cells transfected with Gpr151. CONCLUSIONS: Our results indicate that despite the very large upregulation in the DRG after a nerve injury model of neuropathic pain, the Gpr151 orphan receptor does not appear to be involved in the modulation of pain-related behaviours. Further, galanin is unlikely to be an endogenous ligand for Gpr151.

The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2.

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1-3 gene products). There is a wealth of data on expression of Gal1-3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs).

Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor.

In order to further investigate the molecular mechanisms that regulate oligodendrocyte (OC) survival, we utilized microarrays to characterize changes in OC gene expression after exposure to the cytokines neurotrophin3, insulin, or leukemia inhibitory factor (LIF) in vitro. We identified and validated the induction and secretion of the neuropeptide galanin in OCs, specifically in response to LIF. We next established that galanin is an OC survival factor and showed that autocrine or paracrine galanin secretion mediates LIF-induced OC survival in vitro. We also revealed that galanin is up-regulated in OCs in the cuprizone model of central demyelination, and that oligodendroglial galanin expression is significantly regulated by endogenous LIF in this context. We also showed that knock-out of galanin reduces OC survival and exacerbates callosal demyelination in the cuprizone model. These findings suggest a potential role for the use of galanin agonists in the treatment of human demyelinating diseases.

Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity.

Galanin was first identified 30 years ago as a "classic neuropeptide," with actions primarily as a modulator of neurotransmission in the brain and peripheral nervous system. Other structurally-related peptides-galanin-like peptide and alarin-with diverse biologic actions in brain and other tissues have since been identified, although, unlike galanin, their cognate receptors are currently unknown. Over the last two decades, in addition to many neuronal actions, a number of nonneuronal actions of galanin and other galanin family peptides have been described. These include actions associated with neural stem cells, nonneuronal cells in the brain such as glia, endocrine functions, effects on metabolism, energy homeostasis, and paracrine effects in bone. Substantial new data also indicate an emerging role for galanin in innate immunity, inflammation, and cancer. Galanin has been shown to regulate its numerous physiologic and pathophysiological processes through interactions with three G protein-coupled receptors, GAL1, GAL2, and GAL3, and signaling via multiple transduction pathways, including inhibition of cAMP/PKA (GAL1, GAL3) and stimulation of phospholipase C (GAL2). In this review, we emphasize the importance of novel galanin receptor-specific agonists and antagonists. Also, other approaches, including new transgenic mouse lines (such as a recently characterized GAL3 knockout mouse) represent, in combination with viral-based techniques, critical tools required to better evaluate galanin system physiology. These in turn will help identify potential targets of the galanin/galanin-receptor systems in a diverse range of human diseases, including pain, mood disorders, epilepsy, neurodegenerative conditions, diabetes, and cancer.

Stabilization of the syndesmosis in the Maisonneuve fracture--a biomechanical study comparing 2-hole locking plate and quadricortical screw fixation.

OBJECTIVE: The aim of this study is to determine whether a 2-hole locking plate has biomechanical advantages over conventional screw stabilization of the syndesmosis in this injury pattern. METHODS: Six pairs of fresh-frozen human cadaver lower legs were prepared to simulate an unstable Maisonneuve fracture. Each limb was compared with its pair; the syndesmosis in one being stabilized with two 4.5-mm quadricortical cortical screws, the other a 2-hole locking plate with 3.2-mm locking screws. The limbs were then mounted on a servohydraulic testing rig and axially loaded to a peak load of 800N for 12000 cycles. Fibula shortening and diastasis were measured. Each limb was then externally rotated until failure occurred. Failure was defined as fracture of bone or metalwork, syndesmotic widening, or axial migration >2 mm. RESULTS: Both constructs effectively stabilized the syndesmosis during the cyclical loading within 0.1 mm of movement. However, the locking plate group demonstrated greater resistance to torque compared with quadricortical screw fixation (40.6 Nm vs. 21.2 Nm, respectively, P value < 0.03). CONCLUSION: A 2-hole locking plate (with 3.2-mm screws) provides significantly greater stability of the syndesmosis to torque when compared with 4.5-mm quadricortical fixation.

Galanin-expression and galanin-dependent sensory neurons are not required for itch.

BACKGROUND: Galanin is a key modulator of nociception, and it is also required for the developmental survival of a subset of C-fibre sensory neurons which are critical to the mediation of neuropathic and inflammatory pain. However, the potential modulatory roles played by galanin, or the galanin-dependent neurons, in pruritoceptive mechanisms underlying the sensation of itch have not been investigated. FINDINGS: Here we report that mice carrying a loss-of-function mutation in the galanin gene (Gal-KO) show no differences in spontaneous behavioural itch responses compared to wild-type (WT) controls. Similarly, the responses to a range of pruritogens are not significantly different between the two genotypes. CONCLUSIONS: These results suggest that neither galanin expression, nor the galanin-dependent subpopulation of sensory neurons is required for itch-related behaviours.

Galanin negatively modulates opiate withdrawal via galanin receptor 1.

RATIONALE: The neuropeptide galanin has been shown to modulate opiate dependence and withdrawal. These effects could be mediated via activation of one or more of the three distinct G protein-coupled receptors, namely galanin receptors 1 (GalR1), 2 (GalR2), and 3 (GalR3). OBJECTIVES: In this study, we used several transgenic mouse lines to further define the mechanisms underlying the role played by galanin and its receptors in the modulation of morphine dependence. First, transgenic mice expressing ß-galactosidase under the control of the galanin promoter were used to assess the regulation of galanin expression in response to chronic morphine administration and withdrawal. Next, the behavioral responses to chronic morphine administration and withdrawal were tested in mice that over-express galanin, lack the GalR1 gene, or lack the GalR2 gene. METHODS: Transgenic and matched wild-type mice were given increasing doses of morphine followed by precipitation of withdrawal by naloxone and behavioral responses to withdrawal were assessed. RESULTS: Both morphine administration and withdrawal increased galanin gene transcription in the locus coeruleus (LC). Increasing galanin levels in the brain reduced signs of opiate withdrawal. Mice lacking GalR1 undergo more severe opiate withdrawal, whereas mice lacking GalR2 show no significant difference in withdrawal signs, compare with matched wild-type controls. CONCLUSIONS: Opiate administration and withdrawal increase galanin expression in the LC. Galanin opposes the actions of morphine which leads to opiate dependence and withdrawal, an effect that is mediated via GalR1.

Axotomy-induced miR-21 promotes axon growth in adult dorsal root ganglion neurons.

Following injury, dorsal root ganglion (DRG) neurons undergo transcriptional changes so as to adopt phenotypic changes that promote cell survival and axonal regeneration. Here we used a microarray approach to profile changes in a population of small noncoding RNAs known as microRNAs (miRNAs) in the L4 and L5 DRG following sciatic nerve transection. Results showed that 20 miRNA transcripts displayed a significant change in expression levels, with 8 miRNAs transcripts being altered by more than 1.5-fold. Using quantitative reverse transcription PCR, we demonstrated that one of these miRNAs, miR-21, was upregulated by 7-fold in the DRG at 7 days post-axotomy. In dissociated adult rat DRG neurons lentiviral vector-mediated overexpression of miR-21 promoted neurite outgrowth on a reduced laminin substrate. miR-21 directly downregulated expression of Sprouty2 protein, as confirmed by Western blot analysis and 3' untranslated region (UTR) luciferase assays. Our data show that miR-21 is an axotomy-induced miRNA that enhances axon growth, and suggest that miRNAs are important players in regulating growth pathways following peripheral nerve injury.

Endogenous galanin protects mouse hippocampal neurons against amyloid toxicity in vitro via activation of galanin receptor-2.

Expression of the neuropeptide galanin is known to be upregulated in the brain of patients with Alzheimer's disease (AD). We and others have shown that galanin plays a neuroprotective role in a number of excitotoxic injury paradigms, mediated by activation of the second galanin receptor subtype (GAL2). In the present study, we investigated whether galanin/GAL2 plays a similar protective role against amyloid-ß(Aß) toxicity. Here we report that galanin or the GAL2/3-specific peptide agonist Gal2-11, both equally protect primary dispersed mouse wildtype (WT) neonatal hippocampal neurons from 250 nM Aß1-42 toxicity in a dose dependent manner. The amount of Aß1-42 induced cell death was significantly greater in mice with loss-of-function mutations in galanin (Gal-KO) or GAL2 (GAL2-MUT) compared to strain-matched WT controls. Conversely, cell death was significantly reduced in galanin over-expressing (Gal-OE) transgenic mice compared to strain-matched WT controls. Exogenous galanin or Gal2-11 rescued the deficits in the Gal-KO but not the GAL2-MUT cultures, confirming that the protective effects of endogenous or exogenous galanin are mediated by activation of GAL2. Despite the high levels of endogenous galanin in the Gal-OE cultures, the addition of exogenous 100 nM or 50 nM galanin or 100 nM Gal2-11 further significantly reduced cell death, implying that GAL2-mediated neuroprotection is not at maximum in the Gal-OE mice. These data further support the hypothesis that galanin over-expression in AD is a neuroprotective response and imply that the development of a drug-like GAL2 agonist might reduce the progression of symptoms in patients with AD.

Characterisation of the nociceptive phenotype of suppressible galanin overexpressing transgenic mice.

The neuropeptide galanin is widely expressed in both the central and peripheral nervous systems and is involved in many diverse biological functions. There is a substantial data set that demonstrates galanin is upregulated after injury in the DRG, spinal cord and in many brain regions where it plays a predominantly antinociceptive role in addition to being neuroprotective and pro-regenerative. To further characterise the role of galanin following nerve injury, a novel transgenic line was created using the binary transgenic tet-off system, to overexpress galanin in galaninergic tissue in a suppressible manner. The double transgenic mice express significantly more galanin in the DRG one week after sciatic nerve section (axotomy) compared to WT mice and this overexpression is suppressible upon administration of doxycycline. Phenotypic analysis revealed markedly attenuated allodynia when galanin is overexpressed and an increase in allodynia following galanin suppression. This novel transgenic line demonstrates that whether galanin expression is increased at the time of nerve injury or only after allodynia is established, the neuropeptide is able to reduce neuropathic pain behaviour. These new findings imply that administration of a galanin agonist to patients with established allodynia would be an effective treatment for neuropathic pain.

The expression of ELK transcription factors in adult DRG: Novel isoforms, antisense transcripts and upregulation by nerve damage.

ELK transcription factors are known to be expressed in a number of regions in the nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fails to bind ETS binding sites and yet can activate transcription by an indirect mechanism. The Elk3 and Elk4 genes are overlapped by co-expressed Pctk2 (Cdk17) and Mfsd4 genes, respectively, with the potential formation of Elk3/Pctaire2 and Elk4/Mfsd4 sense-antisense mRNA heteroduplexes. After peripheral nerve injury the Elk3 mRNA isoforms are each upregulated approximately 2.3-fold in DRG (P<0.005), whereas the natural antisense Pctaire2 isoforms show only a small increase (21%, P<0.01) and Elk1 and Elk4 mRNAs are unchanged.

Intra-neural administration of fractalkine attenuates neuropathic pain-related behaviour.

There is increasing evidence that a number of cytokines and their receptors are involved in the processes that lead to the development and maintenance of neuropathic pain states. Here we demonstrate that levels of CX3CR1 (the receptor for the chemokine fractalkine) mRNA in lumbar dorsal root ganglia (DRG) increase 5.8-fold 7 days after sciatic nerve axotomy, and 1.7- and 2.9-fold, 3 and 7 days respectively, after the spared nerve injury (SNI) model of neuropathic pain. In contrast, no significant change in the levels of fractalkine mRNA is apparent in the DRG after axotomy or SNI. The increase in CX3CR1 mRNA is paralleled by a 3.9- and 2.1-fold increase in the number of CX3CR1-positive macrophages in the DRG 7 days after axotomy and SNI, respectively. Expression of CX3CR1 in macrophages is also markedly increased in the sciatic nerve proximal to site of injury, by 25.7-fold after axotomy and 16.2-fold after SNI, 7 days after injury. Intra-neural injection into the sciatic nerve of 400 ng or 100 ng of fractalkine in adult 129OlaHsd mice significantly delayed the development of allodynia for 3 days following SNI. Further, CX3CR1 knockout (KO) mice display an increase in allodynia for three weeks after SNI compared to strain-matched Balb/c controls. Taken together, these results suggest an anti-allodynic role for fractalkine and its receptor in the mouse.

Characterization of an enhancer region of the galanin gene that directs expression to the dorsal root ganglion and confers responsiveness to axotomy.

Galanin expression markedly increases in the dorsal root ganglion (DRG) after sciatic nerve axotomy and modulates pain behavior and regeneration of sensory neurons. Here, we describe transgenic mice expressing constructs with varying amounts of sequence upstream of the murine galanin gene marked by LacZ. The 20 kb region upstream of the galanin gene recapitulates the endogenous expression pattern of galanin in the embryonic and adult intact DRG and after axotomy. In contrast, 1.9 kb failed to drive LacZ expression in the intact DRG or after axotomy. However, the addition of an additional 2.7 kb of 5' flanking DNA (4.6 kb construct) restored the expression in the embryonic DRG and in the adult after axotomy. Sequence analysis of this 2.7 kb region revealed unique 18 and 23 bp regions containing overlapping putative Ets-, Stat-, and Smad-binding sites, and adjacent putative Stat- and Smad-binding sites, respectively. Deletion of the 18 and 23 bp regions from the 4.6 kb construct abolished the upregulation of LacZ expression in the DRG after axotomy but did not affect expression in the embryonic or intact adult DRG. Also, a bioinformatic analysis of the upstream regions of a number of other axotomy-responsive genes demonstrated that the close proximity of putative Ets-, Stat-, and Smad-binding sites appears to be a common motif in injury-induced upregulation in gene expression.

The sodium channel Nav1.5a is the predominant isoform expressed in adult mouse dorsal root ganglia and exhibits distinct inactivation properties from the full-length Nav1.5 channel.

Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by approximately 5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.

Mice deficient for galanin receptor 2 have decreased neurite outgrowth from adult sensory neurons and impaired pain-like behaviour.

Expression of the neuropeptide galanin is markedly up-regulated within the adult dorsal root ganglia (DRG) following peripheral nerve injury. We have previously demonstrated that galanin knockout (Gal-KO) mice have a developmental loss of a subset of DRG neurons. Galanin also plays a trophic role in the adult animal, and the rate of peripheral nerve regeneration and neurite outgrowth is reduced in adult Gal-KO mice. Here we describe the characterization of mice with an absence of GalR2 gene transcription (GalR2-MUT) and demonstrate that they have a 15% decrease in the number of calcitonin gene-related peptide (CGRP) expressing neuronal profiles in the adult DRG, associated with marked deficits in neuropathic and inflammatory pain behaviours. Adult GalR2-MUT animals also have a one third reduction in neurite outgrowth from cultured DRG neurons that cannot be rescued by either galanin or a high-affinity GalR2/3 agonist. Galanin activates extracellular signal-regulated kinase (ERK) and Akt in adult wild-type (WT) mouse DRG. Intact adult DRG from GalR2-MUT animals have lower levels of pERK and higher levels of pAkt than are found in WT controls. These data suggest that a lack of GalR2 activation in Gal-KO and GalR2-MUT animals is responsible for the observed developmental deficits in the DRG, and the decrease in neurite outgrowth in the adult.

Novel isoforms of the sodium channels Nav1.8 and Nav1.5 are produced by a conserved mechanism in mouse and rat.

The voltage-gated sodium channel Na(v)1.8 is only expressed in subsets of neurons in dorsal root ganglia (DRG) and trigeminal and nodose ganglia. We have isolated mouse partial length Na(v)1.8 cDNA clones spanning the exon 17 sequence, which have 17 nucleotide substitutions and 12 predicted amino acid differences from the published sequence. The absence of a mutually exclusive alternative exon 17 was confirmed by sequencing 4.1 kilobases of genomic DNA spanning exons 16-18 of Scn10a. A novel cDNA isoform was identified, designated Na(v)1.8c, which results from alternative 3'-splice site selection at a CAG/CAG motif to exclude the codon for glutamine 1031 within the interdomain cytoplasmic loop IDII/III. The ratio of Na(v)1.8c (CAG-skipped) to Na(v)1.8 (CAG-inclusive) mRNA in mouse is approximately 2:1 in adult DRG, trigeminal ganglion, and neonatal DRG. A Na(v)1.8c isoform also occurs in rat DRG, but is less common. Of the two other tetrodotoxin-resistant channels, no analogous alternative splicing of mouse Na(v)1.9 was detected, whereas rare alternative splicing of Na(v)1.5 at a CAG/CAG motif resulted in the introduction of a CAG trinucleotide. This isoform, designated Na(v)1.5c, is conserved in rat and encodes an additional glutamine residue that disrupts a putative CK2 phosphorylation site. In summary, novel isoforms of Na(v)1.8 and Na(v)1.5 are each generated by alternative splicing at CAG/CAG motifs, which result in the absence or presence of predicted glutamine residues within the interdomain cytoplasmic loop IDII/III. Mutations of sodium channels within this cytoplasmic loop have previously been demonstrated to alter electrophysiological properties and cause cardiac arrhythmias and epilepsy.

Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior.

The neuropeptide galanin is expressed in the dorsal root ganglia (DRG) and spinal cord and is thought to be involved in the modulation of pain processing. However, its mechanisms of action are complex and poorly understood, as both facilitatory and inhibitory effects have been described. To understand further the role played by galanin in nociception, we have generated two transgenic lines that overexpress galanin in specific populations of primary afferent DRG neurons in either an inducible or constitutive manner. In the first line, a previously defined enhancer region from the galanin locus was used to target galanin to the DRG (Gal-OE). Transgene expression recapitulates the spatial endogenous galanin distribution pattern in DRG neurons and markedly overexpresses the peptide in the DRG after nerve injury but not in the uninjured state. In the second line, an enhancer region of the c-Ret gene was used to constitutively and ectopically target galanin overexpression to the DRG (Ret-OE). The expression of this second transgene does not alter significantly after nerve injury. Here, we report that intact Ret-OE, but not Gal-OE, animals have significantly elevated mechanical and thermal thresholds. After nerve damage, using a spared nerve-injury model, mechanical allodynia is attenuated markedly in both the Gal-OE and Ret-OE mice compared with WT controls. These results support an inhibitory role for galanin in the modulation of nociception both in intact animals and in neuropathic pain states.

The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons.

Expression of the neuropeptide galanin is markedly upregulated within the adult dorsal root ganglion (DRG) after peripheral nerve injury. We demonstrated previously that the rate of peripheral nerve regeneration is reduced in galanin knock-out mice, with similar deficits observed in neurite outgrowth from cultured mutant DRG neurons. Here, we show that the addition of galanin peptide significantly enhanced neurite outgrowth from wild-type sensory neurons and fully rescued the observed deficits in mutant cultures. Furthermore, neurite outgrowth in wild-type cultures was reduced to levels observed in the mutants by the addition of the galanin antagonist M35 [galanin(1-13)bradykinin(2-9)]. Study of the first galanin receptor (GalR1) knock-out animals demonstrated no differences in neurite outgrowth compared with wild-type animals. Similarly, use of a GalR1-specific antagonist had no effect on neuritogenesis. In contrast, use of a GalR2-specific agonist had equipotent effects on neuritogenesis to galanin peptide, and inhibition of PKC reduced neurite outgrowth from wild-type sensory neurons to that observed in galanin knock-out cultures. These results demonstrate that adult sensory neurons are dependent, in part, on galanin for neurite extension and that this crucial physiological process is mediated by activation of the GalR2 receptor in a PKC-dependent manner.