Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.

Completion of the Human Genome Project and recent developments in proteomics make it possible to systematically generate affinity reagents to a large portion of the proteome. Recently an antibody-based human protein atlas covering many organs including four areas of the brain has been released (www.proteinatlas.org). Due to the heterogeneity, size, and availability of tissue a more thorough analysis of the human brain is associated with considerable difficulties. Here we applied 120 antibodies raised against 112 human gene products to the smaller rat brain, a rodent animal model, where a single section represents a 'superarray' including many brain areas, and consequently allowing analysis of a huge number of cell types and their neurochemicals. Immunoreactive structures were seen in the investigated brain tissue after incubation with 56 antibodies (46.6%), of which 25 (20.8%) showed a clearly discrete staining pattern that was limited to certain areas, or subsets of brain cells. Bioinformatics, pre-adsorption tests and Western blot analysis were applied to identify non-specific antibodies. Eleven antibodies, including such raised against four 'ambiguous' proteins, passed all validation criteria, and the expression pattern and subcellular distribution of these proteins were studied in detail. To further explore the potential of the systematically generated antibodies, all 11 antibodies that passed validation were used to analyze the spinal cord and lumbar dorsal root ganglia after unilateral transection of the sciatic nerve. Discrete staining patterns were observed for four of the proteins, and injury-induced regulation was found for one of them. In conclusion, the study presented here suggests that a significant portion (10%) of the antibodies generated to a human protein can be used to analyze orthologues present in the rodent brain and to produce a protein-based atlas of the rodent brain. It is hoped that this type of antibody-based, high throughput screening of brain tissue from various rodent disease models will provide new information on the brain chemical neuroanatomy and insights in processes underlying neurological pathologies.

Deletion of the neuropeptide Y Y1 receptor affects pain sensitivity, neuropeptide transport and expression, and dorsal root ganglion neuron numbers.

Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity.

Galaninergic mechanisms at the spinal level: focus on histochemical phenotyping.

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.

CCK-ergic mechanisms in sensory systems.

The histochemical analysis of cholecystokinin (CCK) systems in sensory systems has revealed involvement of CCK-ergic mechanisms both at the spinal level and in the viscero-sensory vagal pathway, with distinct differences between these two systems as well as between species. Thus, the CCK1 receptor is particularly abundant in rat nodose ganglion neurons which express the food intake-suppressing cocaine- and amphetamine-regulated transcript (CART) peptide(s), representing a likely link between gastrointestinal CCK and central feeding-regulatory centers. In contrast, rat dorsal root ganglions have lower numbers of CCK1 receptor mRNA-positive neurons, and CART is only expressed sparingly in this system. The CCK2 receptor is normally almost absent from both systems but is strongly upregulated after peripheral nerve injury, suggesting a role in regenerative and trophic phenomena as well as, at the spinal level, in nerve injury-induced pain. In man and monkey the CCK1 receptor seems important in the dorsal horn under normal conditions, indicating distinct species differences.

Effect of peripheral nerve injury on dorsal root ganglion neurons in the C57 BL/6J mouse: marked changes both in cell numbers and neuropeptide expression.

Several types of changes have been reported to occur in dorsal root ganglia following peripheral nerve injury, including loss of neurons and increases and decreases in peptide expression. However, with regard to loss of neurons, results have not been consistent, presumably due to different quantitative methodologies employed and species analyzed. So far, most studies have been conducted on rats; however, with the fast development of the transgenic techniques, the mouse has become a standard model animal in primary sensory research. Therefore we used stereological methods to determine the number of neurons, as well as the expression of galanin message-associated peptide, a marker for galanin-expressing neurons, neuropeptide Y, and calcitonin gene-related peptide in lumbar 5 dorsal root ganglia of both control C57 BL/6J mice and in mice subjected to a 'mid-thigh' sciatic nerve transection (axotomy). In control animals the total number of lumbar 5 dorsal root ganglion neurons was about 12000. Seven days after axotomy, 24% of the dorsal root ganglion neurons were lost (P<0.001), and 54% were lost 28 days after axotomy (P<0.001). With regard to the percentage of peptide-expressing neurons, the results obtained showed that both galanin message-associated peptide (from <1% to about 21%) and neuropeptide Y (from <1% to about 16%) are upregulated, whereas calcitonin gene-related peptide is downregulated (from about 41% to about 14%) following axotomy. Results obtained with retrograde labeling of the axotomized dorsal root ganglion neurons indicate that the neuropeptide regulations may be even more pronounced, if the analysis is confined to the axotomized dorsal root ganglion neurons rather than including the entire neuron population. We also applied conventional profile-based counting methods to compare with the stereological data and, although the results were comparable considering the trends of changes following axotomy, the actual percentage obtained with the two methods differed markedly, both for neuropeptide Y- and, especially, for galanin message-associated peptide-positive neurons. These present results demonstrate that marked species differences exist with regard to the effect of nerve injury on dorsal root ganglion neurons. Thus, whereas no neuron loss is seen in rat up to 4 weeks after a 'mid-thigh' transection [Tandrup et al. (2000) J. Comp. Neurol. 422, 172-180], the present results indicate a dramatic loss already after 1 week in mouse. It is suggested that the proximity in physical distance of the lesion to the cell body is a critical factor for the survival of the target-deprived neurons. Finally, stereological methodology seems warranted when assessing the total number of neurons as well as changes in peptide regulations after axotomy in mouse.

Expression and regulation of cholecystokinin and cholecystokinin receptors in rat nodose and dorsal root ganglia.

Cholecystokinin (CCK) is an important satiety factor, acting via the vagus nerve to influence central feeding centers. CCK binding sites have been demonstrated in the vagal sensory nodose ganglion and within the nerve proper. Using in situ hybridization, expression of the CCK(A) and (B) receptors (Rs), as well as of CCK itself, was studied in the normal nodose ganglion (NG), and after vagotomy, starvation and high-fat diet. CCK(A)-R mRNA expression in dorsal root ganglia (DRGs) was also explored. In the NG, 33% of the neuron profiles (NPs) contained CCK(A)-R mRNA and in 9% we observed CCK(B)-R mRNA. CCK mRNA was not found in normal NGs. Peripheral vagotomy decreased the number of CCK(A)-R mRNA-expressing NPs, dramatically increased the number of CCK(B)-R mRNA, and induced CCK mRNA and preproCCK-like immunoreactivity in nodose NPs. No significant differences in the number of NPs labelled for either mRNA species were detected following 48 h food deprivation or in rats fed a high-fat content diet. In DRGs, 10% of the NPs expressed CCK(A)-R mRNA, a number that was not affected by either axotomy or inflammation. This cell population was distinct from neurons expressing calcitonin gene-related peptide mRNA. These results demonstrate that the CCK(A)-R is expressed by both viscero- and somatosensory primary sensory neurons, supporting a role for this receptor as a mediator both of CCK-induced satiety and in sensory processing at the spinal level. The stimulation of CCK and CCK(B)-R gene expression following vagotomy suggests a possible involvement in the response to injury for these molecules.

Intrathecal administration of PNA targeting galanin receptor reduces galanin-mediated inhibitory effect in the rat spinal cord.

Peptide nucleic acids (PNA) are nucleic acid analogues containing neutral amide backbone, forming stable and tight complexes with complementary DNA/RNA. However, it is unclear whether unmodified PNA can efficiently penetrate neuronal tissue in order to act as antisense reagent. Here we show that intrathecal (i.t.) injection of an unmodified antisense PNA complementary to the rat galanin receptor type 1 (GalR1) mRNA is able to block the inhibitory effect of i.t. administered galanin on spinal nociceptive transmission. Autoradiographic ligand binding studies using [125I]galanin show that the unmodified PNA is able to reduce the density of galanin binding sites in the dorsal horn. Thus, unmodified PNA applied i.t. appears to function as an effective antisense reagent in rat spinal cord in vivo.

Effect of peripheral nerve lesion and lumbar sympathectomy on peptide regulation in dorsal root ganglia in the NGF-overexpressing mouse.

Galanin is a peptide normally expressed at low levels both in sensory and in sympathetic neurons. It is strongly upregulated after peripheral nerve lesions, and it has been proposed that nerve growth factor (NGF) plays a role in this regulation. In the present study the effect of both sciatic nerve transection and lumbar sympathectomy on galanin in lumbar dorsal root ganglia (DRGs) was examined in mice overexpressing NGF (NGFOE) in the skin under the keratin promoter. The superior cervical ganglia (SCG) were also studied. In the DRG pericellular baskets containing tyrosine hydroxylase- (TH) and galanin-like immunoreactivity (LI) were found, mostly in the same fibers. Galanin-positive baskets were also found in the trigeminal ganglia. However, only single neuropeptide Y (NPY)-positive baskets were observed within the DRGs. No marked difference in number of galanin-positive neurons was seen between wild-type and NGFOE mice. After sciatic nerve transection galanin was upregulated in DRG neurons to about the same extent in NGFOE mice as in wild-type mice. Galanin-, but not TH-LIs decreased in the pericellular baskets. After lumbar sympathectomy both galanin- and TH-immunoreactive baskets disappeared, suggesting a sympathetic origin. In the SCG the very low galanin mRNA levels were strongly increased after lesion of the carotid nerves, both in wild-type and in NGFOE mice. However, whereas NPY mRNA levels decreased in the SCG after axotomy in the wild-type mice, there was a distinct increase in the NGFOE mice. Our results show that high NGF levels in skin induce formation of pericellular baskets in DRGs expressing galanin- and TH-LI and that galanin in these baskets is strongly influenced by peripheral axotomy. However, overexpression of NGF did not markedly influence galanin expression in DRG neurons, neither normally nor after nerve lesions. Finally, expression of NPY in sympathetic ganglia is differently regulated in NGFOE compared to wild-type mice.

Distribution of alpha2-adrenoceptor mRNAs in the rat lumbar spinal cord in normal and axotomized rats.

Using riboprobe in situ hybridization we have studied the distribution of alpha2A-, alpha2B- and alpha2c-adrenoceptor (AR) mRNAs in the lumbar spinal cord in normal rats and after peripheral axotomy. A strong alpha2A- and alpha2C AR mRNA labelling was found in motoneurons and other cells in the ventral horns. In the dorsal horns strong alpha2A-AR mRNA labelling was found in all layers and in the lateral spinal nucleus, whereas alpha2C-AR mRNA was found in lower numbers of cells in various layers. The alpha2B-AR mRNA signal was only detected in some small cells superficially in the dorsal horn. With regard to axotomy only a marginal effect was observed for alpha2C-AR mRNA in the ventral horn. The results suggest that alpha2-ARs are involved both in sensory and motor processing.

Regulation of galanin and neuropeptide Y in dorsal root ganglia and dorsal horn in rat mononeuropathic models: possible relation to tactile hypersensitivity.

The expression of galanin and neuropeptide Y in rat lumbar 5 (L5) dorsal root ganglia and dorsal horn (L4-5) was studied after four types of peripheral nerve injury using immunohistochemistry and in situ hybridization. The possible correlation between these two peptides and tactile allodynia-like behaviour was analysed as well. The models employed were the Gazelius (photochemical lesion) and Seltzer and Bennett (constriction lesions) models, as well as complete sciatic nerve transection (axotomy). Two weeks after surgery, the Gazelius model rats more frequently displayed a greater tactile allodynia than the rats from the Seltzer and Bennett models. Tactile allodynia was not observed in any of the axotomized rats. A marked increase in the number of galanin-immunoreactive and galanin messenger RNA-positive neuron profiles was observed in ipsilateral dorsal root ganglia in all types of models. The increase in allodynic rats (Gazelius, Seltzer and Bennett models) was less pronounced than that after axotomy. In addition, in the Bennett model the number of galanin-immunoreactive neurons was significantly lower in allodynic rats as compared to non-allodynic rats, and the same tendency, but less obvious was found in the Seltzer model. Furthermore, an increase in galanin-immunoreactive fibres was found in the superficial laminae of the ipsilateral dorsal horn in all lesion models, especially in lamina II. A dramatic increase in the number of neuropeptide Y and neuropeptide Y messenger RNA-positive neuron profiles was also found in the ipsilateral dorsal root ganglia in all models, but no significant difference was found in peptide levels between allodynic and non-allodynic rats in any of the models. The present results suggest that the levels of endogenous galanin may play a role in whether or not allodynia develops in the Bennett model.

Intrathecal galanin alleviates allodynia-like behaviour in rats after partial peripheral nerve injury.

We have previously suggested that the neuropeptides galanin and galanin message-associated peptide (GMAP) may have an inhibitory role in spinal nociception. The present study examined the effects of intrathecal (i.t.) administration of these two peptides on allodynia-like behaviours in response to mechanical and cold stimulation in rats after photochemically induced ischaemic peripheral nerve injury. I.t. galanin significantly alleviated the mechanical- and cold-allodynia-like behaviours in nerve injured rats, and was not associated with motor impairment or sedation. I.t. GMAP relieved mechanical allodynia much less than galanin. I.t. M-35, a high-affinity galanin receptor antagonist, did not significantly alter the response of the rats to mechanical or cold stimulation. At 1 or 2 weeks postinjury, around 15% of dorsal root ganglion (DRG) neuron profiles showed galanin-like immunoreactivity. These profiles were mostly small sized. Although the number of galanin positive cells was thus increased in the DRG in the present model, the increase was substantially less than after complete sciatic nerve section, as previously shown. The present results showed that spinal administration of galanin inhibited some abnormal pain-like behaviours in rats after partial peripheral nerve injury. These results further support an inhibitory function for galanin in nociception. However, endogenous galanin may not play a significant role in suppressing nociceptive input after partial ischaemic peripheral nerve injury, as the upregulation of galanin is moderate.

Effect of peripheral axotomy on dorsal root ganglion neuron phenotype and autonomy behaviour in neuropeptide Y-deficient mice.

The lumbar 5 (L5) dorsal root ganglia (DRGs) were studied in neuropeptide tyrosine (NPY)-deficient (-/-) and wild type (+/+) mice after unilateral sciatic nerve transection using in situ hybridization and immunohistochemistry. NPY, galanin and two NPY receptors (Y-Rs) were analyzed as well as self-mutilation behaviour (autotomy) and nociceptive thresholds. No difference between wild type and NPY-deficient mice was seen in the tail-flick or hot plate test. However, -/- mice showed a much stronger autotomy behaviour than wild type mice. NPY was not found in L5 DRGs in -/- mice, not even after axotomy. Galanin was upregulated to the same extent after axotomy in NPY-deficient and wild type mice. Y1- and Y2-R mRNAs were found mainly in small DRG neuron profiles. Both receptor mRNAs were downregulated after axotomy, to about the same extent in NPY-deficient as in wild type mice. In control and contralateral ganglia the mRNA levels of both receptors were lower in NPY-deficient mice than in wild type mice. The contralateral Y2-R mRNA levels did not reach control values in the NPY-deficient mice, as they did in the wild type mice. In both strains the Y1-R protein was decorating the somatic plasmalemma. The present results suggest that lack of NPY may cause exaggerated autotomy, a self-mutilation behaviour possibly related to pain sensation, in agreement with the described analgesic effect of NPY. Although significant differences in levels of Y1- and especially Y2-R mRNAs were observed between wild type and NPY-deficient mice, they were only moderate. These findings suggest that expression, regulation, localization and possible function of Y1- and Y2-Rs are not dependent on presence of the endogenous ligand. Also, deletion of NPY does not seem to influence the expression of the partly coexisting peptide galanin.

Down-regulation of mu-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy.

To understand the role of opioids and their receptors in chronic pain following peripheral nerve injury, we have studied the mu-opioid receptor in rat and monkey lumbar 4 and 5 dorsal root ganglion neurons and the superficial dorsal horn of the spinal cord under normal circumstances and after peripheral axotomy. Our results show that many small neurons in rat and monkey dorsal root ganglia, and some medium-sized and large neurons in rat dorsal root ganglia, express mu-opioid receptor-like immunoreactivity. Most of these neurons contain calcitonin gene-related peptide. The mu-opioid receptor was closely associated with the somatic plasmalemma of the dorsal root ganglion neurons. Both mu-opioid receptor-immunoreactive nerve fibers and cell bodies were observed in lamina II of the dorsal horn. The highest intensity of mu-opioid receptor-like immunoreactivity was observed in the deep part of lamina II. Most mu-opioid receptor-like immunoreactivity in the dorsal horn originated from spinal neurons. A few mu-opioid receptor-positive peripheral afferent terminals in the rat and monkey dorsal horn were calcitonin gene-related peptide-immunoreactive. In addition to pre- and post-junctional receptors in rat and monkey dorsal horn neurons, mu-opioid receptors were localized on the presynaptic membrane of some synapses of primary afferent terminals in the monkey dorsal horn. Peripheral axotomy caused a reduction in the number and intensity of mu-opioid receptor-positive neurons in the rat and monkey dorsal root ganglia, and of mu-opioid receptor-like immunoreactivity in the dorsal horn of the spinal cord. The decrease in mu-opioid receptor-like immunoreactivity was more pronounced in the monkey than in the rat dorsal root ganglia and spinal cord. It is probable that there was a parallel trans-synaptic down-regulation of mu-opioid-like immunoreactivity in local dorsal horn neurons of the monkey. These data suggest that one factor underlying the well known insensitivity of neuropathic pain to opioid analgesics could be due to a marked reduction in the number of mu-opioid receptors in the axotomized sensory neurons and in interneurons in the dorsal horn of the spinal cord.

Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors.

By using immunofluorescence methodology, extensive galanin (GAL) and GAL message-associated peptide (GMAP)-positive terminal networks were observed in the hippocampal formation. The majority of the GAL/GMAP fibers were dopamine beta-hydroxylase- (DBH) positive, that is, they were noradrenergic. This finding was established with GAL/GMAP-DBH double-staining and with 6-hydroxy-dopamine treatment, which totally abolished all fibers in which GAL/GMAP and DBH coexisted. Also, reserpine treatment caused a marked depletion of GAL. No evidence for GAL/GMAP coexistence with 5-hydroxytryptamine was obtained. In the ventral hippocampus, GAL/GMAP-, DBH-negative fibers were seen in the stratum oriens, the anterior stratum radiatum, along the granule cell layer and in the strata oriens and alveus. In the locus coeruleus (LC), around 80% of the GMAP-positive neurons contained neuropeptide tyrosine (NPY), and about 40% of the NPY-positive neurons expressed GMAP. GAL-R1 receptor mRNA was expressed in Barrington's nucleus (close to the LC), but was not detected in the hippocampal formation/dorsal cortical areas. GAL-R2 receptor mRNA was found in the granule cell layer in the dentate gyrus. The present results show that most, but not all, immunohistochemically detectable GAL/GMAP in the hippocampal formation/dorsal cortex is present in noradrenergic nerve terminals originating in the LC, which has a robust GAL/GMAP synthesis. The functional role of GAL may be related to noradrenaline, possibly by a presynaptic action. However, the presence of GAL in other systems and of GAL-R2 receptor mRNA in granule cells also indicates other targets.

Galanin in ascending systems. Focus on coexistence with 5-hydroxytryptamine and noradrenaline.

Galanin can be synthesized in several ascending systems including cholinergic forebrain neurons, serotonergic dorsal raphe neurons, and the noradrenergic locus coeruleus system. Recent immunohistochemical studies suggest that of these three systems, the locus coeruleus neurons express the highest levels of galanin and that in cortex and hippocampus galanin peptide can only be detected in the noradrenergic projections. Electrophysiologic studies show that galanin hyperpolarizes both serotonergic dorsal raphe neurons and noradrenergic locus coeruleus neurons at fairly high concentrations (10(6)-10(-7) M). In addition, galanin at low concentrations (10(-9) M) enhances the 5-HT- and noradrenaline-induced hyperpolarization. Consequently, a galanin antagonist could attenuate an inhibitory tone on both dorsal raphe and locus coeruleus neurons and thus perhaps exert antidepressant activity.

Regulation of expression of galanin and galanin receptors in dorsal root ganglia and spinal cord after axotomy and inflammation.

Galanin can normally be detected only in a few dorsal root ganglion (DRG) neurons, but it is dramatically upregulated after peripheral nerve injury in both rat and monkey. Galanin is stored in large dense core vesicles, which after axotomy are often found close to the membrane of afferent nerve endings in the dorsal horn. In the monkey there is an increase in galanin in many nerve terminals in the superficial dorsal horn after axotomy, but such an increase is more difficult to detect in the rat. Galanin is also present in local dorsal horn neurons, where it is upregulated by peripheral inflammation. Both galanin-R1 and galanin-R2 receptor mRNAs are expressed in rat DRGs, mainly in, respectively, large and small DRG neurons. Galanin-R1 receptor mRNA is downregulated in DRG neurons after axotomy, and a small decrease in galanin-R2 receptor mRNA levels can also be seen. After peripheral tissue inflammation galanin-R1 receptor mRNA levels decrease and galanin-R2 receptor mRNA levels increase. The present results show that galanin and galanin receptors are present in sensory and local dorsal horn neurons and are regulated by nerve injury and inflammation. Galanin may therefore be involved in processing of pain information, primarily exerting analgesic effects. Whereas local dorsal horn neurons represent a defense system against inflammatory pain, we have proposed that a second defense system, against neuropathic pain, is intrinsic to DRG neurons.

Expression and regulation of galanin-R2 receptors in rat primary sensory neurons: effect of axotomy and inflammation.

Using in situ hybridization, we studied galanin-R2 receptor (GAL-R2-R) mRNA in rat lumbar 5 dorsal root ganglia (DRGs) at different time points after peripheral tissue inflammation and sciatic nerve transection (axotomy). About 25% of all normal DRG neuron profiles were GAL-R2-R mRNA-positive, and the majority was of the small type. In normal DRGs GAL-R2-R mRNA often (approximately 80%) colocalized with CGRP mRNA and sometimes (approximately 20%) with GAL-R1-R mRNA. There was a strong increase in the number and labeling intensity of GAL-R2-R mRNA-positive neuron profiles after peripheral tissue inflammation with a peak at 3 days, as well as a long-lasting decrease after axotomy. These results, together with the previously shown regulation of GAL and the GAL-R1-R, suggest that GALergic mechanisms participate in complex adaptive responses in DRGs after inflammation and nerve injury.

Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos.

Using in situ hybridization and immunohistochemistry the expression of, respectively, prepro-galanin (pre-pro-GAL) mRNA and GAL receptor-1 mRNA, as well as GAL-like and GAL message-associated peptide-like immunoreactivities, were studied in rats from embryonic day 14 (E14) to postnatal day 1. GAL expression was observed already at E14 in trigeminal and dorsal root ganglion neurons and at E15 in the sensory epithelia in developing ear, eye, and nose, as well as at E19 during bone formation. Also, GAL receptor-1 mRNA was expressed in the sensory ganglia of embryos but appeared later than the ligand. These findings suggest that GAL and/or GAL message-associated peptide may have a developmental role in several sensory systems and during bone formation.

Evidence for galanin receptors in primary sensory neurones and effect of axotomy and inflammation.

Using in situ hybridization, mRNA coding for the galanin R1 receptor (GAL-R1) was demonstrated in > 20% of all lumbar 4 and 5 dorsal root ganglion neurone profiles, mainly constituting small and medium sized neurones. Almost all of these neurones expressed CGRP mRNA as revealed by analysis of adjacent sections and of the same section using a double-labelling technique. GAL-R1 mRNA levels were down-regulated transiently by inflammation, and more strongly by peripheral nerve injury. These results suggest that DRG neurones are sensitive to galanin and that GAL-R1 receptors are involved in sensory processing.