Marc Landry

Subtypes Y1 and Y2 of the Neuropeptide Y Receptor Are Respectively Expressed in Pro-Opiomelanocortin- and Neuropeptide-Y-Containing Neurons of the Rat Hypothalamic Arcuate Nucleus

The arcuate nucleus of the hypothalamus houses a number of neurochemically different cell populations. Among these, a dense cluster of small neuropeptide-Y (NPY)-expressing neurons is located in its ventromedial subdivision and a pro-opiomelanocortin (POMC)-expressing neuron population in its ventrolateral part. Furthermore, both neuropeptide Y Y1 and Y2 receptors (Y1-Rs and Y2-Rs) are expressed in the arcuate nucleus. Here we analyse the co-expression of NPY and POMC/adrenocorticotropic hormone with the Y1-R and Y2-R in arcuate neurons using immunohistochemistry and in situ hybridization. Many, but not all, POMC neurons expressed Y1-R mRNA and protein. Conversely, several Y1-R-positive, POMC-negative neurons were found. NPY-positive nerve terminals were found in close apposition to Y1-R-like immunoreactivity localized close to the dendritic and somatic cell membranes. Y2-R mRNA was found in almost all NPY mRNA-expressing neurons, but also in a group of NPY mRNA-negative cells. These results show that the POMC neurons are targets for NPY, which is presumably present in, and released from, fibres originating in the ventromedial arcuate nucleus and which may play a role in NPY-induced feeding. Release of NPY, and possible coexisting messengers, may be controlled by presynaptic Y2-R expressed in NPY neurons. Taken together, the findings support the division of Y1-Rs and Y2-Rs into post- and presynaptic receptors, respectively.

Neuropeptide Y: some viewpoints on a multifaceted peptide in the normal and diseased nervous system

Using immunohistochemical and in situ hybridization methodologies the localization of neuropeptide tyrosine (NPY) and two of its receptors, the Y1- and the Y2-receptor (R), has been analysed in various tissues in normal animals and animals subjected to different experimental procedures as well as animals with a genetic and an acquired disease. (1) Dorsal root ganglion (DRG) neurons are discussed with special focus on the effect of peripheral nerve injury. In normal DRG neurons NPY cannot be detected, whereas Y1-R mRNA and Y1-R-like immunoreactivity (LI) are strongly expressed. The Y1-Rs decorate the membrane of the cell soma and are not transported peripherally into the axonal branches. Y2-R mRNA levels are low. After axotomy there is a marked increase in NPY, a decrease in Y1-Rs and an increase in Y2-Rs. The Y2-R is transported centrifugally. These findings suggest that NPY-ergic mechanisms participate in the adaptive changes of sensory neurons in response to injury. (2) Using specific antibodies the cellular and subcellular localization of the Y1-R protein have been analysed in cerebral blood vessels. The results demonstrate high concentrations of receptors in smooth muscle cells around pial arterioles with lower numbers in large vessels on the basal surface of the brain. In many regions the receptors disappear after the arterioles have entered the brain tissue. At the ultrastructural level the receptors are found both on the endothelial and peripheral side of the muscle cells as well as laterally, where muscle cells oppose each other. The receptor protein is often associated with small vesicles. No NPY-positive nerve fibers were found around the Y1-R-rich arterioles, but they were only seen around the arteries with low Y1-R levels. The Y1-R-rich arterioles were, however, seen close to numerous NPY-positive fibers originating from central interneurons. These findings raise the possibility that centrally originating NPY can influence cerebral blood flow, possibly by stimulating NPY-Rs on the peripheral side of the muscle cells. However, also blood borne NPY, released under special conditions, such as stress from sympathetic nerves and the adrenal medulla and transported with blood, may stimulate receptors on the endothelial side of the smooth muscle cells. (3) In the arcuate nucleus Y1- and Y2-Rs are found, whereby the Y1-Rs are located in its ventro-medial portion and co-localized with POMC peptides, and the Y2-R in its ventromedial part, partly co-localized with NPY. NPY nerve endings makes synaptic contact with the POMC/Y1-R-positive neurons. In a mouse model for genetic anorexia very high levels of NPY were observed in arcuate neurons as compared to control mice. However, NPY mRNA levels were not different between the two groups. Taken together these findings are in good agreement with the view that NPY in the arcuate nucleus plays an important role in regulating feeding behaviour. (4) After intracerebral prion inoculation in mice an upregulation of NPY mRNA levels was observed in CA3 pyramidal neurons, and this effect was seen at a time point just before the first behavioural symptoms were manifested. At approximately the same time there was a dramatic decrease in Y2-R binding in strata oriens and radiatum of the CA1 region of the hippocampus, whereas in other regions no changes or much smaller changes were observed. Also, there was only a very slight decrease in Y2-R mRNA levels in CA3 neurons. It thus appears as if the prion disease prevents ligand binding to the Y2-R, perhaps by influencing traffic of receptor proteins, possibly at the level of cell membrane-associated caveolae, which have been implicated in the conversion of normal protein to scrapie protein. It is possible that these changes in NPY-ergic mechanisms may underlie some of the central symptoms associated with the prion disease. (ABSTRACT TRUNCATED)

Effect of Axotomy on Expression of NPY, Galanin, and NPY Y1 and Y2 Receptors in Dorsal Root Ganglia and the Superior Cervical Ganglion Studied with Double-Labeling in Situ Hybridization and Immunohistochemistry

Using double-labeling techniques for both in situ hybridization and immunohistochemistry some peptides and peptide receptors were studied quantitatively in a sensory and a sympathetic ganglion after axotomy. In the lumbar 5 dorsal root ganglion (DRG) normally no neuropeptide Y- and only a few galanin-positive cell bodies are seen. Following complete transection of the sciatic nerve around 60% of all neuropeptide Y (NPY) neuron profiles (NPs) were galanin positive (+) and 33-44% of all galanin NPs were NPY(+). A good agreement between immunohistochemistry and in situ hybridization was observed for NPY and galanin. NPY Y1- and Y2-receptor (R) mRNAs were found in around 40% of all NPY mRNA(+) NPs, and more than half of the Y1-R mRNA(+) NPs and two-thirds of the Y2-R mRNA(+) NPs were NPY(+). In addition, more than one-third of the galanin mRNA-containing NPs showed colocalization with NPY receptor mRNAs and up to 70% of the Y2-R mRNA(+) NPs also expressed galanin mRNA. In the control superior cervical ganglion (SCG) 10% of the NPY(+) NPs were Y2-R mRNA(+), and 85% of the Y2-R(+) NPs were NPY mRNA(+), and the corresponding percentages after axotomy were around 35 and 45%, respectively. Following axotomy of the carotid nerves around half of all NPY(+) NPs were galanin(+), and conversely around 50% of all galanin NPs were NPY(+) at the mRNA level, whereas much lower percentages (15 and 9%, respectively) were observed with immunohistochemistry. These results demonstrate that double-labeling procedures are valid tools to quantitatively evaluate coexistence situations in sensory and sympathetic ganglia, showing a high degree of coexistence for NPY and galanin in axotomized neurons both in the lumbar 5 DRG and in the SCG. However, the immunohistochemical analysis in the SCG demonstrated much lower numbers of peptide-positive neurons than seen with in situ hybridization, suggesting that the latter technique is more sensitive. The fact that a considerable number of neurons express NPY together with Y1- and/or Y2-Rs indicates that both receptors may act as autoreceptors, the Y1-R presumably at the level of the cell body and the Y2-R on nerve terminals in the dorsal horn and/or the periphery. The present results also show that in both sensory and sympathetic neurons there is a strong upregulation of the Y2-R after nerve injury, suggesting a possible role in trophic and regenerative events.

Regulation of Expression of Galanin and Galanin Receptors in Dorsal Root Ganglia and Spinal Cord after Axotomy and Inflammation a

Abstract: 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 ner ve terminals in the superficial dorsal horn after axotomy, but such an inc rease 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 axo‐tomy, 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.

Galanin-R1 receptor in anterior and mid-hypothalamus: distribution and regulation.

The distribution and regulation of galanin‐R1 receptor (GAL‐R1‐R) mRNA has been studied in the anterior and mid‐diencephalon by using in situ hybridization. Moreover, possible colocalization of GAL‐R1‐R mRNA and prepro‐galanin or vasopressin mRNAs has been analyzed at the cellular level using double in situ hybridization methodology. Many nuclei in the hypothalamus expressed GAL‐R1‐R mRNA, including the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Strong expression was also seen in the same sections in various areas outside of the diencephalon. The distribution patterns are similar to those described in earlier studies. Double labeling experiments showed GAL‐R1‐R mRNA in vasopressin neurons in the PVN and SON. Moreover, GAL‐R1‐R mRNA and prepro‐galanin mRNA were colocalized in several hypothalamic nuclei. GAL‐R1‐R mRNA levels showed a high degree of plasticity. Thus, salt loading resulted in a marked increase in GAL‐R1‐R mRNA levels in the PVN and SON and a moderate decrease was seen during lactation. In contrast, hypophysectomy caused a decrease in GAL‐R1‐R mRNA levels. Differential effects of colchicine were recorded with a decrease of GAL‐R1‐R mRNA in the magnocellular hypothalamic neurons. After salt loading or during lactation, GAL‐R1‐R mRNA and prepro‐galanin mRNA were regulated in parallel, whereas their levels changed in opposite directions after hypophysectomy and colchicine injection. In conclusion, GAL‐R1‐Rs are present in several hypothalamic nuclei, partly in neurons synthesizing galanin. The receptors are regulated in a specific fashion in the various nuclei, depending on the stimulus applied. The results suggest that the effect of galanin in the hypothalamus partly depends on the state of receptor expression. J. Comp. Neurol. 399:321–340, 1998.

Subcellular localization of preprogalanin messenger RNA in perikarya and axons of hypothalamo-posthypophyseal magnocellular neurons : An in situ hybridization study

The subcellular compartmentalization and axonal transport of oxytocin and vasopressin messenger RNAs have recently been reported in the rat hypothalamo-posthypophyseal system using in situ hybridization. So far, no data are available concerning the intracellular distribution of co-localized peptide transcripts, for example of galanin, which is synthesized in the vasopressinergic magnocellular neurons of the rat and which is up-regulated in these neurons under different conditions, including salt loading and colchicine injection. In the present study, using non-radioactive in situ hybridization and immunohistochemistry at the light and electron microscope levels, preprogalanin messenger RNA and galanin-like immunoreactivity were localized in the hypothalamo-posthypophyseal system. After salt loading, preprogalanin transcripts were found throughout the perikaryal cytoplasm, especially in the peripheral cytoplasm and in the perinuclear area. Since immunohistochemistry also showed galanin-like immunoreactivity preferentially in the perinuclear area of control rats, galanin synthesis may occur mainly in this cytoplasmic domain. Preprogalanin messenger RNA was also clustered in dendrites containing rough endoplasmic reticulum. The use of a new in situ hybridization method involving tyramide signal amplification, based on catalysed reporter deposition, allowed visualization of preprogalanin messenger RNA in axonal projections running through the internal layer of the median eminence after salt loading, but not in control or in colchicine-injected animals. The negative results obtained after colchicine injection indicate that the mechanism of messenger RNA transport may require an intact cytoskeleton. The labelling was found in non-dilated axon segments as well as in a subset of axonal swellings in the rostral aspect of the median eminence, but was restricted to a few swellings in its caudal part, with no labelling in the posterior pituitary. Thus, preprogalanin messenger RNA was segregated in the axons. The functional significance of messenger RNAs exportation into axons is not known, but our results suggest that this phenomenon may not be limited to the two principal magnocellular hormone messenger RNAs, but may also involve co-existing peptide messenger RNAs.

Galanin expression in adult human dorsal root ganglion neurons: Initial observations

Human dorsal root ganglia (DRGs) were obtained during various procedures and processed for single and double in situ hybridisation using oligonucleotide probes complementary to three peptide mRNAs. Some postmortem ganglia were also analysed. In donor (unlesioned) DRGs 12.5% of the neuron profiles (NPs) were galanin mRNA-positive (mRNA(+)), 47.5% calcitonin gene-related peptide (CGRP) mRNA(+) and 32.7% substance P mRNA(+). The corresponding percentages for cervical/thoracic DRGs from patients suffering from severe brachial plexus injury were 32.8%, 57.4% and 34.5%, respectively. In these DRGs a high proportion of the galanin mRNA(+) NPs contained CGRP mRNA and substance P mRNA. In DRGs from a patient with migraine-like pain a comparatively small proportion expressed galanin, whereas in DRGs from a herpes zoster patient galanin mRNA(+) NPs were comparatively more frequent. The results from human postmortem DRGs revealed only weak peptide mRNA signals. The present results demonstrate that galanin is expressed in DRGs not only in a number of animal species including monkey as previously shown, but also in a considerable proportion of human DRG neurons, often together with CGRP and substance P, and mostly in small neurons. Thus, galanin may play a role in processing of sensory information, especially pain, in human DRGs and dorsal horn. However, to what extent a similarly dramatic upregulation of galanin expression can be seen after peripheral nerve lesion in man, as has been reported for rat, mouse and monkey, remains to be analysed.

The effect of NGF, BDNF and bFGF on expression of galanin in cultured rat dorsal root ganglia.

Peripheral nerve injury causes a marked change in expression of the neuropeptide galanin in dorsal root ganglion (DRG) neurons. We have used DRG cell cultures to study whether growth factors, especially nerve growth factor (NGF), play a role in this regulation. Adult rat DRG cultures seem to represent a suitable model for this study, since the neurons are axotomized during culture preparation and are known to survive independently of added neurotrophic factors. The effect of NGF, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) was studied on the expression of galanin and galanin message-associated peptide (GMAP)-like immunoreactivities using immunohistochemistry, as well as of prepro-galanin (ppGAL) mRNA levels using radioactive and non-radioactive in situ hybridization. The results show that 100, but not 20 or 50 ng/ml NGF, as well as 10 ng/ml BDNF cause a 40% decrease in the number of GMAP expressing neurons in 72 h cell cultures. A 50% decrease was observed after treatment with 10 ng/ml bFGF. The high dose needed and the modest effect suggest that NGF is not a major factor involved in galanin regulation, whereas BDNF and bFGF may have a role. Moreover, the strong upregulation of galanin/GMAP and ppGAL mRNA levels in the untreated cultures indicates that DRG neurons in vitro have a phenotype similar to DRG neurons after axotomy, i.e. a phenotype distinctly different from normal DRG neurons.

Cloning of a second nm23-M1 cDNA: expression in the central nervous system of adult mouse and comparison with nm23-M2 mRNA distribution

Nm23 has been identified as a gene family encoding different isoforms of the nucleoside diphosphate kinase. This protein is a key enzyme in the control of cellular concentrations of nucleoside triphosphates. Moreover, it has been shown to play important roles in various cellular functions such as differentiation and metastasis. In the present study, a second cDNA for nucleoside diphosphate kinase A (Nm23-M1) was isolated from a cDNA library of mouse embryonic stem cells. This clone encodes the same putative 152 aminoacids long protein as an already published cDNA but is longer in both its 5 and 3 untranslated regions. Tissue and cellular distribution of nm23-M1 mRNA was investigated by using Northern blot analysis and in situ hybridization. Nm23-M1 transcripts were found to be widely distributed throughout the mouse central nervous system with prominent expression in several restricted areas. No differences were noticed between the distribution of long and short transcripts. Furthermore, a similar pattern of expression was described in the central nervous system for nm23-M2 mRNA, encoding a second isoform of the nucleoside diphosphate kinase. However, the transcript of this isoform displayed a wider distribution and was expressed in all organs analysed by northern blotting. The possible involvement of nm23-M1 in differentiation of mouse nervous system is further discussed.

Leukemia inhibitory factor regulates galanin/galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology

After transection of the sciatic nerve there is a dramatic increase in both galanin/galanin message-associated peptide-like immunoreactivities and preprogalanin messenger RNA levels in rat and mouse lumbar 4 and 5 dorsal root ganglion neurons. There is strong evidence that after nerve injury leukemia inhibitor factor is a key molecule in the control of peptide expression both in sympathetic neurons and in dorsal root ganglion neurons, although the cells of origin of endogenous leukemia inhibitory factor remain to be established. We have therefore studied the effect of leukemia inhibitory factor on galanin expression in 72 h cultured dorsal root ganglion neurons from normal mice, leukemia inhibitory factor-deficient and heterozygous mice with immunohistochemistry and in situ hybridization. In cultures of leukemia inhibitory factor-deficient (-/-) mice only 13% of the dorsal root ganglion neurons expressed galanin message-associated peptide and in cultures from heterozygous (+/-) and wild-type (+/+) mice the corresponding figures were, respectively, 24 and 40%. After addition of leukemia inhibitory factor (10 or 50 ng/ml) to the culture medium, the number of neurons expressing galanin message-associated peptide was increased (up to 41%) in cultures from (-/-) animals after the high concentration and reached similar values in cultures from heterozygous animals incubated with the low concentration. These findings were supported by parallel analysis of prepro-galanin messenger RNA levels, where similar transcript levels and effects in the various cultures were observed in the non-radioactive in situ hybridization experiments. These results support the hypothesis that leukemia inhibitory factor is an important regulator of galanin/galanin message-associated peptide expression following axotomy, and may therefore be involved in the defence mechanisms against neuropathic pain at the level of dorsal root ganglion neurons.