David Wynick

Distribution of galaninergic immunoreactivity in the brain of the mouse

The distribution of galaninergic immunoreactive (‐ir) profiles was studied in the brain of colchicine‐pretreated and non‐pretreated mice. Galanin (GAL)‐ir neurons and fibers were observed throughout all encephalic vesicles. Telencephalic GAL‐ir neurons were found in the olfactory bulb, cerebral cortex, lateral and medial septum, diagonal band of Broca, nucleus basalis of Meynert, bed nucleus of stria terminalis, amygdala, and hippocampus. The thalamus displayed GAL‐ir neurons within the anterodorsal, paraventricular, central lateral, paracentral, and central medial nuclei. GAL‐ir neurons were found in several regions of the hypothalamus. In the midbrain, GAL‐ir neurons appeared in the pretectal olivary nucleus, oculomotor nucleus, the medial and lateral lemniscus, periaqueductal gray, and the interpeduncular nucleus. The pons contained GAL‐ir neurons within the dorsal subcoeruleus, locus coeruleus, and dorsal raphe. In the medulla oblongata, GAL‐ir neurons appear in the anterodorsal and dorsal cochlear nuclei, salivatory nucleus, A5 noradrenergic cells, gigantocellular nucleus, inferior olive, solitary tract nucleus, dorsal vagal motor and hypoglossal nuclei. Only GAL‐ir fibers were seen in the lateral habenula nucleus, substantia nigra, parabrachial complex, cerebellum, spinal trigeminal tract, as well as the motor root of the trigeminal and facial nerves. GAL‐ir was also observed in several circumventricular organs. The widespread distribution of galanin in the mouse brain suggests that this neuropeptide plays a role in the regulation of cognitive and homeostatic functions. J. Comp. Neurol. 434:158–185, 2001.

Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury

The neuropeptide galanin has been identified as a potential neurotransmitter/neuromodulator within the central nervous system. In the present study, the role of endogenous galanin in nociceptive processing in the nervous system has been analysed by using mice carrying a targeted mutation in the galanin gene. Supporting this, the effect of chronic administration of exogenous galanin on nociceptive sensory inputs has been assayed in adult rats. In the absence of peripheral nerve injury, the sensitivity to threshold noxious stimuli is significantly higher in galanin mutant mice than wild‐type controls. Following peripheral nerve injury, in conditions under which endogenous galanin levels are elevated, spontaneous and evoked neuropathic pain behaviours are compromised in mutant mice. Conversely, chronic intrathecal delivery of exogenous galanin to nerve‐intact adult rats is associated with persistent behavioural hypersensitivity, a significant increase in c‐fos expression and an increase in PKCγ immunoreactivity within the spinal cord dorsal horn. The present results demonstrate that a relationship exists between the degree of nerve injury‐induced galanin expression and the degree of behavioural hypersensitivity, and show that galanin may play a role in nociceptive processing in the spinal cord, with interrelated inhibitory and excitatory effects.

Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage.

Expression of the neuropeptide galanin is up‐regulated in many brain regions following nerve injury and in the basal forebrain of patients with Alzheimers disease. We have previously demonstrated that galanin modulates hippocampal neuronal survival, although it was unclear which receptor subtype(s) mediates this effect. Here we report that the protective role played by galanin in hippocampal cultures is abolished in animals carrying a loss‐of‐function mutation in the second galanin receptor subtype (GalR2‐MUT). Exogenous galanin stimulates the phosphorylation of the serine/threonine kinase Akt and extracellular signal‐regulated kinase (ERK) in wild‐type (WT) cultures by 435 ± 5% and 278 ± 2%, respectively. The glutamate‐induced activation of Akt was abolished in cultures from galanin knockout animals, and was markedly attenuated in GalR2‐MUT animals, compared with WT controls. In contrast, similar levels of glutamate‐induced ERK activation were observed in both loss‐of‐function mutants, but were further increased in galanin over‐expressing animals. Using specific inhibitors of either ERK or Akt confirms that a GalR2‐dependent modulation in the activation of the Akt and ERK signalling pathways contributes to the protective effects of galanin. These findings imply that the rise in endogenous galanin observed either after brain injury or in various disease states is an adaptive response that reduces apoptosis by the activation of GalR2, and hence Akt and ERK.

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.

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.

Symptomatic Secondary Hormone Syndromes in Patients with Established Malignant Pancreatic Endocrine Tumors

Over a five-year period, we measured concentrations of gut hormones in plasma samples from 353 patients in whom diagnoses of pancreatic endocrine tumors were subsequently confirmed. A median of 19 months (range, 7 to 120) after the initial diagnosis, 24 of these patients (6.8 percent) had elevated concentrations of other hormones in association with new clinical symptoms. In 13 of these patients (8 with glucagonomas, 3 with tumors secreting vasoactive intestinal polypeptide, and 2 with insulinomas), hypergastrinemia developed along with the clinical features of a gastrinoma; 5 patients died of gastrointestinal perforation or bleeding, apparently caused by this second tumor. We conclude that patients with pancreatic endocrine tumors, regardless of their initial clinical picture, require continued surveillance for new elevations of hormones.

The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal.

Much research has focused on pathways leading to opiate addiction. Pathways opposing addiction are more difficult to study but may be critical in developing interventions to combat drug dependence and withdrawal. Galanin decreases firing of locus coeruleus neurons, an effect hypothesized to decrease signs of opiate withdrawal. The current study addresses whether galanin affects morphine withdrawal signs by using a galanin agonist, galnon, that crosses the blood–brain barrier, and mice genetically engineered to under- or overexpress galanin peptide. Galnon significantly decreased morphine withdrawal signs in C57BL/6 mice. Further, knockout mice lacking galanin showed exacerbated morphine withdrawal signs, suggesting that endogenous galanin normally counteracts opiate withdrawal. Transgenic mice overexpressing galanin in noradrenergic neurons also showed decreased morphine withdrawal signs, suggesting a possible neuroanatomical locus for these effects of galanin. Both c-fos immunoreactivity, a marker of neuronal activity, and phosphorylation of tyrosine hydroxylase at Ser-40, a marker of cAMP levels, are decreased in the locus coeruleus by galnon treatment after morphine withdrawal, suggesting a possible molecular mechanism for the behavioral effects of galanin. These studies suggest that galanin normally acts to counteract opiate withdrawal and that small molecule galanin agonists could be effective in diminishing the physical signs of withdrawal.

Galanin acts as a trophic factor to the central and peripheral nervous systems.

The neuropeptide galanin is widely, but not ubiquitously, expressed in the adult nervous system. Its expression is markedly up-regulated in many neuronal tissues after nerve injury or disease. Over the last 10 years, we have demonstrated that the peptide plays a developmental survival role to subsets of neurons in the peripheral and central nervous systems with resulting phenotypic changes in neuropathic pain and cognition. Galanin also appears to play a trophic role to adult sensory neurons following injury, via activation of GalR2, by stimulating neurite outgrowth. Furthermore, galanin also plays a neuroprotective role to the hippocampus following excitotoxic injury, again mediated by activation of GalR2. Most recently, we have shown that galanin expression is markedly up-regulated in multiple sclerosis (MS) lesions and in the experimental autoimmune encephalomyelitis (EAE) model of MS. Over-expression of galanin in transgenic mice abolishes disease in the EAE model, whilst loss-of-function mutations in galanin or GalR2 increase disease severity. In summary, these studies demonstrate that a GalR2 agonist might have clinical utility in a variety of human diseases that affect the nervous system.

Microdialysis in freely moving mice: determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice

In the present study, we describe micro-surgical methods for simultaneous implantation of a microdialysis probe and an intraventricular injection cannula via their respective guide cannulas into the mouse brain. Basal and stimulated release of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (NA) was determined in the ventral hippocampus of freely moving mice. NA and 5-HT were determined in one run by a newly developed HPLC method based on precolumn derivatization with benzylamine and fluorescence detection. The mice with a loss-of-function mutation of the galanin gene (KO) and the mice that over-expressed galanin (OE) were studied. No significant differences in basal, potassium-stimulated or scopolamine-induced extracellular ACh levels were observed in 4-month-old wild-type (WT) and KO mice. In the aged, 10-month-old animals, the basal extracellular ACh levels were significantly reduced in both WT and KO groups. Galanin (1 nmol i.c.v.) caused a significant reduction of basal extracellular NA by about 40% in both WT and galanin OE mice, however, in the latter group the effect was delayed by almost 2 h. A 10-min forced swimming stress caused a higher increase in release of NA and 5-HT in the OE group than in the corresponding WT mice. Finally, venlafaxin (10 mg/kg i.p.) increased extracellular NA to 400% of the control values in the CBA mice, but only to 250% in the C57BL mice. It is concluded that galanin may play an important role in the cholinergic mechanisms underlying cognitive disorders. Furthermore, modulation by galanin and by behavioral activation, of NA and 5-HT neurotransmission in galanin over-expressing mice indicates its possible role in the aetiology of mood disorders.

The role of galanin as a multi-functional neuropeptide in the nervous system.

The neuropeptide galanin is expressed developmentally in the DRG and is rapidly up-regulated 120-fold after peripheral nerve section in the adult. The generation and study of galanin knockout mice has indicated that the peptide is critical to the development and function of specific subsets of neurons in the central and peripheral nervous system. These data have important implications for the understanding, and potential therapeutic treatment, of sensory neuropathies and a number of neurological diseases, including Alzheimers disease and epilepsy.