Isabella Gavazzi

Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro

Different subpopulations of adult primary sensory neurons in the dorsal root ganglia express receptors for different trophic factors, and are therefore potentially responsive to distinct trophic signals. We have compared the effect of the neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and NT‐3, and of glial cell line‐derived neurotrophic factor (GDNF) on neurite outgrowth in dissociated cultures of sensory neurons from the lumbar ganglia of young adult rats, and attempted to establish subset‐specific effects of these trophic factors. We analysed three parameters of neurite growth (percentage of process‐bearing neurons, length of longest neurite and total neurite length), which may correlate with particular types of axon growth in vivo, and may therefore respond differently to trophic factor presence. Our results showed that percentage of process‐bearing neurons and total neurite length were influenced by trophic factors, whilst the length of the longest neurite was trophic factor independent. Only NGF and GDNF were found to enhance significantly the proportion of process‐bearing neurons in vitro. GDNF was more effective than NGF on small, IB4– neurons, which are known to develop GDNF responsiveness early in postnatal development. NGF, and to a much lesser extent GDNF, enhanced the total length of the neurites produced by neurons in culture. BDNF exerted an inhibitory effect on growth, and both BDNF and NT‐3 could partially block some of the growth‐promoting effects of NGF on specific neuronal subpopulations.

EphB receptors and ephrin-B ligands regulate spinal sensory connectivity and modulate pain processing.

Eph receptor tyrosine kinases and their ephrin ligands are involved in crucial aspects of nervous system circuit assembly during development, but their functional roles in the mature nervous system are poorly understood. We investigated their role in pain processing, using a combination of immunohistochemical, behavioral, biochemical and primary cell culture techniques. Here we report an in vivo role for EphB–ephrinB interactions as modulators of synaptic efficacy in the spinal cord, contributing to sensory abnormalities in persistent pain states.

BDNF modulates sensory neuron synaptic activity by a facilitation of GABA transmission in the dorsal horn.

Topical application of brain-derived neurotrophic factor (BDNF) to the adult rat isolated dorsal horn with dorsal root attached preparation inhibited the electrically evoked release of substance P (SP) from sensory neurons. This effect of BDNF was dose dependent (EC(50) 250 pM) and reversed by the tyrosine kinase inhibitor, K-252a. BDNF-induced inhibition of SP release was blocked by the GABA(B) receptor antagonist CGP 55485 but not by naloxone. Acute application of BDNF significantly increased potassium-stimulated release of GABA in the dorsal horn isolated in vitro and this effect was blocked by K-252a. Intrathecal injection of BDNF into the rat lumbar spinal cord induced a short-lasting increase in hindpaw threshold to noxious thermal stimulation that was blocked by CGP 55485 and was associated with activation of ERK in dorsal horn. These data suggest that exogenous BDNF can indirectly modulate primary sensory neuron synaptic efficacy via facilitation of the release of GABA from dorsal horn interneurons.

Semaphorin-neuropilin-1 interactions in plasticity and regeneration of adult neurons

Abstract. During development, axonal growth cones are guided to their appropriate targets by many attractive and repulsive cues. It has become increasingly clear over the last few years that how the growth cone responds to these cues depends both on the molecular nature of the cue and on the internal state of the neuron. The unexpected result is that the same molecule can act as an attractor or as a repellent. A number of guidance cues used by neurons during development are retained in the adult nervous system, where their function is often still unclear. Most of these molecules are implicated in plasticity in the adult nervous system and can play a role (sometimes maladaptive) in neuronal regeneration after injury. A group of axonal guidance cues that has been well studied in development is the semaphorin family of secreted and membrane-anchored proteins, which has been implicated in axon steering, fasciculation, branching and synapse formation. This review focuses on semaphorin-3A (probably the best-characterized semaphorin) and its receptors (in particular neuropilin-1) in the adult nervous system and argues that semaphorin-3A plays a role in the maintenance and regeneration of adult sensory neurons.

EphrinB2 induces tyrosine phosphorylation of NR2B via Src-family kinases during inflammatory hyperalgesia

In recent years a role for EphB receptor tyrosine kinases and their ephrinB ligands in activity-dependent synaptic plasticity in the CNS has been identified. The aim of the present study was to test the hypothesis that EphB receptor activation in the adult rat spinal cord is involved in synaptic plasticity and processing of nociceptive inputs, through modulation of the function of the glutamate ionotropic receptor NMDA (N-methyl-D-aspartate). In particular, EphB receptor activation would induce phosphorylation of the NR2B subunit of the NMDA receptor by a Src family non-receptor tyrosine kinase. Intrathecal administration of ephrinB2-Fc in adult rats, which can bind to and activate EphB receptors and induce behavioral thermal hyperalgesia, led to NR2B tyrosine phosphorylation, which could be blocked by the Src family kinase inhibitor PP2. Furthermore animals pre-treated with PP2 did not develop behavioral thermal hyperalgesia following EphrinB2-Fc administration, suggesting that this pathway is functionally significant. Indeed, EphB1-Fc administration, which competes with the endogenous receptor for ephrinB2 binding and prevents behavioral allodynia and hyperalgesia in the carrageenan model of inflammation, also inhibited NR2B phosphorylation in this model. Taken together these findings support the hypothesis that EphB–ephrinB interactions play an important role in NMDA-dependent, activity-dependent synaptic plasticity in the adult spinal cord, inducing the phosphorylation of the NR2B subunit of the receptor via Src family kinases, thus contributing to chronic pain states.

Peripheral, but not central, axotomy induces neuropilin-1 mRNA expression in adult large diameter primary sensory neurons

Neuropilin‐1 (NP‐1) is a component of the receptor for semaphorin3a (Sema3a), a member of a large family of molecules with widespread expression and demonstrable influence (via their ability to repel growing axons) on nervous system development. Recent studies have shown that some types of adult mammalian neurons retain the capacity to respond to Sema3a, particularly in relation to neuronal injury and regeneration. Although variations in expression of Sema3a mRNA have been revealed in neurons in both the central and peripheral nervous systems in this context, relatively little is known about NP‐1 expression patterns. In this study we investigated the expression of NP‐1 mRNA in adult dorsal root ganglion (DRG) neurons in intact and lesioned animals. We compared the effect of unilateral lesioning of the sciatic nerve or unilateral dorsal rhizotomy at lumbar levels L4/5, and bilateral dorsal funiculus lesioning at thoracic levels T10/11 on NP‐1 mRNA expression in the cell bodies of lumbar DRGs. A significantly increased level of NP‐1 mRNA expression was detected only following sciatic nerve lesioning (P < 0.001), but not after rhizotomy or dorsal funiculus lesioning. Furthermore, this upregulation was mainly confined to large diameter neurons of DRGs at lumbar levels L4/5, which provide the main sensory contribution to the sciatic nerve. These results suggest a role for NP‐1 in the axonal response to peripheral nerve injury, which may be specific to a particular subset of primary sensory neurons. J. Comp. Neurol. 423:492–499, 2000.

Nociceptor-expressed ephrin-B2 regulates inflammatory and neuropathic pain

BackgroundEphB receptors and their ephrin-B ligands play an important role in nervous system development, as well as synapse formation and plasticity in the adult brain. Recent studies show that intrathecal treatment with EphB-receptor activator ephrinB2-Fc induced thermal hyperalgesia and mechanical allodynia in rat, indicating that ephrin-B2 in small dorsal root ganglia (DRG) neurons and EphB receptors in the spinal cord modulate pain processing. To examine the role of ephrin-B2 in peripheral pain pathways, we deleted ephrin-B2 in Nav1.8+ nociceptive sensory neurons with the Cre-loxP system. Sensory neuron numbers and terminals were examined using neuronal makers. Pain behavior in acute, inflammatory and neuropathic pain models was assessed in the ephrin-B2 conditional knockout (CKO) mice. We also investigated the c-Fos expression and NMDA receptor NR2B phosphorylation in ephrin-B2 CKO mice and littermate controls.ResultsThe ephrin-B2 CKO mice were healthy with no sensory neuron loss. However, pain-related behavior was substantially altered. Although acute pain behavior and motor co-ordination were normal, inflammatory pain was attenuated in ephrin-B2 mutant mice. Complete Freunds adjuvant (CFA)-induced mechanical hyperalgesia was halved. Formalin-induced pain behavior was attenuated in the second phase, and this correlated with diminished tyrosine phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor subunit NR2B in the dorsal horn. Thermal hyperalgesia and mechanical allodynia were significantly reduced in the Seltzer model of neuropathic pain.ConclusionsPresynaptic ephrin-B2 expression thus plays an important role in regulating inflammatory pain through the regulation of synaptic plasticity in the dorsal horn and is also involved in the pathogenesis of some types of neuropathic pain.

Levels of NGF protein do not correlate with changes in innervation of the rat iris in old age

We have examined the hypothesis that alterations in target levels of NGF protein lead to selective changes in peripheral nerve fibres in maturity and old age. NGF levels were assayed using 2-site ELISA in the iris of young and old rats. NGF levels in the iris showed no significant change in old age and did not correlate with age-related changes in the density of NGF-sensitive irideal sympathetic and sensory nerve fibres. Following sympathectomy, NGF levels increased substantially in old but not in young irises, possibly because the lower density of aged sensory nerves resulted in reduced capacity for NGF uptake. Content and uptake of NGF in sympathetic (SCG) neurones was shown to be reduced in old age, while NGF content of the sensory (trigeminal) ganglion increased in old age. Thus NGF levels in neurones, as well as targets, fail to correlate with peripheral nerve fibre growth or atrophy.

A peripheral nervous system actin-binding protein regulates neurite outgrowth

Difference cloning has identified a Villin‐like mRNA transcript expressed selectively in peripheral sensory and sympathetic neurons. Pervin, the encoded 820‐amino acid protein, has 60% identity with Villin and is the rat homologue of Advillin. Coimmunoprecipitation studies demonstrate that Pervin and actin interact in vivo. Transfection of COS‐7 epithelial cell lines demonstrates colocalization of epitope‐tagged Pervin with green fluorescent protein‐actin and results in an increase in process formation. This effect is abolished when the putative actin‐bundling headpiece of Pervin is deleted. Biolistic transfection of primary cultures of rat dorsal root ganglion sensory neurons also results in increased neurite outgrowth with FLAG‐tagged Pervin. Deletion of the actin‐bundling headpiece inhibits normal neurite growth. These data suggest that Pervin may play a significant role in regulating process outgrowth in peripheral neurons through a mechanism that involves the activity of an actin‐bundling domain.

Involvement of EphB1 Receptors Signalling in Models of Inflammatory and Neuropathic Pain

EphB receptors tyrosine kinases and ephrinB ligands were first identified as guidance molecules involved in the establishment of topographical mapping and connectivity in the nervous system during development. Later in development and into adulthood their primary role would switch from guidance to activity-dependent modulation of synaptic efficacy. In sensory systems, they play a role in both the onset of inflammatory and neuropathic pain, and in the establishment of central sensitisation, an NMDA-mediated form of synaptic plasticity thought to underlie most forms of chronic pain. We studied wild type and EphB1 knockout mice in a range of inflammatory and neuropathic pain models to determine 1), whether EphB1 expression is necessary for the onset and/or maintenance of persistent pain, regardless of origin; 2), whether in these models cellular and molecular changes, e.g. phosphorylation of the NR2B subunit of the NMDA receptor, increased c-fos expression or microglial activation, associated with the onset of pain, are affected by the lack of functional EphB1 receptors. Differences in phenotype were examined behaviourally, anatomically, biochemically and electrophysiologically. Our results establish firstly, that functional EphB1 receptors are not essential for the development of normal nociception, thermal or mechanical sensitivity. Secondly, they demonstrate a widespread involvement of EphB1 receptors in chronic pain. NR2B phosphorylation, c-fos expression and microglial activation are all reduced in EphB1 knockout mice. This last finding is intriguing, since microglial activation is supposedly triggered directly by primary afferents, therefore it was not expected to be affected. Interestingly, in some models of long-term pain (days), mechanical and thermal hyperalgesia develop both in wild type and EphB1 knockout mice, but recovery is faster in the latter, indicating that in particular models these receptors are required for the maintenance, rather than the onset of, thermal and mechanical hypersensitivity. This potentially makes them an attractive target for analgesic strategies.