Adrian Pini

Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia

In neurodegenerative conditions and following brain trauma it is not understood why neurons die while astrocytes and microglia survive and adopt pro-inflammatory phenotypes. We show here that the damaged adult brain releases diffusible factors that can kill cortical neurons and we have identified histone H1 as a major extracellular candidate that causes neurotoxicity and activation of the innate immune system. Extracellular core histones H2A, H2B H3 and H4 were not neurotoxic. Innate immunity in the central nervous system is mediated through microglial cells and we show here for the first time that histone H1 promotes their survival, up-regulates MHC class II antigen expression and is a powerful microglial chemoattractant. We propose that when the central nervous system is degenerating, histone H1 drives a positive feedback loop that drives further degeneration and activation of immune defences which can themselves be damaging. We suggest that histone H1 acts as an antimicrobial peptide and kills neurons through mitochondrial damage and apoptosis.

Chemorepulsion of developing motor axons by the floor plate.

In the developing nervous system, motor axons grow away from the ventral midline floor plate, suggesting that the latter might be a source of repulsive axonal guidance cues. In donor to host transplantation experiments, ectopic pieces of floor plate were positioned between chick hindbrain motor neurons and their exit points. Immunohistochemistry and retrograde axonal labeling techniques demonstrated that motor axons diverted from their normal pathways to avoid grafted floor plate, often traversing abnormally long circuitous trajectories to reach exit points. When ventral explants of rat hindbrain and spinal cord were cocultured at a distance from floor plate explants within collagen gel matrices, the outgrowth of motor axons was dramatically reduced from explant borders that faced the floor plate. Thus, the floor plate secretes diffusible repulsive cues in vitro that may exclude motor axons from the midline during development.

The effects of locally applied capsaicin on conduction in cutaneous nerves in four mammalian species

1 By examination of compound action potentials in the saphenous nerve of the anaesthetized rat it has been shown that capsaicin causes a rapid, dose‐dependent, failure of conduction in many C‐fibres when applied directly to the nerve. A large reduction in C‐fibre conduction occurs with concentrations as low as 110 μM. After a 15–30 min exposure to capsaicin, only partial recovery occurs in 1 h. 2 Similar block of C‐fibre conduction occurs in the ferret. However, only smaller, reversible, reductions in C‐fibre conduction were seen in the guinea‐pig and rabbit, even at the highest concentration of capsaicin used (33 mM). 3 A small reduction in the Aδ component of the compound action potential occurred in all four species. In the rat and ferret the effects were much less than those on C‐fibres. 4 At high doses, small reversible effects were also seen on the fastest conducting Aαβ component of the compound action potential in the rat, rabbit and guinea‐pig; no effects were seen on the Aαβ fibres in the ferret. 5 Decreases in amplitude of the compound action potential were accompanied by some slowing of conduction in most cases. The slowing was less than 5% except for the rat Aαβ and C‐fibres and the ferret C‐fibres where 9–15% changes occurred at the highest doses of capsaicin. 6 Opening the connective tissue sheath of the nerve did not significantly increase the effectiveness of capsaicin.

Extracellular Ig domains 1 and 2 of Robo are important for ligand (Slit) binding.

Robo, the receptor for the midline repellent Slit, is a member of the cell adhesion molecule (CAM) Ig superfamily. We have recently demonstrated that members of the Robo family (Robo1 and Robo2) interact homophilically and heterophilically, thereby functioning to promote neurite outgrowth. Here, we describe a series of in vitro experiments to dissect the Robo ligand-interacting domains by deleting specific extracellular regions of the Robo1 molecule, generating a series of mutant proteins. Using these, we demonstrate that Ig domains 1 and 2 of Robo1 are important for Robo-Slit interaction and provide functional data using the Slit-mediated olfactory bulb repulsion assay. To investigate whether homophilic binding properties of Robo are domain specific, we used Robo1-Fc mutant deletion proteins in an aggregation assay and observed a reduction in homophilic binding when any one Ig or all the fibronectin domains were deleted, although homophilic binding was never completely abolished.

Long‐Term Reduction in the Number of C‐Fibre Nociceptors Following Capsaicin Treatment of a Cutaneous Nerve in Adult Rats

The structure and function of C‐ and A‐fibres have been studied 3–12 months following a single 30 min exposure of the saphenous nerve of adult rats to 1% capsaicin. Examination of nerve cross‐sections showed that the number of C‐fibres was reduced by 36%, but A‐fibres were unaffected. The remaining C‐fibres included many that were unusually small in size. The changes were not restricted to the treatment site but were found over at least a further 10 mm proximal and distal to it. In treated nerves, the C‐fibre component of the compound action potential was reduced in size relative to the Aαβ component. Single unit studies revealed that an unusually high proportion of C‐fibres had no cutaneous receptive field (54%, compared with 28% in controls). There was no such change for A‐fibres. The conduction velocity range for C‐fibre units in treated nerves was almost normal, but this sample contained an unusually low proportion of polymodal nociceptors. Allowing for the fall in total numbers and the reduction in units with cutaneous receptive fields, it was calculated that overall the numbers of C‐polymodal nociceptor units had fallen by 74%. No significant changes in number had occurred in the other classes of C‐afferents. The reduction in the numbers of nociceptive C‐fibres is likely to be the direct cause of the reduction in pain responses and in neurogenic inflammation that several groups have reported following treatment of adult rats with capsaicin. Reduction in the numbers of C‐fibre is also likely to be the reason for reduced neuropeptide levels seen in tissues innervated by treated nerves.

On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion

The vomeronasal projection conveys information provided by pheromones and detected by neurones in the vomeronasal organ (VNO) to the accessory olfactory bulb (AOB) and thence to other regions of the brain such as the amygdala. The VNO-AOB projection is topographically organised such that axons from apical and basal parts of the VNO terminate in the anterior and posterior AOB respectively. We provide evidence that the Slit family of axon guidance molecules and their Robo receptors contribute to the topographic targeting of basal vomeronasal axons. Robo receptor expression is confined largely to basal VNO axons, while Slits are differentially expressed in the AOB with a higher concentration in the anterior part, which basal axons do not invade. Immunohistochemistry using a Robo-specific antibody reveals a zone-specific targeting of VNO axons in the AOB well before cell bodies of these neurones in the VNO acquire their final zonal position. In vitro assays show that Slit1-Slit3 chemorepel VNO axons, suggesting that basal axons are guided to the posterior AOB due to chemorepulsive activity of Slits in the anterior AOB. These data in combination with recently obtained other data suggest a model for the topographic targeting in the vomeronasal projection where ephrin-As and neuropilins guide apical VNO axons, while Robo/Slit interactions are important components in the targeting of basal VNO axons.

C‐fibre Function During the 6 Weeks Following Brief Application of Capsaicin to a Cutaneous Nerve in the Rat

The functional properties of afferent fibres, especially C‐fibres, have been studied over the 46 days following application of capsaicinoids to a segment of the saphenous nerve in the rat. After 1–3 days, approximately half of the C‐afferents were found to be non‐conducting at or immediately distal to the treatment site. By 6–9 days there was some recovery of conduction at the treatment site, but few of the conducting fibres were excitable from the skin. From 12 days onwards, C‐fibre conduction in the segment of nerve both proximal and distal to the treatment site was reduced and of the conducting fibres relatively few had cutaneous receptive fields. In the sample of C‐fibres with cutaneous receptive fields the proportion of polymodal nociceptors was reduced markedly compared with control values. Analysis of the numbers of surviving units shows a large effect on C‐polymodal nociceptors, but no significant change in the numbers of any other type of afferent unit. At 2 weeks, when C‐fibre numbers were reduced to about one third, there was a virtual abolition of antidromic vasodilatation and an 85% fall in skin substance P levels. The synthetic capsaicinoid, NE‐21610, had similar actions to capsaicin and was more potent in depleting skin substance P levels. These results confirm that capsaicinoids produce a selective local lesion on nociceptive C‐fibres in rat cutaneous nerves. In addition, the transient and partial nature of functional recovery indicates that capsaicin may also cause a longer‐term toxic action that inhibits significant regeneration.

Axon guidance: Following the Eph plan

Recently discovered Eph family receptors and their ligands appear likely to provide the cytochemical tags that Sperry speculated enable axons projecting from the retina to find their correct targets in the brain.

Axon guidance. Growth cones say no.

A recently characterized protein stops developing axons from growing; a gene family encoding related molecules has also been described.