Use of genetically engineered transgenic mice to investigate the role of galanin in the peripheral nervous system after injury.

The neuropeptide galanin is present at high levels within the dorsal root ganglia (DRG) and spinal cord during development and after peripheral nerve damage in the adult. This pattern of expression suggests that it may play a role in the adaptive response of the peripheral nervous system (PNS) to injury. Several experimental paradigms have demonstrated that galanin modulates pain transmission, particularly after nerve injury. In our laboratory we have used a transgenic approach to further elucidate the functions of galanin within the somatosensory system. We have generated mice which over-express galanin (either inducibly after nerve injury, or constitutively), and knock-out (KO) mice, in which galanin is absent in all cells, throughout development and in the adult. Analysis of the nociceptive behaviour of the galanin over-expressing animals, before and after nerve injury, supports the view that galanin is an inhibitory neuromodulator of spinal cord transmission. In apparent contradiction to these findings, galanin KO animals fail to develop allodynia and hyperalgesia after nerve injury. However, further studies have shown that galanin is critical for the developmental survival of a subset of small diameter, unmyelinated sensory neurons that are likely to be nociceptors. This finding may well explain the lack of neuropathic pain-like behaviour after injury in the KO animals. Furthermore, the developmental survival role played by galanin is recapitulated in the adult where the peptide is required for optimal neuronal regeneration after injury, and in the hippocampus where it plays a neuroprotective role after excitotoxic injury.

Stabilization of neurites in cerebellar granule cells by transglutaminase activity: identification of midkine and galectin-3 as substrates.

The formation of covalent isopeptide cross-links between cell surface protein molecules by the enzyme transglutaminase C influences cell adhesion and morphology. Retinoid-inducible cross-linking activity associated with this enzyme is present in the developing rat cerebellar cortex [Perry M. J. M. et al. (1995) Neuroscience 65, 1063-1076]. A monoclonal antibody was used to localize transglutaminase C to granule neurons in the developing cerebellar cortex. The enzyme was inducible by retinoic acid both in granule neurons cultured from postnatal rat cerebellar cortex and in cells of the embryonic dorsal rhombic lip, which contain granule neuron precursors. A possible biological function for transglutaminase activity was investigated in living granule neurons, cultured on a biomatrix substratum, studied by time-lapse cinematographic analysis using the transglutaminase inactivator RS-48373-007. Inhibition of cross-linking activity did not influence the number of neurites formed by granule neurons, but caused the destabilization of neurites during the initial outgrowth period, seen as an increase in the number of growth cone retractions and the onset of premature axon collateral formation (bifurcation). Inactivation of cross-linking activity prevented the formation of fascicles between neurites only when cells were cultured on a biomatrix surface. Two glial proteins involved in cell-extracellular matrix interactions, midkine and galectin-3, were identified as putative substrates for granule neuron transglutaminase. The results suggest that covalent cross-link formation by transglutaminase C or a related enzyme generates multimeric molecular forms of glial-derived proteins, and plays a role in stabilizing newly formed neurites. A possible non-pathological role for transglutaminase in the control of axon collateral branching by developing granule neurons in the cerebellar cortex is discussed.

Transglutaminase forms midkine homodimers in cerebellar neurons and modulates the neurite-outgrowth response.

Midkine is a prominent acyl donor substrate for the protein cross-linking enzyme transglutaminase type 2 in rat brain neurons. Transglutaminase type 2 and midkine immunoreactivity are regionally colocalized in developing cerebellar cortex. Monomeric midkine is present in the embryonic dorsal rhombic lip which gives rise to the cerebellar cortex. A high-molecular weight (29-30 kDa) midkine appears during postnatal cerebellar development. The presence of the high-molecular weight midkine in cultured cerebellar cortical interneurons is dependent upon culture conditions. Transglutaminase catalyzes the calcium-dependent cross-linking of midkine predominantly into 29-30 kDa dimers. Dimer-formation of midkine in vitro and in cultured neurons is reduced in the presence of a transglutaminase inactivator. Neurons plated onto previously cross-linked midkine exhibit larger growth cones and enhanced neurite outgrowth compared to those plated onto monomeric midkine alone.

Transglutaminase C in cerebellar granule neurons: regulation and localization of substrate cross-linking.

Covalent cross-linking of cell surface proteins by the calcium-dependent enzyme transglutaminase C may be implicated in cell-cell interactions and growth regulation. We demonstrate the presence of the enzyme in rat cerebellar cortex during postnatal development. Transglutaminase C was induced in cerebellar granule neurons in culture by retinoic acid, dibutyryl- and 8-bromo-cyclic AMP analogues and by cultivation on a biomatrix substratum. Cyclic AMP analogues stimulated transglutaminase activity in protein synthesis-dependent and -independent phases. The enzyme was distributed at focal adhesion sites on the axon. By calcium-dependent covalent incorporation of the primary amine acceptor substrate, 5-(biotinamido)pentylamine, an increase in the Ca(2+)-dependent cross-linking of at least 11 substrate proteins in the presence of retinoic acid and dibutyryl-cyclic AMP was detected. Of these substrates, a subset was labelled on the surface of living granule neurons. A low-molecular-weight substrate, p18, was tentatively identified as the retinoic acid-inducible neurite-promoting factor, midkine. Transglutaminase-mediated amine incorporation, midkine and isopeptide cross-links were co-localized to axonal adhesion sites. The results provide evidence of transglutaminase C-catalysed protein cross-linking activity in cerebellar granule neurons and its possible implication in cell-substratum interactions.

Cost of locomotion and heat balance during rest and running from 0 to 55 degrees C in a patas monkey.

A young patas monkey was run on a treadmill for 15-20 min at speeds in excess of 15 km times h-1 over a range of laboratory air temperatures from 0 to 55 degrees C. Cost of locomotion for the monkey was 83% that predicted for a running mammal of similar weight, 4 kg. At the highest steady-state running of speeds, the patas monkey's energy expenditure was 11 times the resting rate. Heat storage and respiratory evaporation during running each accounted for less than 16% of the heat production. Cutaneous evaporation, mainly sweating, was the major means of heat loss at high ambient temperatures and during heavy exercise. Maximum sweat rates of 0.5 mg times cm-2 times min-1 found in these experiments approach the sweat rates of humans. Whole-body dry thermal conductance increased 2-3 times the rest value at a given temperature. Environmentally gained heat contributed about two-thirds of the total heat load on the patas monkey at rest at 53 degrees C. At these same temperatures, environmental and metabolic heat loads were about equal when the animal ran at moderately high speeds (11-13.4 km times h-1).