Faculty Bibliography

Ras signal transduction pathways have been implicated as key regulators in neuroplasticity and synaptic transmission in the brain. These pathways can be modulated by Ras guanyl nucleotide exchange factors, (GEF) which activate Ras proteins by catalysing the exchange of GDP for GTP. Ras guanyl nucleotide-releasing protein (RasGRP), a recently discovered Ras GEF, that links diacylglycerol and probably calcium to Ras signaling pathways, is expressed in brain as well as in T-cells. Here, we have used a highly selective monoclonal antibody against RasGRP to localize this protein within the striatum and related forebrain structures of developing and adult rats. RasGRP immunolabeling was found to be widespread in the mature and developing rat forebrain. Most notably, it presented a prominent patchy distribution throughout the striatum at birth and at all postnatal ages examined. These patches were found to correspond with the striosomal compartment of the striatum, as identified by micro-opioid receptor labeling in the adult. RasGRP-immunoreactivity was also observed in the matrix-like compartment surrounding these patches/striosomes but appeared later in development and was always weaker than in the patches. In both striatal compartments, RasGRP was exclusively expressed by medium-sized spiny neurons and showed no preference for neurons that project either directly or indirectly to the substantia nigra. At the ultrastructural level, immunogold labeling of RasGRP was confined to the cell bodies and dendritic shafts of these output neurons. We conclude that the prominent expression of RasGRP in striosomes may be of significance for diacylglycerol signaling in the striatum, and could be of importance for the processing of limbic-related activity within the basal ganglia.

AIMS/HYPOTHESIS/OBJECTIVE:This study compared the effects of streptozotocin-induced diabetes in rats with those of two pro-oxidant interventions; a diet deficient in vitamin E and treatment with primaquine. METHODS:Measurements were made by the classic motor and sensory conduction velocity deficits and by indicators of the breakdown of small fibre phenotype i.e., sciatic nerve content of nerve growth factor and the neuropeptides, substance P and neuropeptide Y. RESULTS:As with diabetes, the pro-oxidant interventions decreased conduction velocities (though the effect of vitamin E deficiency was not significant), the sciatic nerve content of nerve growth factor and the neuropeptides (all percentages refer to the mean value for the appropriate control groups). In diabetes, nerve growth factor was depleted to 50% in the control rats (p < 0.05); oxidative stress depleted nerve growth factor to 64% (primaquine; p < 0.05) and 81% (vitamin E deficient; not significant) of controls. Substance P was depleted to 51% in the control rats (p < 0.01) with depletions to 74% and 72% (both p < 0.01) by oxidative stress; equivalent depletions for neuropeptide Y were 38% controls in diabetes (p < 0.001) and 67% (primaquine; p < 0.001) and 74% (vitamin E deficient; p < 0.05) for oxidative stress. CONCLUSION/INTERPRETATION/CONCLUSIONS:The relative magnitudes of these changes suggest an effect in diabetes of oxidative stress, coupled with some other cellular event(s). This is supported by the effects of a diester of gamma-linolenic acid and alpha-lipoic acid, which completely prevented the effects on the pro-oxidant interventions on conduction velocity, nerve growth factor and neuropeptide contents, but was only partially preventative in diabetes.

We examined the effects of pressure ejected 3, 4-methylenedioxymethamphetamine (MDMA) from a micropipette on direct chemically stimulated release, and on electrically stimulated serotonin (5-HT) or dopamine (DA) release in the caudate putamen (CPu), nucleus accumbens (NAc), substantia nigra pars reticulata (SNr), and the dorsal raphé nucleus (DRN) brain slices of rat, using fast cyclic voltammetry (FCV). MDMA is electroactive, oxidising at +1100 mV. When the anodic input waveform was reduced from +1.4 to +1.0 volt, MDMA was not electroactive. Using this waveform, pressure ejection of MDMA did not release 5-HT or DA in brain slices prepared from any of the nuclei studied. MDMA significantly potentiated electrically stimulated 5-HT release in the SNr and DA release in CPu. In the DRN or in the NAc, MDMA was without effect on peak electrically stimulated 5-HT or DA release. The rates of neurotransmitter uptake, expressed as t(1/2), were in all cases significantly decreased after MDMA. The results indicate that MDMA, unlike (+)amphetamine, is not as a releaser of DA or 5-HT, it is a potent inhibitor of both DA and 5-HT uptake.

We compared a conventional method of measurement of sciatic motor and sensory nerve conduction velocity, with a novel procedure that measures conduction in an 8-mm segment of the rat sural nerve. Conventional procedures gave reductions in velocity of 20% and 14% for motor and sensory fibers, respectively, whereas sural sensory fibers showed a 40% reduction (P <0.05). Changes were attenuated by treatment with either an aldose reductase inhibitor or a gamma-linolenic acid-alpha-lipoic acid conjugate, such that values from conventional procedures were not significantly different from controls and the sural sensory deficit halved. Putative motor fibers of the sural nerve showed no conduction velocity deficit in diabetic rats. Measurement of chronaxie and rheobase in sural sensory fibers revealed mild reductions in excitability in diabetics, with prevention of the chronaxie change by the treatments. Thus, measurement of sensory conduction in distal nerve segments show more profound defects in diabetic rats and may give a truer picture of preventive drug efficacy.

Aberrant neurofilament phosphorylation occurs in many neurodegenerative diseases, and in this study, two animal models of type 1 diabetes--the spontaneously diabetic BB rat and the streptozocin-induced diabetic rat--have been used to determine whether such a phenomenon is involved in the etiology of the symmetrical sensory polyneuropathy commonly associated with diabetes. There was a two- to threefold (P < 0.05) elevation of neurofilament phosphorylation in lumbar dorsal root ganglia (DRG) of diabetic rats that was localized to perikarya of medium to large neurons using immunocytochemistry. Additionally, diabetes enhanced neurofilament M phosphorylation by 2.5-fold (P < 0.001) in sural nerve of BB rats. Neurofilaments are substrates of the mitogen-activated protein kinase (MAPK) family, which includes c-jun NH2-terminal kinase (JNK) or stress-activated protein kinase (SAPK1) and extracellular signal-regulated kinases (ERKs) 1 and 2. Diabetes induced a significant three- to fourfold (P < 0.05) increase in phosphorylation of a 54-kDa isoform of JNK in DRG and sural nerve, and this correlated with elevated c-Jun and neurofilament phosphorylation. In diabetes, ERK phosphorylation was also increased in the DRG, but not in sural nerve. Immunocytochemistry showed that JNK was present in sensory neuron perikarya and axons. Motoneuron perikarya and peroneal nerve of diabetic rats showed no evidence of increased neurofilament phosphorylation and failed to exhibit phosphorylation of JNK. It is hypothesized that in sensory neurons of diabetic rats, aberrant phosphorylation of neurofilament may contribute to the distal sensory axonopathy observed in diabetes.

1. Fast cyclic voltammetry was used to investigate the effect of 7-OH-DPAT (7-hydroxy-N,N-di-n-propyl-2-aminotetralin), a putative D3 receptor agonist, on electrically stimulated endogenous dopamine release in slices of rat nucleus accumbens. 2. 7-OH-DPAT inhibited single pulse stimulated dopamine release in a concentration-dependent manner with a maximum inhibition of 95.5%. Analysis of concentration-response curves to 7-OH-DPAT showed that they were biphasic, with the high affinity component contributing 18.0% to the total inhibition and the low affinity component 77.5%. 7-OH-DPAT exhibited a 560 fold selectivity between the high and low affinity components (0.015 nM compared to 8.4 nM). 3. Concentration-response curves to the non-selective D2/D3 agonist, apomorphine, were monophasic. The maximum inhibition was 93.1% and the EC50 value 82 nM. 4. The selective D2 antagonist, haloperidol (30 nM), antagonized the low affinity component of the concentration-response cuve to 7-OH-DPAT whilst the high affinity component was essentially unaffected. The pKB values calculated for the high and low affinity components were 7.89 and 9.45 respectively. 5. In conclusion, these results demonstrate that 7-OH-DPAT inhibits stimulated dopamine release by acting at two different sites. Furthermore, the results are consistent with the hypothesis that the high and low affinity components of the concentration-response curve to 7-OH-DPAT may reflect activation of functional D3 and D2 release-regulating autoreceptors respectively. However, the possibility that the biphasic nature of the curve may reflect different subtypes of the D2 receptor cannot be excluded.