Serine proteases play a key function in extracellular processes affecting central nervous system plasticity. Recently, the role of extracellular proteolytic processes in regulating synaptic structure and function has been described. However, to date direct evidence linking extracellular serine protease activity with drug-related behavioural changes has not been documented. Importantly, in a screening for genes induced after drug treatment we found that urokinase plasminogen-type activator (uPA) was strongly regulated by cocaine in several protocols of drug administration. Cocaine-induced up-regulation could be verified on microarray analysis under several protocols of drug administration, then further fully confirmed by means of qRT-PCR. As a result, we chose to investigate further uPA function in the mesolimbic dopaminergic pathway, a major target area of cocaine and drugs of misuse. Our approach was based on the characterization of cocaine-induced behavioural changes following lentiviral vector delivery of a doxycycline-regulated uPA expression cassette (or of its mutated form), into specific rat brain areas (the hippocampus, the nucleus accumbens and the ventral tegmental area). We show that doxycycline-dependent over-expression of uPA in these regions yields a 10-to 12.3-fold increase in locomotor activity after cocaine administration. These behavioural effects were completely abolished when the active site of the protease was point-mutated and used as a dominant negative. The physiological relevance of these drastic behavioural changes is discussed.

Beefheart aconitase, isolated under aerobic conditions, has been studied with Mossbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mossbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably ofthe In the past few years, it has been recognized that many features ofFe-S proteins cannot be understood in terms ofthe structural types known for these proteins-i.e., FeS4, [2Fe-2S], [4Fe-4S], or 2[4Fe-4S].The occurrence of 3Fe clusters has been reported for a few proteins, of which ferredoxin I of Azotobacter vinelandii (1), ferredoxin II ofDesulfovibrio gigas (2), and beefheart aconitase (3) are probably the most intensively studied. X-ray diffraction studies on A. vinelandii ferredoxin (Fd) show that these clusters have a [3Fe-3S] core (4). The decisive spectroscopic evidence for the presence of a [3Fe-3S] center has been the observation ofan EPR signal at g = 2.01 in oxidized samples together with the unique Mossbauer spectra that these structures yield in the reduced state (5).Beef heart aconitase has properties not found in other proteins thought to contain [3Fe-3S] clusters. As obtained on routine purification, it is enzymatically inactive, but it can be reactivated by a number oftreatments; all ofwhich have in common the reduction of the Fe-S cluster (6, 7). Although inclusion of iron in the activation medium yields the highest activities, iron does not appear to be an obligatory ingredient of such media; activities up to 70% of maximum can be induced by some reducing agents alone (6, 7). We have now studied, by M6ssbauer spectroscopy, aconitase samples reduced and activated in a variety of ways. Only samples reduced and activated with dithionite (' 30% of maximal activity) have the Mossbauer features characteristic of [3Fe-3S] centers. Those activated with dithiothreitol/Fe2", dithionite/Fe2", or dithiothreitol alone display 60-100% activity and show entirely different Mossbauer features. We propose that the latter activation procedures convert the [3Fe-3S] cluster into a structure that has a core.In the present report, we limit ourselves to observations concerned with the process and consequences of reductive activation and oxidative deactivation of the enzyme. Conditions pertaining to the catalytic action of the enzyme will be considered elsewhere. MATERIALS AND METHODSAconitase was purified from beef heart as reported (8) with minor modifications. Analytical methods and EPR spectroscopy were as in ref. 9 and yielded results as in refs. 8 and 7 respectively. Activations were carried out anaerobically; for routine assays (10), 0.1 mM ferrous ethylene diammonium sulfate and 5 mM dithiothreitol were used, and Mbssbauer and EPR samples were prepared as desired for the specific purpose. Anaerobic procedures followed the outlines ofref. 11 with appropriate modifications.Although enzymatic assays for aconitase activity are simple and reproducible, it must be kept in mind that drawing ...

Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. We now report that EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. In addition, we show that ephrin-B2 expression is upregulated by cocaine and amphetamine in adult mice, suggesting that ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults as well.

CD81, a tetraspanin transmembrane protein involved in cell adhesion, is up-regulated in the mesolimbic dopaminergic pathway 24 h following acute administration of high doses of cocaine [Brenz-Verca et al., (2001) Mol. Cell. Neurosci., 17, 303±316]. Further evidence consecutive with this observation and based on microarray analysis are presented here. In addition, a regulatable lentivirus was developed bearing the rat CD81 gene under the control of a tetracycline inducible system. This lentivirus vector was stereotaxically injected into the ventral tegmental area (VTA) of two groups of animals, one fed water (expressing CD81) and the other Doxycycline solution (which down-regulates CD81 expression) and locomotor activity after chronic cocaine administration (10 mg/kg daily) was monitored. After 2 weeks, the groups were inverted, animals receiving water were placed on Doxycycline and the second group was placed on water. In all cases highly a signi®cant increase (3.2-fold) in locomotor activity was observed in animals expressing CD81 in the VTA vs. animals placed on Doxycycline. Similar studies where CD81 was delivered into the nucleus accumbens (NAcc) resulted in signi®cantly higher effects (30%), in accordance with microarray data and our previous reports, yielding a 4.2-fold increase in locomotor activity. No change was observed under similar conditions in control animals, which were injected a regulatable lentivirus expressing GFP. These ®ndings suggest that CD81 expression in the mesolimbic dopaminergic pathway contributes to behavioural changes associated with cocaine sensitization. This study provides a powerful approach for evaluating a gene function in vivo in a single animal under various paradigms, even on gene candidates, which display small changes of expression.

Aquatic insects are a common and important subsidy to terrestrial systems, yet little is known about how these inputs affect terrestrial food webs, especially around lakes. Mývatn, a lake in northern Iceland, has extraordinary midge (Chironomidae) emergences that result in large inputs of biomass and nutrients to terrestrial arthropod communities. We simulated this lake-to-land resource pulse by collecting midges from Mývatn and spreading their dried carcasses on 1-m2 plots at a nearby site that receives very little midge deposition. We hypothesized a positive bottom-up response of detritivores that would be transmitted to their predators and would persist into the following year. We sampled the arthropod community once per month for two consecutive summers. Midge addition resulted in significantly different arthropod communities and increased densities of some taxa in both years. Detritivores, specifically Diptera larvae, Collembola, and Acari increased in midge-addition plots, and so did some predators and parasitoids. Arthropod densities were still elevated a year after midge addition, and two years of midge addition further increased the density of higher-order consumers (e.g., Coleoptera and Hymenoptera). Midge addition increased arthropod biomass by 68% after one year and 108% after two years. By manipulating the nutrient pulse delivered by midges we were able to elucidate food web consequences of midge deposition and spatial and temporal dynamics that are difficult to determine based on comparative approaches alone. Resources cross ecosystem boundaries and are assimilated over time because of life-history strategies that connect aquatic and terrestrial food webs and these systems cannot be fully understood in isolation from each other.