Parkinson’s disease is a disorder mainly characterized by progressive degeneration of dopamine producing neurons in the substantia nigra of the midbrain. The most commonly used treatment strategy is to pharmacologically restore the lost function by the administration of the dopaminergic precursor L-DOPA. Another treatment strategy is to replace the degenerated neurons with immature fetal ventral mesencephalic tissue, or ultimately stem cell-derived tissue. Grafting trials have, however, revealed poor reinnervation capacity of the grafts, leaving much of the striata dopamine-denervated. An additional drawback is the upcoming of dyskinesia (involuntary movements), a phenomenon also observed during L-DOPA treatment of Parkinson’s disease patients. Attempts to characterize nerve fiber formation from dopamine neurons have demonstrated that the nerve fibers are formed in two morphologically diverse outgrowth patterns, one early outgrowth seen in the absence of astrocytes and one later appearing outgrowth seen in co-existence with astrocytes. The overall objective of this thesis has been to study the dopaminergic outgrowth including guidance of nerve fiber formation, and to look into the mechanisms of L-DOPA-induced dyskinesia. The first paper in this thesis characterizes the different outgrowth patterns described above and their relation to different glial cells. The study demonstrated the two different outgrowth patterns to be a general phenomenon, applying not only to dopamine neurons. Attempts of characterization revealed no difference of origin in terms of dopaminergic subpopulations, i.e. A9 or A10, between the outgrowth patterns. Furthermore, the “roller-drum” technique was found optimal for studying the dual outgrowth sequences. The second and the third paper also utilized the “roller-drum” technique in order to promote both patterns of neuronal fiber formation. The effects of glial cell line-derived neurotrophic factor (GDNF) on the formation of dopamine nerve fibers, was investigated. Cultures prepared from gdnf knockout mice revealed that dopaminergic neurons survive and form nerve fiber outgrowth in the absence of GDNF. The dopaminergic nerve fibers exhibited an outgrowth pattern consistent with that previous observed in rat. GDNF was found to exert effect on the glial-associated outgrowth whereas the non-glial-associated was not affected. Astrocytic proliferation was inhibited using cytosine β-D-arabinofuranoside, resulting in reduced glial-associated outgrowth. The non-glial-associated dopaminergic outgrowth was on the other hand promoted, and was retained over longer time in culture. Furthermore, the non-glial-associated nerve fibers were found to target the fetal frontal cortex. Different developmental stages were shown to promote and affect the outgrowths differently. Taken together, these data indicate and state the importance of astrocytes and growth factors for neuronal nerve fiber formation and guidance. It also stresses the importance of fetal donor age at the time for transplantation. The fourth and fifth studies focus on L-DOPA dynamics and utilize in vivo chronoamperometry. In study four, 6-OHDA dopamine-depleted rats were exposed to chronic L-DOPA treatment and then rated as dyskinetic or non-dyskinetic. The electrochemical recordings demonstrated reduced KCl-evoked release in the intact striatum after chronic L-DOPA treatment. Time for maximal dopamine concentration after L-DOPA administration was found to be shorter in dyskinetic animals than in non-dyskinetic animals. The serotonergic nerve fiber content in the striatum was evaluated and brains from dyskinetic animals were found to exhibit significantly higher nerve fiber density compared to non-dyskinetic animals. Furthermore, the mechanisms behind the conversion of L-DOPA to dopamine in 6-OHDA dopamine-depleted rats were studied. Local administration of L-DOPA in the striatum increased the KCl-evoked dopamine release in the intact striatum. Acute application of L-DOPA resulted sometimes in a rapid conversion to dopamine, probably without vesicle packaging. This type of direct conversion is presumably occurring in non-neuronal tissue. Furthermore, KCl-evoked dopamine releases were present upon local application of L-DOPA in the dopamine-depleted striatum, suggesting that the conversion to dopamine took place elsewhere, than in dopaminergic nerve fibers. In conclusion, these studies state the importance of astrocytes for neuronal nerve fiber formation and elucidate the complexity of L-DOPA conversion in the brain.

Parkinson’s disease is a progressive neurodegenerative disorder, characterized by a depletion of the dopaminergic neurons in the substantia nigra. The cause of the disease is yet unknown but age, oxidative stress, and neuroinflammation are some of the features involved in the degeneration. In addition, substantial cell death of noradrenergic neurons occurs in the locus coeruleus (LC). Noradrenaline has been suggested to protect the dopamine neurons from oxidative stress and neuroinflammation. The main treatment of Parkinson’s disease is Levo-dopa, although severe side effects arise from this therapy. Hence, grafting fetal ventral mesencephalic (VM) tissue into the adult striatum has been evaluated as an alternative treatment for Parkinsons’s disease. However, the survival of the grafted neurons is limited, and the dopamine-denervated striatum does not become fully reinnervated. Therefore, elucidating factors that enhance dopamine nerve fiber formation and/or survival of the grafted neurons is of utmost importance. To investigate dopamine nerve fiber formation and the interactions with glial cells, organotypic VM tissue cultures were utilized. Two morphologically different nerve fiber outgrowths from the tissue slice were observed. Nerve fibers were initially formed in the absence of migrating astrocytes, although thin vimentin-positive astrocytic processes were detected within the same area. A second, persistent nerve fiber outgrowth was observed associated with migrating astrocytes. Hence, both of these nerve fiber outgrowths were to some extent dependent on astrocytes, and appeared as a general feature since this phenomenon was demonstrated in β-tubulin, tyrosine hydroxylase (TH), and aldehyde dehydrogenase A1 (ALDH1)-positive nerve fibers. Neither oligodendrocytes (NG2-positive cells), nor microglia (Iba-1-positive cells) exerted any effect on these two neuronal growths. Since astrocytes appeared to influence the nerve fiber formation, the role of proteoglycans, i.e. extracellular matrix molecules produced by astrocytes, was investigated. β-xyloside was added to the cultures to inhibit proteoglycan synthesis. The results revealed a hampered astrocytic migration and proliferation, as well as a reduction of the glia-associated TH-positive nerve fiber outgrowth. Interestingly, the number of cultures displaying the non-glia-mediated TH-positive nerve fibers increased after β-xyloside treatment, although the amount of TH-protein was not altered. Thus, proteoglycans produced by astrocytes appeared to be important in affecting the dopamine nerve fiber formation. The noradrenaline neurons in LC have been suggested to protect dopamine neurons from damage. Therefore, the interaction between VM and LC was evaluated. Using the intraocular grafting method, fetal VM and LC were grafted either as single grafts or as VM+LC co-grafts. Additionally, the recipient animals received 2% blueberry-enriched diet. The direct contact of LC promoted graft volume and survival of TH-positive neurons in the VM grafts. The number of dopamine neurons, derived preferably from the A9 (ALDH1/TH-positive) was increased, whereas the dopamine neurons from the A10 (calbindin/TH-positive) were not affected. A dense dopamine-β-hydroxylase (DBH)-positive innervation was correlated to the improved survival. Blueberry-enriched diet enhanced the number of TH-positive neurons in VM, although the graft size was not altered. The combination of blueberries and the presence of LC did not yield additive effects on the survival of VM grafts. The attachment of VM or the addition of blueberries did not affect the survival of TH-positive neurons in LC grafts. The number of Iba-1-positive microglia was decreased in co-grafted VM compared to single VM transplants. The addition of blueberries reduced the number of Iba-1-positive microglia in single VM transplants. Hence, the direct contact of LC or the addition of blueberries enhanced the survival of VM grafts. Taken together, these data demonstrate novel findings regarding the importance of astrocytes for the nerve fiber formation of dopamine neurons. Further, both the direct attachment of LC or antioxidant-enriched diet promote the survival of fetal VM grafts, while LC is not affected.

Neurologically normal individuals show a leftward spatial bias and tend to collide with objects on the right side more frequently than on the left. The upper visual field is associated with extrapersonal space, and mediated by the ventral stream through parvocellular projections. The lower visual field is associated with peripersonal space, and mediated by the dorsal stream through magnocellular projections. Upper and lower visual field differences have been observed in perceptual asymmetries but results have been mixed. Object- and space-based coordinates also both influence the leftward bias; however their relative contributions are unknown as similar spatial conditions are often collapsed across. More left-side collisions emerged on a route following task in the lower visual field and more right-side collisions were seen in the upper visual field (Thomas, Stuckel, Gutwin, & Elias, 2009). Left-handers made more right-side collisions in the central condition, whereas right-handers showed no bias. Leftward biases on the greyscales task were stronger in the lower visual field; however no distance-based differences were observed (Thomas & Elias, 2010). A stronger spatial bias was found on the greyscales task, whereas a stronger object-based bias was found on the object luminosity task (Thomas & Elias, in press). When individual spatial conditions were examined, the image chosen most often was always located in the lower field. Stimulus type and spatial location interacted to determine which coordinate type contributes more strongly to leftward biases. We also found that the leftward bias on the greyscales task was stronger in the lower visual field during prolonged presentation and in the upper visual field during brief presentation. A global motion task was created to preferentially engage magnocellular projections to the dorsal stream. Isoluminant red/green and blue/yellow colour tasks, which preferentially engage parvocellular projections to the ventral stream, were also created. Leftward biases were seen on the greyscales and motion tasks. On an isoluminant colour task, biases were significantly weakened, suggesting leftward biases exhibited by neurologically normal people are mediated by magnocellular projections to the dorsal stream and this preferential processing leads to a lower visual field advantage on the greyscales task.

The involvement of dopamine (DA) in the emotional and psychiatric disturbances associated with schizophrenia and psychomotor stimulant abuse is well known; however, the mechanism by which DA mediates fear expression and anxiety is not well defined. Accordingly, the objective of the present thesis was to determine the fear-motivational functions of DA neurons in the ventral tegmental area (VTA) and to examine the role of DA in fear extinction using the potentiated startle paradigm. In Experiment 1, it was observed that electrical stimulation of the VTA produced a pronounced increase in the amplitude of the acoustic startle reflex. In subsequent experiments fear-potentiated startle was assessed following axon-sparing N-methyl-D-aspartic acid (NMDA) lesions of the VTA and after bilateral intra-VTA infusion of the DA D_2/3 receptor agonist quinpirole (Experiments 2-4). The NMDA lesions resulted in substantial cell loss in the medial ventral tegmentum and blocked fear-potentiated startle. Similarly, inhibition of DA neuronal activity associated with locally-administered quinpirole suppressed the expression of the conditioned fear-induced increase in startle amplitudes. The quinpirole results implicate DA neuronal functioning in fear motivation. To explore further the involvement of DA in aversive emotional behavior, pharmacological experiments were conducted in which the effects of peripherally-administered DA agonist drugs on fear extinction were assessed. Subjects in Experiment 5 received an acute injection of either cocaine hydrochloride (40.0 mg/kg), d-amphetamine sulphate (5.0 mg/kg), the D_2/3 agonist quinpirole hydrochloride (5.0 mg/kg), or the D₁-type agonist SKF 38393 (5.0 mg/kg) during a single extinction session following fear acquisition. Animals treated with cocaine, d-amphetamine, and SKF 38393 exhibited fear-potentiated startle, whereas quinpirole treatment failed to alter fear extinction to the nonreinforced conditioned stimulus (CS). Also, it was revealed using a within-subjects design in Experiment 6 that cocaine administration reinstated fear-potentiated startle following extinction. Taken together, the results of the present experiments suggests fundamental role for DA and DA D₁ receptors in fear expression. It was proposed that VTA DA neurons gate levels of aversive emotional arousal within the amygdala-based fear system.

Neurologically normal individuals show a leftward spatial bias and tend to collide with objects on the right side more frequently than on the left. The upper visual field is associated with extrapersonal space, and mediated by the ventral stream through parvocellular projections. The lower visual field is associated with peripersonal space, and mediated by the dorsal stream through magnocellular projections. Upper and lower visual field differences have been observed in perceptual asymmetries but results have been mixed. Object- and space-based coordinates also both influence the leftward bias; however their relative contributions are unknown as similar spatial conditions are often collapsed across. More left-side collisions emerged on a route following task in the lower visual field and more right-side collisions were seen in the upper visual field (Thomas, Stuckel, Gutwin, & Elias, 2009). Left-handers made more right-side collisions in the central condition, whereas right-handers showed no bias. Leftward biases on the greyscales task were stronger in the lower visual field; however no distance-based differences were observed (Thomas & Elias, 2010). A stronger spatial bias was found on the greyscales task, whereas a stronger object-based bias was found on the object luminosity task (Thomas & Elias, in press). When individual spatial conditions were examined, the image chosen most often was always located in the lower field. Stimulus type and spatial location interacted to determine which coordinate type contributes more strongly to leftward biases. We also found that the leftward bias on the greyscales task was stronger in the lower visual field during prolonged presentation and in the upper visual field during brief presentation. A global motion task was created to preferentially engage magnocellular projections to the dorsal stream. Isoluminant red/green and blue/yellow colour tasks, which preferentially engage parvocellular projections to the ventral stream, were also created. Leftward biases were seen on the greyscales and motion tasks. On an isoluminant colour task, biases were significantly weakened, suggesting leftward biases exhibited by neurologically normal people are mediated by magnocellular projections to the dorsal stream and this preferential processing leads to a lower visual field advantage on the greyscales task.

The mesolimbic dopamine system, including dopaminergic projections from the ventral tegmental area (VTA) to nucleus accumbens (NAc), is critically involved in the development of addiction to many drugs of abuse, including cocaine (CA). Although there is an attractive hypothesis that the modifications of mesolimbic reward circuit following repeated drug exposure are responsible for cocaine-addicted causes behaviors change, however, our understanding in the underlying molecular mechanisms at the neural circuit level is still in its infancy. It has been suggested PKMzeta, a constitutively active atypical isoform of PKC, plays a critical role in spatial memory formation and long-term synaptic potentiation in hippocampus. To define the relationship among PKMzeta, CA-induced synaptic long-term potentiation and CA addiction, we examined the regulation of PKMzeta after CA administration in Sprague-Dawley rat. We found single CA injection elicits an increase in PKMzeta protein expression in the VTA region. The increase was first observed 10 min after CA administration and lasted for 7 days, the longest sampling time point of our experimental design. The PKMzeta protein expression can also be induced in 10 minutes while incubating the acute isolated brain slice with CA, the expression within 1 hr can be eliminated at the present of Chelerythrine (PKC inhibitor) and ZIP (PKMzeta inhibitor) suggests a positive feedback loop. The PKMzeta mRNA expression can be induced within 1 hr, and Actinomycin d (transcription inhibitor) had no effect on the PKMzeta protein expression indicating CA increases PKM£a translation from preexisting PKM£a mRNA. Furthermore,real time PCR-based analysis showed resembling increase profile ofPKM£a mRNA after single CA injection, suggesting a co-upregulation of transcription and translation of PKM£a after CA administration in VTA. Eticlopride (dopamine receptor D2-subtype antagonist) ¡BSCH-23390(dopamine receptor D1-subtype antagonist)¡BH-89 (PKA inhibitor)¡B Wortmannin (PI3K inhibitor)¡BPD98059 (MEK1 inhibitor) decreasedcocaine-induced PKM£a expression within 1 hr in VTA. On the contrary, KN-62 (CaMK II inhibitor) has no obvious effect on PKM£a expression. CA challenge not only induces the PKM£a expression in the VTA region but also in the NAc and hippocampus region. The CA-induced PKM£a expression is more obvious in elder group (>45 days in age) than younger group (18~30 days in age), similar results also showed in the locomotor activity assay. Prenatal CA exposure decreased the postnatal CA-induced PKM£a expression and the locomotor sensitivity in younger group. Overall, results from our current experiments have raised the possibility of PKM£a involvement in CA addiction. How CA regulates PKM£a expression and the context dependence between PKM£a and CA-induced behavior change and synaptic long-term potentiation remains further elucidation.

Addiction is a chronic disease that characterize as habitual or compulsive involvement in an activity despite it¡¦s bring negative consequences. Some of psystimulants such as cocaine or amphetamine cause a strong reinforcing effects even after prolonged abstinence periods. Such illegal drugs not only hurt on the adult health but also result in fetal physiological damage. For example, that babies born to mothers who abuse with cocaine bring prematurely delivered, low birth weights, smaller head circumferences and increased heart disease in adult offspring. Mesolimbic dopamine system include nucleus accumbens (NAc) and ventral tegmental area (VTA) are critical regions for the neural adaptations that contribute to addiction. VTA that receives inputs from a large number of brain regions. For example, it receives glutamatergic inputs from prefrontal cortex, or GABAergic inputs from NAc. It has been known that VTA play a major role in the acquisition and expression of learned addictive behaviors. Results from many neuropharmacological studies in animal models indicate that exposure to cocaine or some other drugs of abuse seems to induce long-term potentiation (LTP) ¢w like changes of synaptic plasticity among neurons in VTA region. LTP was first described in hippocampus, a region that associated with memory formation, and were found widespread events in many mammalian brain sites. In the present time, theories and investigation indicated that memory and addiction might shared the similar neural circuitry and signal pathways. In general, LTP can be separate into two main phases : induction and maintenance phases. Many of molecules participate in induction phase such as calcium/calmodulin-dependent protein kinase II (CaMKII), cyclic AMP (cAMP), phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC). However, until now there was only one molecule has been found associated with LTP maintenance¡Xprotein kinase M£a (PKM£a). PKM£a is a brain specific, constitutively active form of PKC that does not need Ca2+ or diacylglycerol (DAG) for its activation. Molecular evidences showed that PKM£a is translated uniquely by PKM£a mRNA which is generated under the control of an internal promoter in the PKC£a gene. Recently, investigators introduced a PKM£a selective inhibitor¡XZIP, to hippocampus or insular cortex both successful to eliminate long-term spatial memory or conditioned taste aversion (CTA) behavior, respectively, on rat. Therefore, exclude PKM£a by specific inhibitors and then result in abolish long-term synaptic potentiation which had already established seem to be a leading candidate for cure addiction. Here we showed that blocked of PKM£a activity in VTA dopaminergic neuron eliminated mEPSCs or AMPAR/NMDAR ratio increment elicited by cocaine. Otherwise, our results also presented that myristoylatedinhibitory peptide¢wZIP had no effect on spike timing-dependent long-term potentiation in rats previously injected with saline but remarkably restored spike timing-dependent long-term potentiation in VTA dopamine neurons in slices prepared from rats that received single or multiple cocaine exposure. Furthermore, our western blot analyses showed that both single and five consecutive cocaine injections induced a significant increase in PKM£a level in VTA or NAc. Moreover, our ex vivo cocaine incubation results indicated that multiple kinases activation or de novo protein synthesis was required for PKM£a increment. The most important, our data provided the first physiological evidence between PKM£a and drug addiction when intracranial administered specific PKM£a inhibitors to VTA reversed cocaine-induced conditioned-place preference (CPP) behavior. Finally, we investigated the behavioral effect of cocaine-induced locomotor sensitization in an open field apparatus. Our data showed that peri-adolescent (P21) rats exhibited prominently increased in either acute or repeated cocaine-induced locomotor activity than mid-adolescent (P28) and post-adolescent (P41). Interestingly, applied to high dosage cocaine (30 mg/kg) rescued the acute locomotor response in P28 rats but not behavioral sensitization. We further examined the locomotion on rats that were exposed to cocaine in utero after single or multiple cocaine injection. However, cocaine-induced increase in locomotor activity was lower in P21 rats which exposed to cocaine during pregnancy but no significantly difference in P28 rats. Surprisingly, single high dose cocaine treatment caused a marked reduction in locomotor activity on P21 rats prenatally exposed to cocaine. Otherwise, we also provided the first evidences that repeated cocaine injection in pregnant rats induced a significant decreased to KCC2 level in PFC regions prepared from P20 rat. In conclusion, results from our current studies demonstrate for the first time that persistently active PKM£a is necessary in (1) mEPSC facilitation induced by single cocaine exposure; (2) cocaine-induced enhancement in AMPAR/NMDAR ratio; (3) single or repeated cocaine-induced LTP but not in LTP induced by spike-timing stimulation; and (4) cocaine conditioned place preference in the VTA. In addition, our results also present evidence that the expression of PKM£a is increased by either single or repeated cocaine exposure. Furthermore, our behavioral or Western blotting consequence of cocaine treatment in utero was reflected by the diminishion in the sensitivity of locomotor activity in postnatal rats to cocaine and KCC2 level in PFC regions.

Η μελέτη της φυλοσύνδετης μετάλλαξης wisertsl (1-21.7, 7E) της Drosophila melanogaster αποκάλυψε ότι υπεύθυνο για τους φαινότυπους φαγωμένα/τσιμπημένα φτερά, ελαφρώς ανώμαλα μάτια και τη θερμοευαισθησία είναι το γονίδιο CG32711, που ονομάσαμε wiser (wings scalloped-eyes rough). Το γονίδιο wiser είναι απαραίτητο για τη σωστή ανάπτυξη της Drosophila melanogaster. Η μετάλλαξη wisertsl χαρτογραφείται στη 5΄ ρυθμιστική περιοχή του γονιδίου wiser. Στην ίδια περιοχή χαρτογραφείται και η θανατογόνος μετάλλαξη wiserPL26. Παραπέρα μελέτη του γονιδίου wiser με τη χρήση αυτών των δυο μεταλλάξεων και του διαγονιδίου UAS-wiser αποκάλυψε ότι: α) Οι μεταλλάξεις wisertsl και wiserPL26 ενισχύουν το φαινότυπο των μεταλλάξεων Beadex1 και Serrate1. Το γονίδιο wiser αλληλεπιδρά με τα γονίδια Beadex και Serrate, τα οποία εμπλέκονται στην ενεργοποίηση του Notch μονοπατιού σηματοδότησης κατά μήκος του ραχιοκοιλιακού άξονα του φτερού. Η παρατήρηση αυτή δείχνει, ότι το wiser εμπλέκεται στον προσδιορισμό του ραχιοκοιλιακού άξονα. β) Η μετάλλαξη wisertsl σε ομοζυγωτία μειώνει σημαντικά την έκφραση των διαγονιδίων fringe-lacZ, m8-lacZ, wingless-lacZ, vestigial-lacZ και Distalless-lacZ, αλλάζει το πρότυπο έκφρασης του mβ-lacZ και δεν επηρεάζει την έκφραση του apterous-lacZ στους εμβρυικούς δίσκους του φτερού προνυμφών 3ου σταδίου. Τα αποτελέσματα αυτά έδειξαν, ότι το γονίδιο wiser δρα μετά το γονίδιο apterous και πριν το γονίδιο fringe, που είναι τροποποιητής του υποδοχέα Notch. Επομένως, η δράση του Notch εξαρτάται και από το wiser. γ) Εκτοπική έκφραση του διαγονιδίου UAS-wiser με οδηγό το ap-Gal4, έδειξε ότι διασώζει μερικώς το φαινότυπο apterous- αλλά όχι το φαινότυπο Serrate1. δ) Εκτοπική έκφραση του UAS-wiser με οδηγό το dpp-Gal4, επηρεάζει την έκφραση του wingless-lacZ αλλά όχι των apterous, fringe, mβ, m8, vestigial και Distalless στους εμβρυικούς δίσκους φτερού. ε) Η δημιουργία μιτωτικών κλώνων με το σύστημα FRT/FLP, σε θηλυκά άτομα wiserPL26/+ οδήγησε στη δημιουργία κλώνων +/+ και wiserPL26/wiserPL26 διαφορετικού μέγεθος στους εμβρυικούς δίσκους του φτερού. Οι πρώτοι (+/+), έχουν σημαντικά μεγαλύτερο μέγεθος από τους δεύτερους όταν συμβαίνουν στη περιοχή του εμβρυικού δίσκου που θα δώσει το φτερό του ακμαίου ατόμου. Στα ακμαία θηλυκά οι σωματικοί κλώνοι εκδηλώνονται με το φαινότυπο φαγωμένα φτερά. Σωματικοί κλώνοι παρατηρήθηκαν και στα μάτια των ακμαίων. Τα αποτελέσματα των μιτωτικών κλώνων δείχνουν ότι το γονίδιο wiser εμπλέκεται στον πολλαπλασιασμό των κυττάρων. Όλα τα παραπάνω αποτελέσματα, δείχνουν ότι το γονίδιο wiser είναι απαραίτητο για την ανάπτυξη του φτερού, καθώς εμπλέκεται στο σχηματισμό του ραχιοκοιλιακού άξονα και επηρεάζει τον πολλαπλασιασμό των κυττάρων. / The analysis of the X-linked wisertsl (1-21.7, 7E) mutation in Drosophila melanogaster has shown that responsible for the scalloped phenotype and the temperature sensitivity is the CG32711 gene, which we name wiser (wings scalloped-eyes rough). The gene wiser is essential for Drosophila development. The wisertsl mutation is mapped at the 5′ regulatory region of the gene CG32711. The wiserPL26 lethal mutation is mapped in the same region. Using these two mutations and a UAS-wiser transgene we have shown that: a) The wisertsl and wiserPL26 mutations increase the wing scalloping (phenotype) of the mutations Beadex1 and Serrate1. The genes Beadex and Serrate are implicated in the activation of Notch signaling pathway along the dorsal-ventral axis of the wing. This observation indicates that the wiser gene is involved in determination of dorsal-ventral axis. b) The wisertsl mutation in homozygous condition reduces substantially the expression of fringe-lacZ, m8-lacZ, wingless-lacZ, vestigial-lacZ and Distalless-lacZ transgenes, alters the expression pattern of mβ-lacZ and does not affect the expression of apterous-lacZ transgene in the wing imaginal disc. This indicates that the expression of fringe (a modifier of Notch receptor) is regulated by wiser too. c) Ectopic expression of UAS-wiser by the ap-Gal4 driver partially rescues apterous- but not Serrate1 phenotype. d) Ectopic expression of UAS-wiser by the dpp-Gal4 driver affects the expression of wingless and does not affects the expression of apterous, fringe, mβ, m8, vestigial and Distalless in the wing imaginal disc (revealed by the corresponding -lacZ strains). e) Induction of somatic clones with the FRT/FLP system in wiserPL26/+ mutants led to mitotic +/+ and wiserPL26/wiserPL26 clones of different sizes. The first clones were much larger than the second ones in the territory of wing pouch. Adult females with scalloped wings were also produced. These results indicate that the wiser gene is involved in cell proliferation. All the above findings suggest that the wiser gene is essential for wing development and cell proliferation.

The symptoms of Parkinson's disease occur due to degeneration of dopamine neurons in substantia nigra. It has been demonstrated that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor when it comes to protect and enhance survival of dopamine neurons in animal models of Parkinson's disease. The aim of this study was to evaluate short- and long-term effects of GDNF on survival and nerve fiber outgrowth of dopamine cells and astrocytic migration in mouse fetal ventral mesencephalic (VM) tissue. Primary tissue cultures were made of mouse fetal VM tissue and evaluated at 7 and 21 days in vitro (DIV) in terms of dopaminergic nerve fiber outgrowth and astrocytic migration when developed with GDNF present, partially, or completely absent. The results revealed that VM tissue cultured in the absence of GDNF did not exhibit any significant differences in migration of astrocytes or dopaminergic nerve fiber outgrowth neither after 7 DIV nor after 21 DIV, when compared with tissue cultured with GDNF present. Migration of astrocytes and dopaminergic nerve fiber outgrowth reached longer distances when tissue was left to develop for 21 DIV in comparison with 7 DIV. In order to study the long-term effects of GDNF, mouse fetal dopaminergic tissue was transplanted into the ventricles of adult mice and evaluated after 6 months. No surviving dopamine neurons were present in the absence of GDNF. In contrast dopamine neurons developed with GDNF did survive, indicating that GDNF is an essential neurotrophic factor when it comes to long-term dopamine cell survival. More cases have to be assessed in the future in order to strengthen the findings. Thus, transplanted dopamine neurons will be assessed after 3 and 12 months in order to map out when dopamine neurons deprived of GDNF undergo degeneration.

The ventral tegmental area (VTA) and its dopaminergic (DA) mesocorticolimbic projections are thought to be essential in the brain’s reward neurocircuitry. In humans and animal experimental subjects, mild electrical VTA stimulation increases dopamine levels and can induce euphoria. Paradoxically, aversive stimuli activate VTA neurons and forebrain DA activity, and excessive electrical stimulation of the VTA exaggerates fearfulness. Research suggests that experimental manipulation of either the amygdala or the VTA has similar effects on the acquisition and expression of Pavlovian conditioned fear. Recently it was demonstrated that electrical stimulation of the amygdala produced fear extinction deficits in rats. Fear extinction involves the progressive dissipation of conditioned fear responses by repeated non-reinforced exposure to a conditioned stimulus (CS). Maladaptive states of fear in fear-related anxiety disorders, such as post-traumatic stress disorders (PTSD) or specific phobias are thought to reflect fear extinction learning deficits. The primary purpose of the present study was to examine the effects of intra-VTA stimulation on fear extinction learning. Using fear-potentiated startle as a behavioural index of conditioned fear, it was found that 120 VTA stimulations paired or unpaired with non-reinforced CS presentations impaired the extinction of conditioned fear. This effect was not apparent in rats that received electrical stimulation of the substantia nigra (SN), suggesting that not all midbrain regions respond similarly. Electrical stimulation parameters did not have aversive affects because rats failed to show fear conditioning when electrical VTA stimulation was used as the unconditioned stimulus. Also, VTA stimulation did not alter conditioned fear expression in non-extinguished animals. Based on the results it is suggested that VTA activation disinhibited conditioned fear responding. Therefore, VTA neuronal excitation by aversive stimuli may play a role in fear-related anxiety disorders thought to reflect extinction learning deficits.