Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης είναι ομο- ή ετεροπενταμερείς διαμεμβρανικές γλυκοπρωτεΐνες οι οποίες, σχηματίζοντας ιοντικά κανάλια, συμβάλλουν στην λειτουργία του μυικού και νευρικού συστήματος. Η επικείμενη εμπλοκή των υποδοχέων αυτών σε διάφορες παθολογικές καταστάσεις του οργανισμού, επιβάλλει τη γνώση της δομής τους, απαραίτητη προϋπόθεση για την ανάπτυξη εξειδικευμένων θεραπευτικών προσεγγίσεων. Μέχρι τώρα, δεν έχουν δημοσιευτεί υψηλής ανάλυσης πληροφορίες για τη δομή του μορίου του ανθρώπινου υποδοχέα μέσω κρυσταλλογραφίας ακτίνων Χ ή μέσω πειραμάτων πυρηνικού μαγνητικού συντονισμού (NMR). Η απουσία των συγκεκριμένων πληροφοριών, οφείλεται στη φύση του μορίου του υποδοχέα όπως η ύπαρξη των υδρόφοβων διαμεμβρανικών περιοχών, το μέγεθος του μορίου καθώς και η αδυναμία απομόνωσης σε μεγάλες ποσότητες από φυσικές πηγές. Για το λόγο αυτό, είναι αναγκαίο να προκύψουν πρωτεϊνικά μόρια υδατοδιαλυτά, λειτουργικά, με δομή η οποία να πλησιάζει αρκετά τη φυσική διαμόρφωσή τους και σε ποσότητες ικανές ώστε να είναι εφικτές οι δομικές μελέτες τους. Τα τμήματα του νικοτινικού υποδοχέα της ακετυλοχολίνης, τα οποία συγκεντρώνουν τις παραπάνω προϋποθέσεις, είναι τα εξωκυτταρικά αμινοτελικά πολυπεπτίδια των διαφόρων υπομονάδων που τον αποτελούν. Από την άλλη πλευρά, ο οργανισμός που πρόκειται να χρησιμοποιηθεί ως σύστημα ετερόλογης έκφρασης, πρέπει να είναι εύκολος στους χειρισμούς του, γρήγορος, οικονομικός, με μεγάλο επίπεδο έκφρασης (όπως τα βακτήρια π.χ. E.coli) αλλά ταυτόχρονα να έχει την ικανότητα να πραγματοποιεί μεταμεταφραστικές τροποποιήσεις στις πρωτεΐνες που εκφράζει ώστε τα παραγόμενα μόρια να μπορούν να αναδιπλωθούν (όπως τα ευκαρυωτικά κύτταρα). Άρα, για να προκύψουν τα επιθυμητά μόρια, σύμφωνα με τις παραπάνω προϋποθέσεις, εκφράστηκαν τα εξωκυτταρικά τμήματα (ECDs) των ανθρώπινων υπομονάδων α1, α4 και β2 στο υπερκείμενο καλλιέργειας του μεθυλοτροφικού ζυμομύκητα P. pastoris. Αρχικά, μελετήθηκαν τα εξωκυτταρικά τμήματα των α4 και β2 υπομονάδων με διαφορετικούς επιτόπους ώστε να γίνει η επιλογή του καλύτερου συνδιασμού που πρόκειται να χρησιμοποιηθεί για έκφραση των δύο υπομονάδων στο ίδιο σύστημα. Η έκφραση των α4 και β2 ECDs με τον επίτοπο των έξι αμινοξικών καταλοίπων στο καρβοξυτελικό τους άκρο (α4-ECD-6xHis και β2-ECD-6xHis) στο υπερκείμενο καλλιέργειας του P. pastoris, κατέληξε στην απομόνωση μικρών ποσοτήτων πρωτεϊνικών συσσωματωμάτων τα οποία κρίνονται ακατάλληλα για δομικές μελέτες εξαιτίας της υδροφοβικότητας και κατά συνέπεια του μεγέθους τους. Όταν πραγματοποιήθηκε αλλαγή της υδρόφοβης περιοχής μεταξύ των κυστεινών 128 και 142 με την αντίστοιχη υδρόφιλη ολιγοπεπτιδική αλληλουχία της πρωτεΐνης η οποία δεσμεύει ακετυλοχολίνη (AChBP) στις ανασυνδιασμένες πρωτεΐνες, παρατηρήθηκε αισθητή βελτίωση για την β2-ECD υπομονάδα (β2-cysloop-ECD-6xHis) τόσο σε επίπεδο έκφρασης όσο και στο μέγεθος το οποίο σχηματίζει. Ωστόσο, παρόμοια βελτίωση για την α4-cysloop-ECD-6xHis υπομονάδα, παρατηρήθηκε όταν αντικαταστάθηκε ο καρβοξυτελικός επίτοπος των έξι αμινοξικών καταλοίπων ιστιδίνης από τον επίτοπο του οκταπεπτιδίου FLAG (α4-cysloop-ECD-FLAG). Στα πειράματα συνέκφρασης, των δύο υπομονάδων στο ζυμομύκητα P. pastoris που ακολούθησαν, αρχικά πιστοποιήθηκε η συνέκφραση και στη συνέχεια ο σχηματισμός ετεροπολυμερούς των δύο υπομονάδων. Ακόμα, η απογλυκοζυλίωση των α4-cysloop-ECD-FLAG και β2-cysloop-ECD-6xHis όταν εκφράζονται μόνες τους ή όταν εκφράζονται ταυτόχρονα στο υπερκείμενο του P. pastoris, φανέρωσε την αύξηση της ομοιογένειας των μορίων τους. Επιπλέον, ενθαρυντική ήταν η εύρεση της επιθυμητής στοιχειομετρίας 2 α4/ 3 β2 των υπομονάδων βάση της οποίας συμμετέχουν στο σχηματισμό του ετεροπολυμερούς. Ωστόσο, η μάζα του ετεροπολυμερούς των ανασυνδιασμένων α4 και β2 υπομονάδων, όπως προέκυψε από την επεξεργασία των αποτελεσμάτων της χρωματογραφίας μοριακής διήθησης (1191 και 496kDa), απέχει κατά πολύ από την θεωρητικά αναμενόμενη μάζα ετεροπενταμερούς των 175kDa. Μελέτες δυναμικής σκέδασης φωτός σε δείγματα του απομονωμένου ετεροπολυμερούς, επιβεβαίωσαν το παραπάνω αποτέλεσμα. Τα αποτελέσματα της μελέτης μείωσης του μεγέθους των ετεροπολυμερών με απορρυπαντικά, έδειξαν ότι είναι δυνατή η επιθυμητή μείωση του μεγέθους αλλά σε υψηλές τιμές συγκεντρώσεων απορρυπαντικού. Επιπλέον, η ανάγκη για ακόμα υψηλότερες τιμές απορρυπαντικών προς βελτίωση της ομοιογένειας δεν συνίσταται, διότι κατευθύνει τους σχηματισμούς των πρωτεϊνικών μοριών σε ασταθείς καταστάσεις οι οποίες χαρακτηρίζονται από ευμετάβλητες διαμορφώσεις και πολλές φορές καταστροφή των υπό μελέτη πρωτεϊνών. Ακόμα, το γεγονός ότι τα απομονωμέμενα ετεροπολυμερή των α4 και β2 ECDs δεν εμφανίζουν την ικανότητα δέσμευσης νικοτίνης, σε συνδιασμό με τα παραπάνω αποτελέσματα μελέτης τους, οδήγησαν στο συμπέρασμα ότι δεν τηρούν τις προϋποθέσεις των κατάλληλων πρωτεϊνικών δειγμάτων τα οποία προορίζονται για πειράματα μελέτης της δομής τους. Η έκφραση της ανασυνδιασμένης α1-ECD με τον επίτοπο των έξι αμινοξικών καταλοίπων στο καρβοξυτελικό της άκρο (α1-ECD-6xHis) στο υπερκείμενο καλλιέργειας του P. pastoris, κατέληξε στην απομόνωση υδατοδιαλυτού και λειτουργικού μορίου. Οι πληροφορίες των μελετών κυκλικού διχρωισμού σε δείγμα της απομονωμένης ανασυνδιασμένης πρωτεΐνης, δείχνουν ότι πρόκειται για ένα μόριο με διαμόρφωση και μάλιστα, σε επίπεδο δευτεροταγούς δομής, εμφανίζει ένα πλούσιο περιεχόμενο β-πτυχωτής επιφάνειας (40,9%). Ωστόσο, οι δοκιμές κρυστάλλωσης στις οποίες τέθηκαν δείγματα της α1-ECD-6xHis, δεν απέδωσαν το επιθυμητό αποτέλεσμα της κρυστάλλωσης του μορίου. Ακόμα και όταν πραγματοποιήθηκε προσπάθεια μελέτης του μορίου σε διαλύματα (NH4)2SO4 με μεταβλητή συγκέντρωση, pH και θερμοκρασία, στάθηκε αδύνατο να κρυσταλλωθεί το μόριο. Το πρόβλημα της συγκεκριμένης έκφρασης, εντοπίστηκε επιπλέον και στο ποσό της ανασυνδιασμένης πρωτεΐνης που είναι δυνατόν να απομονωθεί. Για να αυξηθεί το επίπεδο έκφρασης, πραγματοποιήθηκαν πειράματα μεταβολής των συνθηκών καλλιέργειας (θερμοκρασία, σύσταση θρεπτικού μέσου) όπως επίσης και των συνθηκών απομόνωσης. Παρατηρήθηκε βελτίωση στην ποσότητα του τελικού απομονωμένου προϊόντος η οποία όμως δεν θεωρείται ικανοποιητική. Για το λόγο αυτό, στη συνέχεια, πραγματοποιήθηκε έκφραση και απομόνωση της α1-ECD με τον επίτοπο του οκταπεπτιδίου FLAG στο αμινοτελικό της άκρο, στο υπερκείμενο καλλιέργειας του ίδιου ζυμομύκητα. Το επίπεδο έκφρασης της συγκεκριμένης ανασυνδιασμένης πρωτεΐνης, ήταν αρκετά ικανοποιητικό (1mg πρωτεΐνης /lt υπερκειμένου καλλιέργειας) και μάλιστα αυξημένο κατά πολύ σε σχέση με τις προηγούμενες εκφράσεις (0,34mg πρωτεΐνης /lt υπερκειμένου καλλιέργειας). Ωστόσο, η αδυναμία ορθής λήψης φάσματος του μορίου μέσω κυκλικού διχρωισμού εξαιτίας αυξημένου θορύβου, στάθηκε η αιτία να σταματήσει η έκφραση της συγκεκριμένης ανασυνδιασμένης πρωτεΐνης. / Nicotinic acetylcholine receptors are homo- or heteropentameric transmembrane glycoproteins which form ion channels and contribute to the function of muscles and neurons. The involvement of these receptors in pathological situations, imposes the knowledge of their structure for the development of specific therapeutic approaches. Till now, there is not sufficient knowledge about the structure of the human acetylcholine receptor through x-ray crystallography or through NMR experiments. The absence of these data, comes from the nature of the receptor molecules such as the existence of hydrophobic transmembrane domains, the size of the molecule. Therefore, it is necessary to obtain hydrophilic and functional protein molecules with native-like structure and in adequate quantities in order to be possible structural studies of these molecules. So, the most suitable domains of the acetylchlonine receptors to be used for these studies, are the extracellular domains. At the beginning, studies of extracellular domains of α4 and β2 subunits with different tags were accomplished in order to choose the best combination to be used in cooexpression experiments in Pichia Pastoris. The expression of the extracellular domains of α4 and β2 tagged with 6xHis in the C-terminus (α4-ECD-6xHis and β2-ECD-6xHis) to the supernatant of the P. pastoris culture, gave very little amounts of protein aggregates which are not suitable for structural studies because of their hydrophobicity. When the hydrophobic region between Cys 128 and Cys 142 was changed with the respective hydrophilic region from Acetylcholine Binding Protein, an increase of expression of the β2-ECD subunit and a decrease of protein aggregation was observed. However, a similar improvement for α4-cysloop-ECD-6xHis subunit was observed after substitution of 6xHis tag with FLAG tag. Thereafter, the cooexpression experiments through P. pastoris, showed the formation of an heteropolymer which is formed by the two different subunits. Moreover, the in vitro deglycosylation of α4-cysloop-ECD-FLAG and β2-cysloop-ECD-6xHis, either they are coexpressed or not, revealed an improvement of homogeneity of these molecules. Besides these results, the determination of the desirable stoichiometry 2 α4/ 3 β2 of the subunits that participate in the heteropolymer, was also encouraging. However, the estimation of protein mass of the α4β2 heteropolymer, through gel filtration chromatography (1191 and 496 kDa), showed that these formations are much bigger than the expected mass of heteropentamers. This result is also confirmed by dynamic light scattering experiments. The results from decreasing the size of heteropolymers in the presence of detergents, showed that this is possible to be achieved in high concentrations of detergents with the danger of denaturing the protein molecules. So, it was obvious that this strategy for studying the structure of the extracellular domain of α4β2 receptors, had to be changed. The expression of the extracellular domain of α1 subunit tagged with 6xHis to the supernatant of P. pastoris culture, led to the isolation of water soluble and functional molecule. Data from circular dicroism studies in a protein sample, showed that α1-ECD has a formation rich in β-sheets (40.9%). However, crystallization trials in samples of α1-ECD-6xHis, gave no protein crystals. Moreover, the yield of the purified protein was too small. In order to achieve an increase of the protein yield, experiments of modifying the temperature and the composition of nutrient were carried out. The result from these experiments showed a slight improvement for the final quantity of protein that is obtained but not satisfactory. For this reason, there were performed expression and isolation of the extracellular domain of α1 subunit with a FLAG tag in the N-terminus, to the supernatant of P. pastoris culture. The quantity of purified protein was dramatically increased (1mg protein/lt of culture supernatant). However, it was not possible to obtain a circular dichroism spectrum without background noise and that was the reason for not continuing the expression of this protein.

Nicotinic acetylcholine receptors (nAChR) are neurotransmitter-gated ion channels that exist as a family of subtypes defined by unique subunit compositions. nAChR containing α6 subunits (α6*-nAChR) have attracted interest because α6 subunits are thought to be localized in brain regions implicated in reward, mood and drug dependence. To provide new information necessary toward a more complete understanding of roles of α6*-nAChR in neuropsychiatric health and disease, three lines of investigation were pursued. A set of stably transfected, human, immortalized cell lines were generated that heterologously express nAChR α6 subunits in combination with other nAChR subunits found in reward brain regions (nAChR subunit combinations α6β2, α6β4, α6β2β3, α6β4β3, α6β2β3α5, α6β4β3α5, α6α4β2β3 and α6α4β4β3). The α6α4β2β3 combination may have a functional response to epibatidine that differs from that of the α4β2 nAChR. A unique binding site was identified in cells transfected with the α6β4β3α5 nAChR subunit combination. Messenger RNA fluorescence in situ hybridization (mRNA FISH) studies established regional and celluar distribution of nAChR α6 subunit mRNA in the mouse brain. The third line of study extended this work to examine potential co-expression of nAChR α6 subunits and glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH) as labels of GABAergic and dopaminergic/catecholaminergic neurons respectively, using tandem mRNA FISH and fluorescence immunohistochemistry. nAChR α6 subunit signal in the substantia nigra (SN) and ventral tegmental area (VTA) was congruent with previous studies. Message was also detected in the amydala, dentate gyrus, striatum, zona incerta, and cingulate, entorhinal, perirhinal, piriform, and prelimbic cortices. nAChR α6 mRNA was coexpressed with GAD in the amygdala, dentate gyrus, striatum, SN, VTA and cingulate, entorhinal, prelimbic and prelimbic cortices. TH was exclusively co-localized with nAChR α6 mRNA in the SN and VTA. Findings suggest extended roles for α6*-nAChR in the brain, particularly in the control of GABAergic neuronal activity and/or GABA release. These studies provide new insights into the composition of α6*-nAChR, the localization and cellular origins of nAChR α6 subunit expression. Data collected suggest roles for α6*-nAChR in many brain regions, including those involved in higher order processes involved in drug dependence and reward, and in modulation of inhibitory neurotransmission.

Agrin triggers the subsynaptic aggregation of acetylcholine receptor (AChR) via activation of the receptor tyrosine kinase MuSK (muscle-specific kinase). At present, the intracellular mechanisms utilized by MuSK to initiate such a complex process remain unknown. In the present study, I first tested if H-ras was involved in the process of synaptogenesis induced by agrin. The data presented suggest that ras could have a role in this process because a dominant inhibitory ras mutant (ras-N17) partially blocked the inductive effects of agrin while two activated ras mutants (ras-V12 and ras-V12-D38) induced agrin-independent AChR clusters. These effects were not due to major alterations in the levels of AChR, though more experiments are required to confirm these preliminary findings. / Second, I investigated whether the adaptor protein Shc was a downstream effector of activated MuSK. MuSK and Shc could be co-immunoprecipitated, but this association was not consistently observed nor was it modulated by agrin at all times. Generally, no alteration in Shc phosphotyrosine content was observed in response to agrin, and when an increase was detected, it was modest. Finally, agrin did not modulate the interaction between Shc and Grb2. Based on these results, I conclude that Shc interaction with MuSK is not regulated by agrin.

M5 muscarinic receptors are associated with dopamine neurons of the ventral tegmental area (VTA) and substantia nigra, and provide an important excitatory input to the mesolimbic dopamine system. Here, I studied locomotion induced by systemic morphine (3, 10, 30 mg/kg, i.p.) in M5 knockout mice of the C57Bl/6 (B6) and CD1 x 129SvJ (129) background strains. M5 knockout mice of both strains showed reduced locomotion in response to 30 mg/kg morphine, while only B6 M5 knockout mice showed reduced locomotion in response to 10 mg/kg morphine. In B6 wild-type mice VTA pre-treatment with the non subtype-selective muscarinic receptor antagonist atropine (3 mg per side), but not the non subtype-selective nicotinic receptor antagonist mecamylamine (5 mg per side), reduced locomotion in response to 30 mg/kg (i.p.) morphine to a similar extent as systemic M5 knockout, suggesting that the reduced morphine-induced locomotion in M5 knockout mice was due to the loss of M5 receptors on VTA dopamine neurons. By contrast, in M5 knockout mice, either intra-VTA atropine or mecamylamine alone increased locomotion by almost 3 times relative to saline, and potentiated morphine-induced locomotion. Therefore, in M5 knockout mice, more clearly than in wild-type mice, blockade of either VTA muscarinic or nicotinic receptors activated locomotion. Infusions of morphine (50 ng) into the VTA increased nucleus accumbens dopamine efflux in urethane-anesthetized wild-type mice. Either M5 knockout or pre-treatment with VTA scopolamine (50 ug) in wild-type mice blocked accumbal dopamine efflux in response to VTA morphine. Therefore, M5 receptors are critical for excitation of dopamine neurons by intra-VTA morphine, suggesting that the reduced locomotion produced by systemic morphine in M5 knockout mice was, in part, due to loss of M5-mediated excitation of VTA dopamine neurons by opiates. The locomotion data also show that in the absence of M5 receptors, cholinergic afferents to mesolimbic dopamine neurons are inhibitory. This supports and extends the conclusions from many studies that non-M5 muscarinic receptors inhibit, and M5 receptors excite, dopamine neurons. Loss of M5-mediated excitation results in reduced acute effects of opiates.

M5 muscarinic receptors are associated with dopamine neurons of the ventral tegmental area (VTA) and substantia nigra, and provide an important excitatory input to the mesolimbic dopamine system. Here, I studied locomotion induced by systemic morphine (3, 10, 30 mg/kg, i.p.) in M5 knockout mice of the C57Bl/6 (B6) and CD1 x 129SvJ (129) background strains. M5 knockout mice of both strains showed reduced locomotion in response to 30 mg/kg morphine, while only B6 M5 knockout mice showed reduced locomotion in response to 10 mg/kg morphine. In B6 wild-type mice VTA pre-treatment with the non subtype-selective muscarinic receptor antagonist atropine (3 mg per side), but not the non subtype-selective nicotinic receptor antagonist mecamylamine (5 mg per side), reduced locomotion in response to 30 mg/kg (i.p.) morphine to a similar extent as systemic M5 knockout, suggesting that the reduced morphine-induced locomotion in M5 knockout mice was due to the loss of M5 receptors on VTA dopamine neurons. By contrast, in M5 knockout mice, either intra-VTA atropine or mecamylamine alone increased locomotion by almost 3 times relative to saline, and potentiated morphine-induced locomotion. Therefore, in M5 knockout mice, more clearly than in wild-type mice, blockade of either VTA muscarinic or nicotinic receptors activated locomotion. Infusions of morphine (50 ng) into the VTA increased nucleus accumbens dopamine efflux in urethane-anesthetized wild-type mice. Either M5 knockout or pre-treatment with VTA scopolamine (50 ug) in wild-type mice blocked accumbal dopamine efflux in response to VTA morphine. Therefore, M5 receptors are critical for excitation of dopamine neurons by intra-VTA morphine, suggesting that the reduced locomotion produced by systemic morphine in M5 knockout mice was, in part, due to loss of M5-mediated excitation of VTA dopamine neurons by opiates. The locomotion data also show that in the absence of M5 receptors, cholinergic afferents to mesolimbic dopamine neurons are inhibitory. This supports and extends the conclusions from many studies that non-M5 muscarinic receptors inhibit, and M5 receptors excite, dopamine neurons. Loss of M5-mediated excitation results in reduced acute effects of opiates.

Thesis (Ph.D.)--Tufts University, 1998. / Adviser: Barry A. Trimmer. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 92-105). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 116-138).