Ph.D. / Omslagstitel: Metabotropic glutamate receptor 4 activation and expression.

Ph.D.

Huntington's disease (HD) is an inherited autosomal-dominant neurodegenerative disease caused by the abnormal expansion of CAG repeats in exon 1 of the huntingtin gene located on chromosome 4. This disease is characterized by the premature loss of medium spiny neurons in the striatum and behavioural deficits that typically manifest at middle-age. Despite the identification of its cause decades ago, there is still no disease modifying treatment available for HD patients. Current evidence indicates that exacerbated glutamate signalling in the striatum plays a key role in the pathophysiology of HD. Within the striatum, metabotropic glutamate receptor (mGluR) 2/3 are predominantly expressed on presynaptic terminals, whereas mGluR5 is predominantly localized to postsynaptic terminals. Here, we show that both the activation of mGluR2/3 and the inhibition of mGluR5 can improve HD symptoms in the zQ175 HD mouse model. Specifically, treating zQ175 HD mice with either the mGluR2/3 agonist LY379268 or the mGluR5 negative allosteric modulator (NAM) CTEP rescues motor deficits, reduces mutant huntingtin aggregate formation, improves neuronal survival and alleviates microglia activation. We also provide evidence that shows sex can influence the progression of HD symptoms and the efficacy of therapeutic agents. We found that chronic administration of LY379268 differentially activated and inactivated cell signalling pathways in male and female zQ175 mice. Furthermore, female zQ175 mice required a longer treatment duration with CTEP than male mice to show improvement in their rotarod performance. Using FDNQ175 mice, a newer HD mouse model derived from the zQ175 line, we demonstrated that female FDNQ175 mice were less susceptible to decline in limb function than male mice but showed higher levels of insoluble mutant huntingtin aggregates at a younger age.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 104-118).

Schizophrenia is a serious psychiatric disorder that affects 1% of the population. Current theories implicate NMDA receptor hypofunction as a contributor to the symptomology and pathological alterations in schizophrenia. Cognitive impairments are increasingly recognized as not only fundamental to schizophrenia, but the strongest predictor of patient functional outcomes. Current antipsychotics do not improve the cognitive symptoms of the disorder; however, recent efforts have resulted in the identification of novel drug targets. One target is metabotropic glutamate receptors as they interact with and modulate NMDA receptors. Another approach focuses on dopamine, the neurotransmitter system targeted by traditional antipsychotics. Tetrahydroprotoberberines, such as D- and L-govadine, are synthetic compounds derived from traditional medicine that have demonstrated efficacy in treating schizophrenic symptoms. The present study assessed the effects of CDPPB (a metabotropic glutamate receptor 5 positive allosteric modulator), D- and L-govadine, and the typical antipsychotic haloperidol on the Paired Associates Learning (PAL) task in rats. The PAL task is impaired in patients with schizophrenia, has been adapted for use with rodents using touchscreen-equipped operant chambers, and has been promoted by MATRICS as a promising behavioural task with the potential to further translational health research in schizophrenia. The objectives of this study were: 1) examine the effects of acute NMDA receptor antagonism with MK-801 as a model for schizophrenia on performance of the PAL task; 2) test the effects of the putative antipsychotics, CDPPB and D- and L-govadine on reversing the effects of NMDA receptor antagonism on the task; and 3) to compare these novel therapeutics to a classic antipsychotic. Two squads of male Long-Evans rats were trained to perform the PAL task in touchscreen-equipped operant chambers. After the rats reached criterion the following treatment schedules were divided between the two squads: 1) vehicle (10% cyclodextrin; i.p.), and CDPPB (1.0, 3.0, and 10.0 mg/kg, i.p.); or 2) vehicle (10% cyclodextrin; i.p.), CDPPB (3.0 mg/kg, i.p.), the NMDA receptor antagonist MK-801 (0.15 mg/kg, i.p.), and CDPPB with MK-801; or 3) vehicle (50% DMSO; s.c.), MK-801, D-govadine (1.0 mg/kg; s.c.), L-govadine (1.0 mg/kg; s.c.) and MK-801 with each isomer of govadine; or 4) vehicle (sodium acetate and acetic acid, pH 5.0, s.c.), and haloperidol (0.05 and 0.1 mg/kg, s.c.). Acute MK-801 significantly reduced the number of trials completed, impaired accuracy, and increased the number of errors in the PAL task. CDPPB had no effect on the PAL task and did not improve the MK-801 induced impairments. Administration of L-govadine, but not D-govadine, prior to MK-801 improved accuracy and reduced errors compared to MK-801 alone. L-govadine alone, but not D-govadine, reduced total responding compared to vehicle. Haloperidol caused a dose-dependent decrease in all activity in the task confounding interpretation of the results in regard to cognition. These data establish disruptive effects of acute MK-801 treatment on PAL task performance and demonstrate that L-govadine is capable of cognitive enhancement in a rodent model of schizophrenia.

Immunohistochemical studies were conducted to assess the subunits of ionotropic and metabotropic glutamate receptor present in the rostral ventrolateral medulla (RVLM) of the rat. Double labeling the medullary sections with polyclonal GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B, mGluR1α, and mGluR2/3 antiserum and monoclonal tyrosine hydroxylase (TH) antiserum revealed nearly all TH immunoreactive (irTH) cells and many TH-negative neurons were immunoreactive to GluR2/3 (irGluR2/3), NMDAR1 (irNMDAR1), and NMDAR2A/B (irNMDAR2A/B). A few RVLM neurons were immunoreactive to GluR1 (irGluR1) and GluR4 (irGluR4), but they were generally TH-negative. Immunoreactivity to mGluR1α (irmGluR1α) appeared to be localized exclusively to fiber-like elements in the RVLM area. Our results show that neurons in the RVLM, including irTH, are endowed mainly with GluR2/3 and NMDAR1 or NMDAR2A/B ionotropic receptor subunits, and that irmGluR1α splice variant appears to be located on nerve fibers ramifying within the RVLM. Moreover, TH-negative neurons in the RVLM appear to bear similar subunits of ionotropic glutamate receptors.

The retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and thalamic relay neurons, undergoes extensive remodeling and refinement in the first few postnatal weeks. While many studies have focused on this process, little is known about the factors that influence excitatory transmission during this dynamic period. A major goal of my dissertation research was to identify mechanisms that regulate glutamate release and clearance at the developing synapse. First, we investigated the role of glutamate transporters and metabotropic glutamate receptors (mGluRs) in shaping excitatory transmission. Early in development, we found presynaptic group II/III mGluRs are present and are activated by glutamate released from RGCs following optic tract stimulation at natural frequencies. This response was found to diminish with age, but glutamate transporters continued to shape synaptic currents throughout development. The finding that glutamate is able to escape the synaptic cleft and bind extrasynaptic high-affinity mGluRs led us to speculate that glutamate might also diffuse to neighboring synapses and bind ionotropic glutamate receptors opposing quiescent release sites. Excitatory currents recorded from immature, but not mature, retinogeniculate synapses display a prolonged decay timecourse. We found evidence that both asynchronous release of glutamate as well as spillover of glutamate between neighboring synapses contributes to these slowly decaying synaptic currents. Furthermore, we uncovered and characterized a novel, purely spillover-mediated current from immature relay neurons, which strongly supports the presence of glutamate spillover between boutons of different RGCs. The results of my studies indicate that far more RGCs contribute to relay neuron firing than would be predicted by the anatomy alone. Finally, in an ongoing study, we investigated the functional role of the neuronal glutamate transporter GLT-1 at the immature retinogeniculate synapse. While GLT-1 has been found in both neurons and glia, excitatory currents at the retinogeniculate synapse were largely unaffected in mice lacking neuronal GLT-1, suggesting non-neuronal glutamate transporters are responsible for the majority of glutamate removal from the developing synapse. Taken together, these results provide insight into the synaptic environment of the developing retinogeniculate synapse and identify a number of mechanisms that shape excitatory transmission during this period of synaptic maturation and refinement.

Aromatase is an enzyme that converts androgens to estrogens. In teleosts, brain aromatase, also known as aromatase B (cy19a1b), is only expressed in radial glial cells (RGCs). This is in contrast to aromatase A, which is expressed in gonads. Estrogens such as estradiol (E2) modulate neurogenesis in the adult teleost brain. Recent studies show that E2 also differentially regulates aromatase B expression in goldfish RGCs. As a result, teleost RGCs are suggested to be involved in regulating neurogenesis. In addition, aromatase B expression in goldfish RGC is under the control of dopamine suggesting that neurons and neurotransmitters can regulate RGC function. Interestingly, goldfish RGC transcriptome data shows the expression of one group of metabotropic glutamate receptors (mGluRs), group III mGluRs, which suggests that glutamate may affect RGC function. In this thesis, I present my findings regarding potential glutamatergic regulation of RGCs. Firstly, I investigated the distribution of glutamatergic synaptic vesicles and RGCs in the female goldfish forebrain. Double-staining immunohistochemistry shows that vesicular glutamate transporter (vGLUT) 1/2-labelled glutamatergic synaptic vesicles are in close anatomical proximity to aromatase B-labelled RGCs, which suggests potential regulation of RGCs by glutamate. Glutamatergic regulation of cyp19a1b, cyclin D1 (ccnd1), cyclin A2 (ccna2), mGluR6b (grm6b), mGluR7 (grm7), and mGluR8b (grm8b) expression in cultured adult female goldfish RGCs was also examined. Results from pharmacological manipulations and qPCR data analysis show that selective activation of group III mGluRs decreased cyp19a1b, ccnd1, and ccna2 mRNA via inhibition of cAMP/PKA signalling. Furthermore, grm7 mRNA is positively regulated by cAMP-dependent signalling. The glutamate analog L-glutamic acid decreased cyp19a1b mRNA and increased ccnd1 and grm6b mRNA in a dose-dependent manner. This suggests that ccnd1 and grm6b expression may be regulated by glutamate receptors other than group III mGluRs, for example, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are expressed in cultured goldfish RGCs. It was found that E2 upregulated cyp19a1b, ccnd1 and grm7 mRNA. However, selective activation of group III mGluRs decreases the stimulatory effect of E2 on ccnd1 expression. My findings show that glutamate finely regulates RGC neurogenic and steroidogenic genes, which may implicate glutamate in the regulation of RGC differentiation, RGC proliferation, and neurogenesis in surrounding cells.

No description available.

of the thesis Glutamate is a main excitatory neurotransmitter in the brain of mammals, which activates both ionotropic and metabotropic glutamate receptors. Ionotropic receptors are responsible for fast synaptic transmission leading to membrane depolarization and Ca2+ influx into the cell. On the other hand mGlu receptors play an important role in regulation of the transmission via heterotrimeric G-proteins and activation of various signaling pathways. Postsynaptically localized group I mGlu receptors (mGluR1, 5) together with ionotropic NMDA and AMPA receptors share common large receptor signaling complexes, or signalosome facilitating glutamate signal transductions. Individual mGluR1 splice variants are differently associated with signalosome including scaffold proteins like PSD-95 which organize postsynaptic density (PSD). Heterodimerization of different mGluR1 splice variants is a focal point of my thesis together with investigation of recently discovered protein IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) and its role in organization of postsynaptic signalosome. Using biochemical, immunocytochemical and functional assays we showed heterodimers of mGluR1a/1b were expressed on the plasma membrane and that heterodimers are fully functional in the recombinant system. Next we showed...