The retina contributes to visual submodalities by anatomically and functionally distinct parallel pathways. In this thesis, the synaptic connectivity of cell types in parallel visual pathways in marmoset (New World primate) and macaque (Old World primate) retinas are studied. / In Chapter 1 the main anatomical and physiological properties of parallel pathways in the primate retina are described. / Diffuse bipolar (DB) cells receive synaptic input from multiple cones and provide output to ganglion cells, but their synaptic connectivity in the inner plexiform layer is not well understood. In Chapter 2, the stratification and synaptic connectivity of DB6 axon terminals are described. It is shown that the axons of DB6 cells stratify in the same region as rod bipolar cells and blue-OFF/yellow-ON ganglion cells. The majority (86%) of their synaptic output is onto amacrine cells; only 14% goes to ganglion cells. The DB6 cells may synapse with amacrine cells in the rod pathway and the blue-OFF/yellow-ON ganglion cells. / The inhibitory neurotransmitter glycine is used by about half of all amacrine cells. Using immunohistochemical methods, the experiments in Chapter 3 investigated whether different bipolar and amacrine cell types differ with respect to the expression of glycine receptor (GlyR) subtypes. Postembedding electron microscopy showed the postsynaptic location of the a1, a2 and a3 subunits of the GlyR. Double immunofluorescence demonstrated that firstly, the three a subunits are clustered at different postsynaptic sites, and secondly that OFF bipolar types are predominantly associated with the a1 subunit, whereas ON bipolar types are predominantly associated with the a2 subunit. This shows that different amacrine cell types at synapses express different types of glycine receptors. / The midget pathway is involved in processing red-green colour vision and high spatial acuity. In Chapter 4, the synaptic connectivity of OFF midget bipolar cells was investigated in the central retina of marmosets and macaque. The OFF midget bipolar cells and their synapses were identified immunohistochemically. Midget ganglion cells in marmosets were retrogradely labelled from the parvocellular layers of the dorsal lateral geniculate nucleus. Consistent with previous studies of Old World primates, it is shown that in marmoset the midget bipolar cells contact midget ganglion cells at a ratio of 1:1. The number of output synapses was quantified for 330 OFF midget bipolar cells (n = 104, dichromatic marmoset; n = 108, trichromatic marmoset; n = 118, macaque). The average number of output synapses per axon terminal was comparable for all animal phenotypes. In all animals the number of output synapses per axon terminal showed a unimodal distribution. Our results suggest that the midget circuitry in central retina is comparable in dichromatic and trichromatic animals. / The midget pathway in mid-peripheral retina has been suggested to involve colour selective wiring of midget bipolar to midget ganglion cells. The question whether there is anatomical evidence for colour selective wiring was addressed in Chapter 5, in double labelled preparations of marmoset retina where OFF midget bipolar and OFF midget ganglion cells were identified. The relationship of the bipolar axon terminal mosaic and the dendritic fields of midget ganglion cells was analysed. No anatomical evidence for colour-selective connectivity in the inner retina of marmosets was found.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 100-123).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 122-130).

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references.

Transient receptor potential (TRP) channels play an important role in sensory and nonsensory functions. TRPVanilloid 1 and TRPVanilloid 4 are proposed to be involved in inflammation-induced pain. TRPV1 is extensively studied and it is specifically involved in inflammatory thermal hypersensitivity. Mechanical hypersensitivity is one of the significant components of nociception. Several receptors have been proposed to underlie mechanosensation. The molecular entities responsible for mechanosensation are not fully understood. In this study, I have characterized the properties of TRPV4, a putative mechanosensitive ion channel expressed in dorsal root ganglion (DRG) neurons and nonsensory tissues. First, I have investigated the expression and function of TRPV4 and TRPV1 in the DRG neuronal cell bodies as well as their central terminals and determined the modulation by protein kinase C (PKC). Both TRPV4 and TRPV1 are expressed in DRG and laminae I and II of the spinal dorsal horn (DH). Ca2+ fluorescence imaging and whole-cell patch-clamp experiments showed that both capsaicin-induced TRPV1 response and 4alpha-phorbol 12, 13-didecanoate (4alpha-PDD)-induced TRPV4 response were observed in a proportion of the same DRG neurons, suggesting their co-expression. Incubation of DRG neurons with phorbol 12, 13-dibutyrate (PDBu), a PKC activator, resulted in a significantly greater potentiation of TRPV4 currents than TRPV1 currents. In HEK cells heterologously expressing TRPV4, PDBu potentiated TRPV4-mediated single-channel current activity. In patch-clamped DH neurons, the application of 4alpha-PDD at the first sensory synapse increased the frequency but not the amplitude of the miniature excitatory postsynaptic currents (mEPSCs), suggesting a presynaptic locus of action. 4alpha-PDD-induced increase in the frequency of mEPSC was further facilitated by PDBu. These results suggest that TRPV4 in the central terminals modulates synaptic transmission and is regulated by PKC. Second, I have studied the mechanosensitivity of TRPV4 in cell-attached patches by applying direct mechanical force via the patch pipette. In TRPV4 expressing HEK cells, the application of negative pressure evoked single-channel current activity in a reversible manner and the channel activity was enhanced after incubation with PDBu. TRPV4 has been shown to be activated by hypotonicity. Here I show that negative pressure exaggerated hypotonicity-induced single-channel current activity. However, in similar experimental conditions, cells expressing TRPV1 did not respond to mechanical force. TRP channels are also expressed in non-sensory regions and the role of these channels is not fully understood. Both TRPV4 and TRPV1 are expressed in the hippocampus. Using whole-cell patch-clamp techniques, I have found that 4alpha-PDD increased the frequency, but not the amplitude of mEPSCs in cultured hippocampal neurons, suggesting a presynaptic site of action. Interestingly, the application of capsaicin had no effect on synaptic transmission in hippocampal neuronal cultures. Finally, I have investigated the expression and function of TRP channels in diabetes because TRP channels have been shown to be involved in peripheral neuropathy as well as vascular complications in diabetes. ROS production plays a critical role in the progress of diabetes. I propose that lower levels of ROS up-regulate the expression TRP channels in the early stages of diabetes, leading to hyperalgesia, and higher levels of ROS or chronic exposure to ROS down-regulate TRP channels in the late stages of diabetes, resulting in hypoalgesia. I have found that the expression of TRPV1 and phospho p38 (p-p38) MAPK was increased in DRG of streptozotocin (STZ)-injected diabetic and non-diabetic hyperalgesic mice. An increase in TRPV1 and p-p38 MAPK levels was induced by STZ or H2O2 treatment in stably TRPV1 expressing HEK cells, suggesting the involvement of STZ-ROS-p38MAPK pathway. TRPV4 has been reported to be involved in vasodilatation by shear stress in blood vessels. Here, I have demonstrated that TRPV4 is expressed in lymphatic endothelial cells (LECs). Treatment with low concentration of H2O2 enhanced the expression of TRPV4 at mRNA and protein levels in LECs, suggesting that mild levels of ROS up-regulate TRPV4 expression. In diabetes, beta cell dysfunction is responsible for decreased insulin release. TRPV4 is expressed in RINm5F (beta cell line), islets and pancreas. It has been shown that hypotonicity induced insulin release in beta cell lines, which was mediated by activation of stretch-activated channels, raising the possibility of the involvement of TRPV4, a mechanosensitive channel. Therefore, I have studied the functional role of TRPV4 in beta cells. Incubation with 4alpha-PDD enhanced insulin release in RINm5F cells, suggesting TRPV4 regulates insulin secretion from pancreatic beta cells. Since TRPV4 expression levels are decreased in diabetes, insulin secretion from beta cells may be impaired. In summary, TRPV1, a thermosensitive channel, and TRPV4, a mechanosensitive channel, contribute to thermal and mechanical hyperalgesia, respectively in the early stage of DPN through their up-regulation by ROS-p38 MAPK and insulin/IGF-1 pathways. Due to the mechanical sensitivity of TRPV4 channel, the up-regulation in the early stage and down-regulation in the late stage may be involved in the development of vascular complications and regulation of insulin release in diabetes.

No description available.

Cannabis has been used medicinally for millennia, but the cannabinoid (CB) field exploded with the identification of its endogenous receptors and endocannabinoids (eCBs). In vitro experimentation established that eCBs alter synaptic plasticity at presynaptic nerve terminals; however, the characterization of the eCB system (ECS) in vivo remains incomplete. This study aimed to determine the mechanism of in vivo eCB-mediated hippocampal synaptic plasticity and to analyze the effects this plasticity had on spatial working memory (SWM). With in vivo recordings of field excitatory postsynaptic potentials (fEPSPs) in anesthetized mice and rats as well as pharmacological manipulation of the ECS and glutamate receptor antagonism, it was found that eCBs, both anandamide (AEA) and 2-arachnidonyl glycerol (2-AG), caused LTD at hippocampal CA3-CA1 synapses. Induction of eCB-LTD occurs via a sequential activation of cannabinoid type-1 receptor (CB1R) and NR2B-containing NMDA receptor (NR2BR) and is expressed through the endocytosis of AMPA receptors (AMPARs). Increased eCB tone also caused an impairment of SWM for over 24 hours in the Delayed Non-Match-To-Sample (DNMTS) T-maze. This study provides the first evidence that an acute administration of eCB degradative enzyme inhibitors not only produces an in vivo LTD at hippocampal CA3-CA1 synapses that requires CB1R, NR2BR, and AMPAR, but also impairs SWM, a phenomenon also caused by an acute injection of exogenous CBs.

Sigma-1 receptors (σ-1Rs) are chaperone-like proteins that are broadly distributed throughout the central nervous system and in other tissues. They have been implicated in several physiological and pathological processes, primarily by their ability to modulate certain voltage- and ligand-gated ion channels. Growing evidence suggests that σ-1Rs regulate the functions of ion channels, such as voltage-gated K+ 1.2 (Kv 1.2) and the human Ether-à-go-go-Related Gene (hERG) ion channels, by modulating their expression, trafficking, and targeting. While it is well documented that σ-1Rs enhance the function of N-methyl-D-aspartate receptors (NMDARs), the mechanisms of this enhancement remain poorly understood. Using biochemical methods, we show that 90 minutes after intraperitoneal (i.p.) injection of σ-1R agonists such as (+)-SKF 10,047 (SKF) or (+)-Pentazocine (PTZ) (2 mg/kg), there is an increase in the expression of GluN2 subunits of NMDARs and postsynaptic density protein-95 (PSD-95) in the rat hippocampus. Following activation of σ-1Rs, co-immunoprecipitation (Co-IP) experiments reveal an increased interaction between σ-1Rs and NMDAR subunits; sucrose gradient centrifugation demonstrates an increase in the protein levels of GluN2 subunits in vesicular compartment; and biotinylation shows an increase in the surface levels of GluN2A-containing NMDARs. Taken together, our results suggest σ-1Rs may enhance NMDARs function by increasing their expression, trafficking, and surface levels. This σ-1R-mediated increase in NMDAR expression and surface levels might be involved in several physiological processes such as learning and memory. Our findings also suggest that σ-1Rs could form a potential target for designing novel antipsychotics.

Apolipoprotein E (APOE) is one of the most important genetic risk factors for late-onset sporadic Alzheimer’s disease (LOAD). APOE is a 35 kDa glycoprotein and ligand known to bind to members of the low-density lipoprotein receptor (LDLR) family, including APOE receptor 2 (apoER2; official gene name LRP8). ApoER2 is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail that can be proteolytic cleaved. In addition, apoER2 is enriched in the brain and plays an important role in synaptic function and plasticity. Interestingly, the ECD of apoER2 contains several ligand binding repeats that are organized into exons with aligning phase junctions, which allows exon skipping during alternative splicing to retain protein fidelity. The amount of alternative spliced isoforms distinguishes apoER2 from the rest of the LDLR family members. In fact, mouse Apoer2 has been identified as one of the top ten neuronal genes related to cell-type exon skipping events. Regarding human APOER2, we have identified over 40 different APOER2 isoforms from human brain using gene-specific primers and amplifying the N- and C-terminal open reading frame of APOER2. The majority of APOER2 variants consist of a diverse array of exon skipping events within the ligand binding domain (LBD). We therefore, hypothesized that human APOER2 splice variants act as functionally divergent isoforms that can influence ligand binding properties, receptor proteolysis and changes to synaptic function. ApoER2 undergoes sequential proteolytic cleavage in response to ligand binding, resulting in the release of C-terminal fragments (CTFs) and transcriptionally active intracellular domain (ICD). We therefore, systematically tested whether the diversity of human N-terminal APOER2 splice variants lacking various LBDs affects APOER2 cleavage and signaling events. We found that alternative splicing of certain APOER2 exons generated different amounts of CTFs compared to full-length APOER2 (APOER2-FL). The pattern was not simply based on the number of ligand binding domains suggesting that excision of certain exons may alter the tertiary structure of the receptor sufficiently to make the receptor more or less accessible to cleavage and generation of CTFs. To further characterize APOER2 splice variants, we specifically examined APOER2 splice variants that generated the highest and lowest amounts of CTF generation compared to APOER2-FL and focused on APOER2 splice variant lacking exons 5-8 (Δ5-8) and lacking exons 4-6 (Δ4-6), respectively. The differential CTF generation of APOER2 Δ5-8 and Δ4-6 reflects the proteolytic release of the APOER2-ICD. This APOER2-ICD mediates transcriptional activation, facilitated by the Mint1 adaptor protein. To investigate whether human N-terminal APOER2 splice variants influence APOE binding and receptor cleavage properties, we used microscale thermophoresis and tested the well-validated human APOE mimetic peptide. We found that specific exons or ligand-binding cassettes differentially affect APOE peptide binding to APOER2 splice variants. In addition, APOE peptide induces generation of APOER2-CTF acutely within one hour. Functionally, we demonstrated that APOER2 is required for spontaneous neurotransmitter release in mature neurons. Loss of mouse Apoer2 robustly decreased miniature event frequency in excitatory synapses compared to heterozygous Apoer2 neurons. We found APOER2-FL fully restored the miniature event frequency in excitatory synapses but not APOER2 Δ5-8. APOER2 Δ4-6 restored the miniature event frequency similar to heterozygous Apoer2 neurons. These results suggest that different human N-terminal APOER2 splice variants have distinct and differential synaptic properties signifying a role of APOER2 splice variants as regulators of synaptic function.

博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University