The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

Ankstesni tyrimai, susiję su laikinėmis sinapsinio efektyvumo pasikeitimo taisyklėmis per nuo veikimo potencialų poravimo priklausantį plastiškumą, praktiškai nekreipė jokio dėmesio į neuronų erdvines savybes. Mes nagrinėjame nuo kalcio/kalmodulino priklausančią kinazę II (CaMKII) ir kalcineuriną (CaN) artimuosiuose ir tolimuosiuose spygliuose bei jų įtaką ilgalaikei potenciacijai ir ilgalaikei depresijai. Tyrimų metu buvo atskleista, kad esant neigiamam veikimo potencialų laiko poravimui tolimoje sinapsėje gali vykti ilgalaikė potenciacija, tuo tarpu artimajame spyglyje visi procesai vyksta remiantis klasikinėmis sinapsinio plastiškumo taisyklėmis. Gauti rezultatai teigia, kad sinapsės vieta dendritiniame medyje yra lemiamas veiksnys nuo veikimo potencialų laikų poravimo priklausančiame sinapsiniame plastiškume. / Previous studies focusing on the temporal rules governing changes in synaptic efficacy during spike timing-dependent plasticity (STDP) have paid little attention to spatial characteristics of neurons. We analyze the activity of calcium-calmodulin dependent protein kinase II (CaMKII) and phosphatase calcineurin (CaN) in proximal and distal spines and their impact on long-term potentiation (LTP) and long-term depression (LTD). During tests we found that at negative timing of action potentials in distal spine synapse can undergo LTP while in proximal spine the processes follow the classic STDP rules. Our results suggest that synapse location within the dendritic tree is a crucial determinant of STDP.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

Mental health of a person depends on the correct functioning of the brain. The brain and the spinal cord contain many types of cells, of which one important type are called the neurons. Neurons are special in the way they connect to each other to form large networks. The chemicals called transmitters are packed at the nerve endings into tiny packets called vesicles and when a signal arrives these vesicles fuse immediately to the attached cell surface and release their contents. The role of the synaptic vesicular transporter proteins is to ensure proper packing of transmitter molecules that can be released upon stimulation. Vesicular packing is an important process. The carrier proteins involved in packing work in coordination to determine the amount and type of transmitters to be packed. Missing a carrier protein from the vesicles might lead to improper packing and inaccurate signaliing. These signaling molecules are known for their implications in many psychiatric and neurological disorders like Alzheimer’s disease, Parkinson’s disease, Schizophrenia, and attention deficit to name just a few.  How a vesicular transporter can affect the modulatory functions of aminergic neurons is the subject of this thesis. This thesis reports on the effects of the loss of a vesicular orphan transporter. Study I demonstrates the localization of this protein to monoaminergic and cholinergic terminals. It reports the effect of the loss of Slc10A4 on vesicular dopamine uptake, synaptic clearance of dopamine and hypersensitivity of animals to dopamine related psychostimulants. Study I also provides evidence for ATP as a possible ligand for SLC10A4 protein. Study II provides data on the clinical relevance of Slc10A4 in playing a protective role against vulnerability to epilepsy. It reports that loss of Slc10A4 renders the animals hypersensitive to cholinergic drugs. Study III provides a closer look at individual cholinergic synapses at neuromuscular junctions in mice lacking Slc10A4. The structural and electrophysiological properties of the NMJ are found compromised because of the loss of this vesicular protein. Taken together, this thesis presents a SV protein’s perspective of viewing at modulation of synaptic transmission.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

<p>The late phase of long-term synaptic potentiation (LTP) at glutamatergic synapses, which is thought to underlie the long lasting memory (at least hours), requires gene transcription in the nucleus. However, it remains elusive how signaling initiated at synapses during induction of LTP is transmitted into the nucleus to commence transcription. Using a combination of two-photon glutamate uncaging and a genetically encoded FRET sensor, I found that induction of synapse-specific LTP at only a few (3-7) dendritic spines leads to pronounced activation of extracellular signal-regulated kinase (ERK) in the nucleus and downstream phosphorylation of transcription factors, cAMP-response element-binding protein (CREB) and E26-like protein-1 (Elk-1). The underlying molecular mechanism of this nuclear ERK activation was investigated: it seems to involve activation of NMDA receptors, metabotrophic glutamate receptors, and the classical Ras pathway. I also found that the spatial pattern of synaptic stimulation matters: spatially dispersed stimulation over multiple dendritic branches activated nuclear ERK much more efficiently than clustered stimulation within a single dendritic branch. In sum, these results suggest that biochemical signals could be transmitted from individual spines to the nucleus following LTP induction and that such synapse-to-nucleus signaling requires integration across multiple dendritic branches.</p> / Dissertation

The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.

The dorsal vagal complex (DVC), containing the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus nerve (DMV), plays a pivotal role in autonomic regulation. Afferent fibers from peripheral organs and higher brain centers synapse in the NTS, which integrates these synaptic connections as well as information from systemically circulating hormones and metabolites. The integrated information is relayed to the dorsal motor nucleus of the vagus nerve (DMV), which in turn, projects motor fibers to elicit parasympathetic control of digestive and other viscera. Physiological functions mediated by the DVC are disrupted in diabetic patients and synaptic plasticity within the DVC has been linked to these complications. N-methyl-D-aspartic acid (NMDA) receptors have been extensively studied for their involvement in synaptic plasticity in a variety of central nervous system disorders; and their activation in the DVC modulates hepatic glucose production and feeding behavior. Although chronic disease can alter NMDA function, changes in DVC expression and/or sensitivity of NMDA receptors in diabetic states has not been addressed. Using whole cell electrophysiology, functional properties of the nuclei in the DVC were investigated in normoglycemic and type 1 diabetic mice. Preterminal NMDA (preNMDA) receptors were discovered to tonically modulate excitatory neurotransmission on terminals contacting DMV neurons. While these preNMDA receptors were not found to differentially modulate tonic excitatory neurotranmission, soma-dendritic NMDA receptor responses of NTS neurons were augmented in type 1 diabetic mice. Through the use single-cell PCR, increased NMDA receptor responses could be correlated to neurons that mediate excitatory neurotransmission and would argue that augmented NMDA receptor responses increase vagal output. In general, enhancing vagal output decreases activity of connected peripheral organs. Molecular approaches were employed to corroborate the observed functional NMDA receptors changes to their protein and mRNA expression levels. Overall, results argue that NMDA receptors are involved in synaptic plasticity in DVC of type 1 diabetic mice to enhance excitatory neurotransmission. This modulation may potentially serve as a physiological counter regulatory mechanism to control pathological disturbances of gastrointestinal homeostatic reflex responses.

No description available.

Nerve cells are organized into complex networks that comprise the building blocks of our nervous system. Neurons communicate by transmitting messenger molecules released from synaptic vesicles. Alterations in neuronal circuitry and synaptic signaling contribute to a wide range of neurological conditions, often with consequences for movement. Intrinsic neuronal networks in the spinal cord serve to coordinate vital rhythmic motor functions. In spite of extensive efforts to address the organization of these neural circuits, much remains to be revealed regarding the identity and function of specific interneuron cell types and how neuromodulation tune network activity. In this thesis, two novel genes initially identified as markers for spinal neuronal populations were investigated: Slc10a4 and Dmrt3. The orphan transporter SLC10A4 was found to be expressed on synaptic vesicles of the cholinergic system, including motor neurons, as well as in the monoaminergic system, including dopaminergic, serotonergic and noradrenergic nuclei. Thus, it constitutes a novel molecular denominator shared by these classic neuromodulatory systems. SLC10A4 was found to influence vesicular transport of dopamine and affect neuronal release and reuptake efficiency in the striatum. Mice lacking Slc10a4 displayed impaired monoamine homeostasis and were hypersensitive to the drugs amphetamine and tranylcypromine. These findings demonstrate that SLC10A4 is capable of modulating the modulatory systems of the brain with potential clinical relevance for neurological and mental disorders. The transcription factor encoded by Dmrt3 was found to be expressed in a population of inhibitory commissural interneurons originating from the dorsal interneuron 6 (dI6) domain in the spinal cord. In parallel, a genome-wide association study revealed that a non-sense mutation in horse DMRT3 is permissive for the ability to perform pace among other alternate gaits. Further analysis of Dmrt3 null mutant mice showed that Dmrt3 has a central role for spinal neuronal network development with consequences for locomotor behavior. The dI6 class has been suggested to take part in motor circuits but remains one of the least studied classes due to lack of molecular markers. To further investigate the Dmrt3-derived neurons, and the dI6 population in general, a Dmrt3Cre mouse line was generated which allowed for characterization on the molecular, cellular and  behavioral level. It was found that Dmrt3 neurons synapse onto motor neurons, receive extensive synaptic inputs from various neuronal sources and are rhythmically active during fictive locomotion. Furthermore, silencing of Dmrt3 neurons in Dmrt3Cre;Viaatlx/lx mice led to impaired motor coordination and alterations in gait, together demonstrating the importance of this neuronal population in the control of movement.