We are intrigued by how electrical and intracellular calcium activity of neurons can regulate neurotransmitter switching, a newly recognized form neuroplasticity, during development and in the mature nervous system.

In the developing Xenopus spinal cord, newly born neurons exhibit characteristic spontaneous calcium activity during a critical period that leads to the specification of distinct neurotransmitter phenotypes, such as glutamate and GABA. Altering electrical and calcium activity of these spinal neurons in vitro and in vivo causes homeostatic re-specification of neurotransmitters. Here we investigated the molecular mechanism by which neuronal activity regulates transmitter re-specification. We demonstrated that neuronal activity acts non-cell-autonomously through the release of brain-derived neurotrophic factor (BDNF) to regulate a glutamate/GABA selector transcription factor tlx3 through activation of a TrkB/MAPK signaling pathway.

We then investigated the role of neuronal activity in transmitter switching in the adult mammalian brain. In the adult rat following long-day photoperiod exposure, the number of dopaminergic neurons decreases while the number of somatostatin neurons increases in the paraventricular nucleus of hypothalamus (PaVN), causing anxiety and depression-like behaviors. Here we demonstrated that long-day photoperiod exposure increases neuronal activity of PaVN dopaminergic neurons prior to transmitter switch. Blocking activity of PaVN dopaminergic neurons abolishes their transmitter switch in response to long-day photoperiod exposure, suggesting an activity-dependent cell-population-autonomous mechanism.

Neurotransmitter switching is associated with behavioral changes in every case that a behavior change has been sought, including levels of anxiety (Dulcis et al., 2013) and motor coordination (Li et al., 2016) in adult rodents (see Spitzer, 2017 for review). To preserve synaptic function, a newly expressed neurotransmitter requires the expression of matching receptors, but the mechanisms by which this is achieved remain unclear. To investigate neurotransmitter-receptor matching in vivo, Xenopus laevis embryos were implanted with drug-loaded agarose beads at the time of closure of the neural tube. Glutamate, a major excitatory neurotransmitter, and the NMDA receptor, an ionotropic glutamate receptor, were chosen as the neurotransmitter-receptor pair in this study. Immunohistochemical analysis revealed that an NMDA receptor subunit GluN1 increased in expression in the trunk muscle plasma membrane in response to glutamate-loaded beads compared to control beads. The upregulation of GluN1 was reproduced with agonists AMPA and NMDA in combination, and suppressed with AP5 (NMDAR antagonist) and NBQX (AMPAR antagonist) in combination, establishing specificity of this receptor upregulation. We established the role of mitogen-activated protein kinases (MAPKs) p38 and jun-N-kinase (JNK) as downstream effectors of glutamate signaling, as well as the role of transcription factor MEF2C as a p38 target, by pharmacological knock-down with morpholinos. We propose a model in which glutamate acts on low numbers of ionotropic glutamate receptors initially expressed on embryonic Xenopus muscle (Borodinsky & Spitzer, 2007), promoting calcium entry that activates MAP kinases p38, JNK and MEF2, leading to upregulation of NMDAR expression.

Neurotransmitter switching is a form of neuroplasticity where exposure to external stimuli causes neurons to lose the expression of one transmitter and start expressing a different one. My thesis investigates neurotransmitter switching induced by beneficial and detrimental stimuli.One week of running (a beneficial stimulus associated with cognitive enhancement) decreases the number of neuropeptide Y (NPY)-expressing neurons and increases the number of vesicular glutamate transporter 1 (VGLUT1)-expressing neurons in the hippocampal dentate gyrus (DG). Using genetic mouse lines to permanently label NPY neurons, I tested whether the same neurons lose NPY and gain VGLUT1. Unlike wild-type mice, the genetic lines used displayed no transmitter change, demonstrating that transgenic lines can prevent DG transmitter switching. In contrast, treatment with phencyclidine or methamphetamine (detrimental stimuli associated with cognitive deficits) causes glutamatergic neurons in the prelimbic cortex (PrL) to reduce VGLUT1 and gain GABA expression. I tested whether neurogenesis contributes to this switch. No neurogenesis was detected in the PrL of drug-treated mice, supporting the conclusion that drugs induce PrL glutamatergic neurons to decrease VGLUT1 and gain GABA expression. Suppressing drug-induced hyperactivity of glutamatergic neurons in the PrL or dopaminergic neurons in the ventral tegmental area (VTA) prevents the glutamate-to-GABA switch. I tested whether silencing VTA dopaminergic neurons during drug treatment changes PrL activity and found that it does not. Finally, I tested if phencyclidine treatment altered neuronal transmitter expression in the ventrolateral orbitofrontal cortex (VLOFC), adjacent to the PrL. The VLOFC showed no glutamate-to-GABA switch, suggesting that phencyclidine’s effect may be PrL-specific.

Cyclic adenosine monophosphate (cAMP) is a second messenger that is derived from adenosine triphosphate (ATP) and is used for many biological signal transduction processes. cAMP is one of the best known intracellular second messengers after calcium (Ca2+). In many organisms, including humans, various types of ligands bind to G-protein-linked cell surface receptors and activate G proteins. Trimeric G proteins are disassembled by hydrolysis of GTP and can activate transmembrane adenylyl cyclases that convert ATP to cAMP. Phosphodiesterases then hydrolyse cAMP into AMP by breaking the phosphodiester bond. As the concentration of cAMP increases, it relays signals by binding to the regulatory subunits of PKA (cAMP-dependant protein kinase), to one of the members of the Epac family, or to cyclic nucleotide-gated (CNG) channels. Tools are available to manipulate or monitor cAMP in live cells, but they suffer a number of limitations that make them hard to use for imaging experiments. Here, I modify some of these tools to improve them for use in these experiments.

Proper integration of sensory feedback with motor programs is crucial for successful action execution. Disruption of sensorimotor integration can contribute to core symptoms of many neuropsychiatric disorders. However, where and how the brain integrates real-time sensory feedback to control ongoing behavior remains largely unknown. My thesis focuses on searching for the proposed integration regions. We successfully established a mouse model to address this question and applied a comprehensive approach with viral racing, optogenetics, chemogenetics, and in vivo and slice electrophysiological recordings. We identified an essential corticostriatal circuit mechanism for sensory feedback-based action sequencing, and our findings may provide critical insights into the pathophysiology of major psychiatric symptoms.

Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.