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.

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease regarded as a multifactorial disorder, a condition caused by multiple factors. With the majority of AD patients aged 65 and older, age is one of the strongest risk factors for AD, but its causes are not fully understood. Previous transcriptomic profiling of AD neurons identified a list of differentially expressed genes unique to AD; YY1, RUNX1, NFIB, and PPARγ emerged as some of the most upregulated transcription factors (TFs) reportedly associated with biological dysfunctions phenotypic of AD in various cell types. However, their function in aged neurons remains unknown. To investigate the possible contributions of YY1, RUNX1, NFIB, and PPARγ to AD pathology, we questioned their ability to reproduce AD-related phenotypes in aged neurons by characterizing transcriptomic changes caused by individual overexpression of each TF. We utilized induced neurons (iNs) produced from the reprogramming of old, healthy, donor-derived fibroblasts directly into neurons as a human-relevant means of capturing the age. All four TFs retain aspects of their previously described functions in old iNs with YY1 regulating the metabolic program, RUNX1 restricting neuronal identity, PPARγ regulating lipid metabolism and inflammation, and NFIB promoting the expression of developmental genes, possibly facilitating oncogenic transformations and contributing to the vulnerable hypo-mature state seen in AD neurons. Further investigation confirmed that YY1 and RUNX1’s transcriptomic effects are functionally translatable, identifying them as potential targets for medical intervention.