Abstract by Cesar Ramon Romero Leguizamon
This thesis contains three independent studies:
The first provides evidence of the functional effects of corticotropin-releasing factor receptor 1 (CRFR1) activation by corticotropin-releasing factor (CRF) and urocortin (Ucn) on neurons of the Laterodorsal Tegmentum (LDT). It is a primary cholinergic nucleus located in the brainstem, part of the reticular activating system that provides the majority of cholinergic input to brain areas related to the control of mood, arousal and addiction.
Stress is a physiological response that seeks to maintain homeostasis in response to a traumatic or harmful situation. The problem is when this situation becomes chronic and/or maladaptive, facilitating the development of multiple psychiatric disorders such as anxiety, depression and substance use disorder (SUD), conditions that present at neurobiological and clinical sex-specific differences.
Then, is it possible that in presence of a stressor, an increase in acetylcholine (ACh) release can be induced in LDT cholinergic neurons, through the activation of CRFR1?
To investigate the cellular effects of the CRF/Ucn-CRFR1 interaction on LDT cells, electrophysiological studies, in-vitro calcium imaging and immunohistochemistry, were performed on brain slices of male and female mice, characterizing the membrane responses and intracellular calcium changes in cholinergic and non-cholinergic LDT neurons.
These neuropeptides generated inward membrane currents in cholinergic neurons as well as an increase in spontaneous excitatory postsynaptic currents (sEPSCs) and in the intracellular calcium levels. These responses involved effects on intracellular signaling pathways, the mitogen-activated protein kinase and extracellular signal-regulated kinase (MAPK/ERK) along with Inositol trisphosphate (IP3) pathway. These effects of depolarization on the cell membrane and increase in intracellular levels of calcium in cholinergic neurons of LDT, can link stress with a hypercholinergic state that leads to the development of psychiatric disorders.
The second study consisted in the conception and elaboration of an in-vitro device for the organotypic culture of brain slices that contains the LDT nucleus. Organotypic culture of brain slices is a widely used technique in neuroscience. Some of the advantages of this in-vitro experimentation model are the preservation of the architecture and interactions of the cultured tissue. It is an alternative that allows reducing the number of animals used in experimental protocols. Multiple functional and pharmacological studies have successfully used this methodology. However it has never been used to culture slices containing the LDT.
LDT is a richly vascularized area; this makes it more vulnerable to hypoxia and early cell death. For this reason, a conventional organotypic culture technique has a high probability of failure. There are several micro-perfusion devices that would be ideal to preserve this delicate tissue, however they have high costs. It was decided to design and create a cost-effective flow system that allows keeping viable brain slices for a longer time.
The device kept the LDT brain slices viable for two weeks, and these were evaluated using cell viability assays, electrophysiology and immunohistochemistry. Additionally slices of animals of different ages were cultured and compared with hippocampal slices cultured under the same conditions. Showing that this is a viable methodology to maintain LDT brain slices in adequate conditions during two weeks of culture.
The third one is an ongoing study, seeks to explore whether our organotypic culture system can be a useful tool to evaluate the effects of chronic exposure to drugs and/or peptides on LDT cultured slices. To achieve this objective, an adaptation of the organotypic culture flow system was performed, through the implementation of a double valve system that allowed us to use two different culture media, the first one a free-cocaine medium and the second supplemented with cocaine (5 μM). Cocaine has been added to the culture medium through two protocols, (i) constant exposure 24 hours a day for 14 days of culture and (ii) 1-hour daily cocaine exposure during 14 days.
To assess the neurotoxic effects of this long-term exposure cell viability tests, electrophysiological recordings, indirect calcium measurement, and immunohistochemistry have been performed. Partially, our results suggest that the adaptation made to our previously validated system is a useful in-vitro tool for chronic exposure to drugs and/or peptides. Additionally, our findings show morphological, metabolic and functional changes induced by chronic cocaine exposure on LDT cells, especially on cholinergic neurons. However, it is necessary to continue conducting more tests that allow us to draw precise conclusions regarding the toxic effects of chronic cocaine exposure on LDT neurons.
In summary, evidence has been produced showing how stressed conditions with high levels of CRF and Ucn have effects on LDT cholinergic neurons that are associated with the generation of the hypercholinergic state. In addition, the organotypic culture of LDT slices has been carried out for the first time, which has allowed us to continue experimental studies implementing this technique.