La Gobernanza del Agua y la Gobernanza de los Humedales.

A significant limitation of this dissertation is that we were unable to show whether dHC or vHC neurons form a meal-related memory. Previous work has shown that it is

possible to measures an animal’s ability to remember where it consumed food (Rubinow et al. 2009), or which arm of a maze food is available in (McDonald and White 1993), but not if the memory of a specific meal drives behavior. Modern neuroscience

techniques can use a combination of genetic and viral interventions that selectively target and “tag” ensembles of neurons that are activated by a learning-event.

Researchers can use optogenetic constructs that inhibit or excite these tagged neurons to block memory or induce a memory (Liu et al. 2012, Ramirez et al. 2013, Ramirez et al. 2015). The targeting of neurons specific to a memory experience allows researchers to determine whether the memory formed during an event drives behavioral changes as opposed to simply HC activity. Although it is hypothesized in this dissertation that HC neurons form-meal related memories that inhibit future intake, it is impossible to determine whether the memory of meal inhibits future intake without specifically manipulating HC-neurons activated by an eating event. For the purposes of this dissertation, if HC neurons that were activated during meal consumption were able to be tagged with an inhibitory optogenetic construct (Liu et al. 2012, Ramirez et al. 2013, Ramirez et al. 2015), it would then be possible to determine whether subsequent inhibition of those neurons would accelerate the onset of the next meal or increase intake during the next meal. This could help determine whether HC neurons form-meal related memories

Inhibition of dHC and vHC neurons shows that these brain regions are necessary to limit intake, but does not show whether activation of these neurons is sufficient to suppress future intake. Limited evidence shows that optogenetic excitation of vHC projections to the medial-prefrontal cortex, LS, or BNST (Sweeney and Yang 2015, Hsu

et al. 2017) decreases total food intake, but this excitation was not limited to the period during or after consumption. To determine whether dHC and vHC neurons are sufficient to delay meal onset and limit future intake, it would be necessary tag HC neurons

activated during meal consumption with an excitatory optogenetic construct (Ramirez et al. 2013, Ramirez et al. 2015) and drive activity in those tagged neurons to potentially delay the onset of the next meal or decrease intake during the next meal.

It is impossible to directly compare the two studies that manipulated Arc

expression in vHC (Chapter 3) and dHC neurons (Chapter 5) due to methodological differences. The vHC Arc manipulation was acute (Arc antisense) and the dHC manipulation was chronic (Arc shRNA). The vHC Arc antisense experiment used a within-subject design (n = 9), whereas the chronic knockdown of Arc using the shRNA used a between-subjects design (Controls: n = 12; Arc shRNA: n = 12). It is therefore possible that the Arc shRNA study is underpowered to detect a difference between the experimental and control group. This is unlikely, however, as sucrose consumption increases Arc expression in dHC neurons (Henderson et al. 2013) more than in vHC neurons (Hannapel et al. 2017) and the Arc shRNA knockdown was greater in dHC neurons (~6.5 fold) than the Arc antisense knockdown in vHC neurons (~2.5 fold). This leaves two possibilities, 1) dHC Arc expression is not critical for inhibiting future intake, or 2) the effectiveness of dHC Arc in inhibiting future intake is much less than in vHC neurons and a study would need significantly more subjects than the number of subjects used in the current work to detect any role for dHC Arc in regulating energy intake.

It remains unknown whether other mechanisms of synaptic plasticity are involved in energy intake and whether dHC neurons utilize different mechanisms of synaptic plasticity than vHC neurons. The current studies selectively targeted Arc because it is considered to be a master regulator of synaptic plasticity (Bramham et al. 2010,

Shepherd and Bear 2011), and sucrose consumption increased Arc expression in dHC and vHC neurons. The pilot gene array study that identified ntf4 as a possible target for manipulation, however, found that sucrose consumption increased 20 other genes more than 1.5 fold in dHC neurons. These 20 genes provide promising targets for future manipulation, such as the inflammatory cytokine TNF-α, which increased ~7 fold by acute sucrose intake in dHC neurons. It unknown exactly how proinfammatory cytokines may contribute to HC regulation of energy intake, but expression of proinflammatory cytokines such as TNF-α is increased in obese patients and can enhance energy expenditure (Ye and McGuinness 2013, Wang and Ye 2015), increase the expression of the anorexigenic leptin receptor (Gan et al. 2012), yet impair HC-dependent memory (Golan et al. 2004, Beilharz et al. 2014, Ohgidani et al. 2016). The gene array only tested the effects of sucrose consumption on synaptic plasticity-related genes and did not determine whether consumption of non-sweetened foods or non-caloric sweeteners would have a similar effect on the pattern of gene expression. Consumption of

saccharin increases Arc expression in dHC neurons more so than sucrose consumption (Henderson et al. 2016). It is therefore possible that dHC neurons do form meal-related memories as consumption of non-sweetened foods or non-caloric sweeteners could increase the expression of other genes critical for synaptic plasticity not tested within this dissertation.