In Vivo Activity Of Hypothalamic Neurons Regulate Feeding Behavior

The hypothalamus is the brain neuroendocrine center, and regulates many physiological functions including basic energy balance, water balance, body temperature and circadian rhythms, etc. Thus, the hypothalamus is also involved in the regulation of a variety of instinctive behaviors such as feeding behavior. Experimental evidence shows that the lateral hypothalamic (LH) area is the "hunger center" while the ventromedial hypothalamus (VMH) is the "satiety center" of the brain. Lesion on lateral hypothalamic area can cause animal anorexia, and ventromedial damage can cause animal overeating. Theoretically, neural activities of these two brain areas participate in the regulation of feeding behavior of animals. It’s known that neuronal coding of action potentials has frequency coding, time coding, populational coding, and firing pattern coding. However, what manner the neurons in the hypothalamic feeding related regions firing action potentials to encode or regulate animal feeding behavior, is still poorly understood. In this study, by using in vivo multi-channel tetrodes recording technique, we studied hypothalamic feeding related neuronal firing patterns during feeding behavior in freely moving mice, and further explored the regulatory mechanisms of the feeding related neural activity.In 26 mice hypothalamus, we recorded 649 well-sorted neurons. After analyses, 67 neurons in vivo firing pattern were related with feeding behavior and named as Feeding Related Neurons (FRN), which represented 10.3% of the whole population. Based on their response features in feeding, we named them Fast Feeding Related Neurons (FFRN) and further separated them into four types. Neurons increased firing during feeding were named as "ON" group, in which Type I increased firing rapidly(n=15,2.3%), Type Ⅱ increased firing gradually (n=14.2.1%). And neurons decreased firing were named as "OFF" group, in which Type III decreased firing rapidly(n=9,1.3%). Type IV decreased firing gradually (n=12.1.8%). Statistical analyses showed the responses of FFRN were closely related with the time of animal feeding (r2=0.77; r2=0.74). regardless of "ON" or "OFF" group. However, the responses of FFRN were not related with food type. (P>0.05) so that different kinds of food can induce similar responses in FFRN. In addition, we found in hypothalamus two types of Slow Feeding Related Neurons (SFRN). Along with feeding behavior, the SFRN firing rate slowly increased were named as SI Type, and those firing rate slowly decreased were named as SD Type. To answer whether FRN were inhibitory neurons or excitatory neurons, we analyzed the spike waveform and basal firing rate of FRN. As a result. Type Ⅰ, Ⅱ and SI had a mixed component of excitatory neuron and inhibitory neuron, while Type Ⅲ,Ⅳ and SD were mostly putative inhibitory GABAergic neurons.Next, in order to study the FRN activity in responses to energy and hormone signals, we investigated recorded hypothalamic neurons activities after delivery of D-Glucose and Leptin. In 8 mice i.p. injected of Glucose, we found some neurons increased firing (n=28,20%). some neurons decreased firing (n=24,17%), and still 88 neurons did not change firing rate. Among them there were 40 FRN,15 showed increase of firing,14 showed decrease of firing and 10 showed no changes. In another 8 mice i.p. injected of Leptin, some neurons increased firing (n=20,13%), some neurons decreased firing (n=13,7%) and rest 149 neurons did not change firing rate. Among them there were 30 FRN,3 of them showed increase of firing, while 21 showed decrease of firing, and 6 of them showed no change. These results implied majority FRN could response to energy signal and expressed Leptin Receptor, which differed from common hypothalamic neuron.Finally, we combined optogenetics with in vivo recording, further investigated how hypothalamus orexin (ORX) neurons regulated hypothalamic FRN. By using lentivirus carry orexin promotor, we expressed light sensitive ion channel Channelrhodopsin-2(ChR2) specifically onto ORX neuron. When ORX neurons were activated by blue laser. Type Ⅰ FRN was activated by orexin release, while Type Ⅳ FRN was inhibited by orexin release. The data demonstrated orexin neuron could regulate neural activity of hypothalamic FRN.Our research on in vivo neural activity of hypothalamic FRN demonstrated the diversity of hypothalamic neuron firing during feeding behavior. The results implied the hypothalamic local neural circuits maybe applying different encoding strategy simultaneously on regulating feeding behavior. The results also revealed complexity of hypothalamic neural network, providing basics for better understanding hypothalamic FRN types, and the mechanism of FRN regulate feeding behavior.