Study On The Memory Function Of Neurons In Auditory Cortex Based On Two-Photon Imaging System In Vivo

How long-term memory is embodied at the microscale level in the brain is a fundamental core scientific question in neuroscience.According to current theories,long-term memory is generally believed to be stored in widely sparse and interconnected neural network systems.However,the specific cellular composition and format of storage for a specific piece of long-term memory remain largely unknown.Over the past 20 years,scientists have developed Immediate Early Genes(IEGs)expression marking techniques,which rely on neural activity levels,to identify the cellular ensembles involved in forming a specific piece of memory.Furthermore,optogenetic/chemogenetic manipulation techniques have investigated the causal relationship between the identified cells and specific memory information.To date,the IEG labeling approach has been widely applied to brain regions such as the hippocampus,amygdala,and prefrontal cortex,which are known to be involved in long-term memory formation.However,it is puzzling that IEG methodology has shown limited applicability to the sensory cortex,which is considered a potential site for the consolidation and storage of long-term memory information.Intriguingly,the cells labeled in the sensory cortex using IEG techniques do not correspond to the sensory memory experiences.This study presents a pioneering investigation into the auditory cortex using a combination of in-vivo two-photon imaging and targeted electrophysiological recording.The study observes the sound-evoked calcium signals of the same group of single neurons during consecutive days of sound-licking associative learning.The two-photon calcium imaging signals are calibrated by using single-cell electrophysiological recordings.Through this methodology,it was discovered and confirmed that a small fraction(1%-5%)of neurons in layer 2/3 of the auditory cortex initially showed no response to the learned complex sound before learning.However,with the progression of daily learning,these neurons suddenly transformed into high-frequency bursting firing responses specific to the learned complex sound as a whole while not responding to the individual pure tones composing the sound.This group of neurons was defined as "Holistic Bursting"(HB)cells.Further experimental work demonstrated that HB cells met all four necessary defining criteria as an “engram” by Richard Wolfgang Semon,a German psychologist in 1905.Firstly,HB cells emerged during learning.Sparse HB cells transformed from normal neurons into HB cells in a zero-to-one burst firing pattern.Secondly,once emerged,HB cells existed in a long-term stable state with functional characteristics.Thirdly,the high-frequency burst firing of HB cells occurred before the animal performed the learned sound-water associative learning behavior(rather than later than licking behavior as a feedback signal).Fourthly,there was a dormancy state in HB cells.After dissociated training sessions in which the learned sound and the water-licking behavior were decoupled,most(66.6%)HB cells entered a dormancy state.They no longer exhibited burst firing responses to the learned sound.However,once the behavior was reassociated with the learning,the silent HB cells recovered burst firing responses.This work directly confirmed the existence of long-term memory engrams as HB cells in the auditory cortex for the first time and also skillfully and retrospectively explained why the IEG method system was not applicable to label memory engrams in the sensory cortex: HB cells as stable and consolidated long-term memory information cellular embodiment,may not be active during the process of memory formation,so IEG expression tools that depended on cell activity were not applicable.Furthermore,due to the developmental requirements of two-photon imaging technology and the research demand to elucidate the mechanisms of long-term memory consolidation and storage at the level of single neurons and single synapses,this study has established a set of research methods encompassing chronic somatic imaging and chronic dendritic imaging for HB cells.Additionally,a next-generation ultrafast two-photon imaging system with a frequency of 10 kHz has been developed and tested.As a proof-of-concept,this system enables quantitative analysis of single-neuron activity in vivo.The above three aspects of the study,which closely complemented each other,constituted the content of this paper.Their primary innovative contribution was the first demonstration of long-term storage memory related neurons in the auditory cortex through chronic two-photon calcium imaging and physiological experiments in vivo.Moreover,it identified the unique burst firing output pattern associated with the memory information and established a solid pathway for further in-depth exploration of the role and mechanisms of single cortical neurons in long-term memory.