Tetrode recording from the hippocampus of behaving mice coupled with four-point-irradiation closed-loop optogenetics: A technique to study the contribution of Hippocampal SWR events to learning

With the advent of optogenetics, it became possible to change the activity of a targeted population of neurons in a temporally controlled manner. To combine the advantages of 60-channel in vivo tetrode recording and laser-based optogenetics, we have developed a closed-loop recording system that allows for the actual electrophysiological signal to be used as a trigger for the laser light mediating the optogenetic intervention. We have optimized the weight, size, and shape of the corresponding implant to make it compatible with the size, force, and movements of a behaving mouse, and we have shown that the system can efficiently block sharp wave ripple (SWR) events using those events themselves as a trigger. To demonstrate the full potential of the optogenetic recording system we present a pilot study addressing the contribution of SWR events to learning in a complex behavioral task

자유롭게 움직이는 생쥐의 해마에서 sharp wave-ripple의 전기생리학적 신호와 칼슘 신호를 동시에 기록하는 방법과 활용

학위논문(박사) -- 서울대학교대학원 : 의과대학 의과학과, 2023. 2. 김상정.Various experiences occur in our daily life in diverse context, and among them, some experiences become a life-long memory while some are easily forgotten. Not only encoding of experiences, this phenomenon of memory selection is heavily dependent on the consolidation of memory. In the investigation of long-term memory formation, hippocampal SWR signal and its neuronal contents have been extensively studied using neuronal decoding analysis of electrophysiological signal to recognize SWRs in the neuronal signals. However, as this signal has low spatial resolution and hard to track neurons across time, it has been difficult to analyze the individual contribution of neurons to task-specific SWRs. In this work, I focused on the investigation of the hippocampal SWRs in spatial aspect not only in temporal aspect, and identification of cellular ensembles consisting of the activity to improve contents of consolidated memory by SWRs. To understand the composition of SWRs and its change by the environment in detail, I divided the research process into two parts. In the first part, to investigate individual hippocampal neuronal activity participating in SWRs activity, I developed a Microdrive array with tetrodes, that combines with UCLA miniscope, a 1-p calcium imaging device. This method enables us to observe SWRs activity not only populational electrophysiological manner, as well as individual cellular activity using calcium indicators from freely behaving animals. The acquired data show that a group of hippocampal neurons was identified to have increased activity on the onset of SWRs, while activities were found to be decreased when SWRs are disrupted. This result implies the potential contribution of individual neuronal activity in the memory consolidation process. In the second part, the calcium transient signals acquired from hippocampal neurons was compared by the environment of the animal. While animals are exploring two different environments, SWRs were detected in real-time and hippocampal neuronal activities were observed simultaneously. From the result, we found that different subsets of neurons are firing during SWRs depending on the environment of the animals, suggesting that SWR signals are collective signals of multiple neurons but their compositions are different by the contents of experience. This result has a potential to improve decoding accuracy when investigating replay contents and neuronal composition. Overall, this thesis covers comprehensive strides from the development of tools to analysis of scientific findings in search of neuronal constitution of memory engraved in the hippocampus.우리의 일상 생활에서는 다양한 경험이 다양한 환경에서 일어난다. 그 중 일부는 평생 지속되는 강렬한 경험이 되기도 하고, 다른 경험들은 기억도 되지 못하고 쉽게 잊혀진다. 이러한 기억의 선택적 저장에는, 경험의 입력 과정(encoding) 뿐 만 아니라 경험의 강화 (consolidation) 과정도 큰 영향을 끼친다. 해마의 sharp wave-ripples (SWRs) 와 그 구성 뉴런들은 장기 기억의 형성 과정을 연구하는 데에 큰 비중을 차지해왔다. 특히 이 뉴런의 신호들은 전기생리학적 방식으로 기록되고 연구되었다. 그러나, 이러한 신호들은 뉴런의 위치에 대한 정보가 부족하고 장기간에 걸쳐 같은 뉴런을 인식할 수 없기 때문에, 특정 환경에서 일어나는 SWRs이 어떠한 뉴런으로 구성되어있는지 연구하는 데에는 어려움이 있었다. 이 논문에서는, 해마의 SWRs을 구성하는 뉴런들을 시간적 측면 뿐 만 아니라 공간적 측면에서도 관찰하여, SWRs로 인해 강화되는 기억을 구성하는 뉴런들을 식별하고자 하였다. SWRs의 구성과 환경에 따른 구성의 변화를 알아보기 위해, 본 연구는 아래의 두 부분으로 나누어 진행되었다. 첫번째 부분에서는 전기생리학적 방법과 칼슘 이미징 방법을 통해, 자유롭게 움직이는 생쥐의 해마에서 발생하는 SWRs을 두 가지 방법으로 기록하고, 그 신호에 따라 변화하는 뇌세포의 활동을 알아보고자 한다. 이를 위하여 살아있는 동물에서 실시간으로 전기신호를 측정할 수 있는 초소형 기구를 만들고, 이를 단광자 칼슘 이미징 장치와 결합시켰다. 이러한 방식을 통해 해마의 SWRs을 전기생리학적 방식과 칼슘 신호의 두 가지 방식으로 기록하였다. 기록 결과, 해마의 뇌세포들의 활동성이 SWRs이 시작됨에 따라 증가하는 것이 확인되었다. 반면, SWRs이 방해된 경우에는 해마 뇌세포의 활동성 증가가 관찰되지 않았다. 이러한 결과는, SWRs을 구성하는 세포들이 기억 강화 과정에 영향을 미칠 수 있음을 암시한다. 두 번째 부분에서는, 해마 뉴런에서 얻어진 칼슘 신호들을 동물들의 실험 환경에 따라 비교하여 보았다. 동물들이 두 개의 다른 환경을 경험하고 있는 동안, 해마에서 발생하는 SWRs 신호가 실시간으로 기록되었다. 그 결과, 동물들이 처한 환경에 따라서, 서로 다른 해마 뉴런으로 구성된 그룹들이 SWRs을 구성하고 있다는 것을 발견하였다. 즉, 해마의 SWRs들은 여러 뉴런의 신호들로 이루어져 있으나 그 각각을 구성하는 뉴런은 경험의 환경에 따라 달라질 수 있음을 보여준다. 또한 이러한 결과는, 기억의 재생 (replay)과 그 구성 뉴런을 식별하기 위한 신호의 해독(decoding) 과정에서의 정확성을 높이는 데에 기여할 수 있음을 보여준다. 정리하면, 이 논문을 통하여, 해마에 저장되어 있는 기억을 구성하는 뉴런들을 식별하기 위한 실험 도구의 개발부터 그 결과 발견한 과학적인 내용의 분석에 이르기까지의 단계들을 포괄적으로 기술하고 소개하고자 한다.Abstract 4 Table of contents 7 List of figures and tables 8 Chapter 1. Introduction 10 Chapter 2. Simultaneous cellular imaging, electrical recording and stimulation of hippocampal activity in freely behaving mice 39 Summary 39 Introduction 41 Materials and Methods 43 Results 53 Discussion 58 Chapter 3. Simultaneous cellular imaging and electrical recording for dissecting neuronal ensemble activity of SWRs in multiple environments 76 Summary 76 Introduction 77 Materials and Methods 78 Results 79 Discussion 81 Chapter 4. Conclusion and future perspective 88 References 92 국문요약 113박

Large-scale neural ensemble recording in the brains of freely behaving mice

Abstract With the availability of sophisticated genetic techniques, the mouse is a valuable mammalian model to study the molecular and cellular basis of cognitive behaviors. However, the small size of mice makes it difficult for a systematic investigation of activity patterns of neural networks in vivo. Here we report the development and construction of a high-density ensemble recording array with up to 128-recording channels that can be formatted as single electrodes, stereotrodes, or tetrodes. This high-density recording array is capable of recording from hundreds of individual neurons simultaneously in the hippocampus of the freely behaving mice. This large-scale in vivo ensemble recording techniques, once coupled with mouse genetics, should be valuable to the study of complex relationship between the genes, neural network, and cognitive behaviors

Pitx3Null Mutant (Striatal Dopamine-Deficient) Mice Have Exaggerated Spiny Projection Neuron Responses to l-DOPA and D1 Agonism and Lack Baseline Striatonigral Spiking

L-3,4 dihidroxyphenylalanine (l-DOPA) strongly stimulates motor activity in parkinsonian patients and animal models of Parkinson\u27s disease. Severe striatal dopamine (DA) loss characterizes Parkinson\u27s disease and its animal models. Given the canonical rate model of Parkinson\u27s Disease pathophysiology based on differences in DA pharmacology manifesting as electrophysiological differences in striatal projection neuron (SPN) spike rates, SPNs should increase spiking during the motor response to l-DOPA. In fact, stimulating specific subsets of these neurons to spike in freely-moving wild type and parkinsonian animals causes or inhibits motor activity as predicted. However, pharmacological effects of DA deficiency, let alone those of DA replacement, on SPN spiking activity in freely-moving animals are poorly studied and ultimately unknown. Showing the activity of SPNs of both in-/direct pathways may help elucidate mechanisms by which l-DOPA increases motor activity to normal and sometimes abnormal levels; such mechanistic information would advance understanding about how DA is such a potent motor stimulant. To this end, I devised a Top-hat u-array (with microdrive) for recording in the striatum while stimulating the reticulated substantia nigra. Using my micro-array, I tested l-DOPA\u27s acute effect on SPN spiking activity within contexts that varied in DA deficiency according to the Pitx3Null mouse\u27s Parkinson\u27s-like gradient of striatal DA denervation. Evidently, chronic DA denervation renders SPNs hyper-responsive to l-DOPA and a D1 agonist, SKF 81297, as indicated by exaggerated SPN spike rate responses biased by low baselines in Pitx3Null mice compared to wild-type mice. However, this may be a motor network effect on spiking as it was found in both dorsal (DA-denervated) and non-dorsal (having residual DA) Pitx3Null striatal regions. Furthermore, antidromically identifying dorsal SPNs allowed us to putatively distinguish a particularly relevant subset (striatonigral, D1-SPNs or d[irect]SPNs) known to elicit movement; serendipitously we also identified putative fibers of passage that strongly resembled striatal interneurons. D1-SPNs in Pitx3Null animals had baselines about an order of magnitude significantly below those in wild-type, and all increased firing more so in Pitx3Null than wild-type mice after drug injections, which lends some credence to the hypothesis that direct pathway SPNs are hyper-responsive during l-DOPA-induced normal and abnormal motor behavior secondary to DA depletion. Furthermore, they uncover a need to incorporate more neural factors in explaining electrophysiological effects attributable to DA denervation and restoration pharmacology. The latter data (putative fibers) tempt the interpretation that cortical axons of passage are being mistaken for striatal fast-spiking interneurons in the literature more often than not

Long-Term Neural Recordings Using MEMS Based Movable Microelectrodes in the Brain

One of the critical requirements of the emerging class of neural prosthetic devices is to maintain good quality neural recordings over long time periods. We report here a novel MEMS (Micro Electro Mechanical Systems) based technology that can move microelectrodes in the event of deterioration in neural signal to sample a new set of neurons. Microscale electro-thermal actuators are used to controllably move microelectrodes post-implantation in steps of approximately 9 μm. In this study, a total of 12 movable microelectrode chips were individually implanted in adult rats. Two of the twelve movable microelectrode chips were not moved over a period of 3 weeks and were treated as control experiments. During the first 3 weeks of implantation, moving the microelectrodes led to an improvement in the average signal to noise ratio (SNR) from 14.61 ± 5.21 dB before movement to 18.13 ± 4.99 dB after movement across all microelectrodes and all days. However, the average root-mean-square values of noise amplitudes were similar at 2.98 ± 1.22 μV and 3.01 ± 1.16 μV before and after microelectrode movement. Beyond 3 weeks, the primary observed failure mode was biological rejection of the PMMA (dental cement) based skull mount resulting in the device loosening and eventually falling from the skull. Additionally, the average SNR for functioning devices beyond 3 weeks was 11.88 ± 2.02 dB before microelectrode movement and was significantly different (p < 0.01) from the average SNR of 13.34 ± 0.919 dB after movement. The results of this study demonstrate that MEMS based technologies can move microelectrodes in rodent brains in long-term experiments resulting in improvements in signal quality. Further improvements in packaging and surgical techniques will potentially enable movable microelectrodes to record cortical neuronal activity in chronic experiments

Rhythmic Action Synchronizes Memory Replay During Reinforcement Learning

Our cognitive abilities - learning from the past, sensing the current environment, planning into the future, executing an action, and infusing value into an experience - all rely on precisely timed and widespread electrical communications across neural networks. The brain’s hippocampal formation receives multimodal input, forges episodic associations, and predicts future state. Oscillating electrical bursts originating from the hippocampus, termed ‘sharp-wave ripples’ (SWR), often contain patterns of previously expressed neural spike sequences, and are necessary for certain forms of learning and memory. The discharge of SWR-replay resonates in remote parts of the brain and displays specific characteristics depending on a subject’s state of awareness and sensory context. In the sleep state, when motoric repertoire is limited, waves of breathing synchronize neural activity in several regions of the brain, including SWRs of the hippocampus. During active sensation of the awake state, cyclic licking dynamically entrains taste-reward networks in subcortical and cortical areas throughout learning. However, the neural correlates linking oromotor movements in the active learning state to the memory system of the hippocampal formation have not yet been established. Given the recurrence of SWR-replay during rhythmic ingestion of reinforcement learning and the hierarchical coupling of orofacial behaviors, we hypothesized that repeated licking could provide the oscillatory framework to synchronize memory reactivation during active learning. We approach this question with new technology development to track licking events at a reward port (P-event) during behavior on a spatial alternation task. Additionally, we developed a modular brain implant to simultaneously record from hippocampal area CA1 and medial entorhinal cortex (MEC) - interconnected brain regions that are crucial to episodic memory processing. Along with the co-modulation of individual neurons by licking and SWRs, we provide the first evidence that SWRs detected in dorsal CA1 synchronize with the phase of P-event cycle during learning. Furthermore, we confirmed that SWRs occurring during licking bouts contain neural reactivation of active navigation and trigger enhanced ripple-frequency power in downstream MEC. These results connect movement with memory and may assist in addressing abnormal ingestion behaviors that negatively affect mental or physical healt

Extensive Cortical Convergence to Primate Reticulospinal Pathways.

Early evolution of the motor cortex included development of connections to brainstem reticulospinal neurons; these projections persist in primates. In this study, we examined the organization of corticoreticular connections in five macaque monkeys (one male) using both intracellular and extracellular recordings from reticular formation neurons, including identified reticulospinal cells. Synaptic responses to stimulation of different parts of primary motor cortex (M1) and supplementary motor area (SMA) bilaterally were assessed. Widespread short latency excitation, compatible with monosynaptic transmission over fast-conducting pathways, was observed, as well as longer latency responses likely reflecting a mixture of slower monosynaptic and oligosynaptic pathways. There was a high degree of convergence: 56% of reticulospinal cells with input from M1 received projections from M1 in both hemispheres; for SMA, the equivalent figure was even higher (70%). Of reticulospinal neurons with input from the cortex, 78% received projections from both M1 and SMA (regardless of hemisphere); 83% of reticulospinal cells with input from M1 received projections from more than one of the tested M1 sites. This convergence at the single cell level allows reticulospinal neurons to integrate information from across the motor areas of the cortex, taking account of the bilateral motor context. Reticulospinal connections are known to strengthen following damage to the corticospinal tract, such as after stroke, partially contributing to functional recovery. Extensive corticoreticular convergence provides redundancy of control, which may allow the cortex to continue to exploit this descending pathway even after damage to one area.SIGNIFICANCE STATEMENT The reticulospinal tract (RST) provides a parallel pathway for motor control in primates, alongside the more sophisticated corticospinal system. We found extensive convergent inputs to primate reticulospinal cells from primary and supplementary motor cortex bilaterally. These redundant connections could maintain transmission of voluntary commands to the spinal cord after damage (e.g., after stroke or spinal cord injury), possibly assisting recovery of function

Theta Rhythmic Clock-Like Activity of Single Units in the Mouse Hippocampus

Theta rhythmic clock-like activity was observed in a small group of hippocampal CA1 neurons in freely behaving mice. These neurons were only persistently activated during theta states of waking exploration and rapid eye movement sleep, but were almost silent during the non-theta state of slow-wave sleep. Interestingly, these cells displayed a theta clock-like simple-spike firing pattern, and were capable of firing one spike per theta cycle during theta states. This is the first report of a unique class of hippocampal neurons with a clock-like firing pattern at the theta rhythm. We speculate that these cells may act as a temporal reference to participate in the theta-related temporal coding in the hippocampus. SIGNIFICANCE STATEMENT Theta oscillations, as the predominant rhythms in the hippocampus during waking exploration and rapid eye movement sleep, may be critical for temporal coding/decoding of neuronal information, and theta-phase precession in hippocampal place cells is one of the best demonstrations of such temporal coding. Here, we show that a unique small class of hippocampal CA1 neurons fired with a theta rhythmic clock-like firing pattern during theta states. These firing characteristics support the notion that these neurons may play a critical role in theta-related temporal coding in the hippocampus

Wireless Simultaneous Stimulation-and-Recording Device (SRD) to Train Cortical Circuits in Rat Somatosensory Cortex

The primary goal of this project is to develop a wireless system for simultaneous recording-and-stimulation (SRD) to deliver low amplitude current pulses to the primary somatosensory cortex (SI) of rats to activate and enhance an interhemispheric cortical pathway. Despite the existence of an interhemispheric connection between similar forelimb representations of SI cortices, forelimb cortical neurons respond only to input from the contralateral (opposite side) forelimb and not to input from the ipsilateral (same side) forelimb. Given the existence of this interhemispheric pathway we have been able to strengthen/enhance the pathway through chronic intracortical microstimulation (ICMS) in previous acute experiments of anesthetized rats. In these acute experiments strengthening the interhemispheric pathway also brings about functional reorganization whereby cortical neurons in forelimb cortex respond to new input from the ipsilateral forelimb. Having the ability to modify cortical circuitry will have important applications in stroke patients and could serve to rescue and/or enhance responsiveness in surviving cells around the stroke region. Also, the ability to induce functional reorganization within the deafferented cortical map, which follows limb amputation, will also provide a vehicle for modulating maladaptive cortical reorganization often associated with phantom limb pain leading to reduced pain. In order to increase our understanding of the observed functional reorganization and enhanced pathway, we need to be able to test these observations in awake and behaving animals and eventually study how these changes persist over a prolonged period of time. To accomplish this a system was needed to allow simultaneous recording and stimulation in awake rats. However, no such commercial or research system exists that meets all requirements for such an experiment. In this project we describe the (1) system design, (2) system testing, (3) system evaluation, and (4) system implementation of a wireless simultaneous stimulation-and-recording device (SRD) to be used to modulate cortical circuits in an awake rodent animal model