Implementing simultaneous calcium imaging and optogenetics in freely moving rodents to investigate the role of local inhibition in place field stability

Place cells are pyramidal neurons in CA1, 2, and 3 of the hippocampus that fire preferentially when an animal is located in a certain position in an environment. The location to which a place cell maximally responds (its place field) is randomly assigned within an environment. Place fields can be re-assigned depending on changes to local sensory cues within an environment, a phenomenon called “remapping”. Research has established that place field formation is affected by input from the local network in the hippocampus. Interneurons in CA1 are known to provide inhibitory input into place cells, but no causal link between local inhibition and place map remapping has been demonstrated. Here, we sought to understand if directly driving local inhibitory networks in CA1 could induce place field remapping. To do this, we used optogenetic stimulation to excite interneurons while concurrently monitoring neural activity using a red-shifted calcium indicator (jRCaMP1b) in freely moving mice. Microendoscopic calcium imaging was performed over multiple days as the mouse explored an open field. After the animal had explored the field for several days, interneurons in CA1 were optogenetically activated at certain positions in the environment. Unfortunately, the low signal to noise level of the red-shifted calcium indicator prevented analysis of this dataset. In lieu of this, we analyzed data from a green fluorescent calcium indicator (GCaMP6f) that we used to initially test out our experimental technique. The results of this analysis revealed aberrant neural activity during calcium imaging sessions, suggesting that the calcium imaging technique may have unexpected, pathological effects on neural activity in the hippocampus. The work described here sets the groundwork for further concurrent use of optogenetics with calcium imaging in investigations into the cellular mechanisms behind place field formation

How does the brain extract acoustic patterns? A behavioural and neural study

In complex auditory scenes the brain exploits statistical regularities to group sound elements into streams. Previous studies using tones that transition from being randomly drawn to regularly repeating, have highlighted a network of brain regions involved during this process of regularity detection, including auditory cortex (AC) and hippocampus (HPC; Barascud et al., 2016). In this thesis, I seek to understand how the neurons within AC and HPC detect and maintain a representation of deterministic acoustic regularity. I trained ferrets (n = 6) on a GO/NO-GO task to detect the transition from a random sequence of tones to a repeating pattern of tones, with increasing pattern lengths (3, 5 and 7). All animals performed significantly above chance, with longer reaction times and declining performance as the pattern length increased. During performance of the behavioural task, or passive listening, I recorded from primary and secondary fields of AC with multi-electrode arrays (behaving: n = 3), or AC and HPC using Neuropixels probes (behaving: n = 1; passive: n = 1). In the local field potential, I identified no differences in the evoked response between presentations of random or regular sequences. Instead, I observed significant increases in oscillatory power at the rate of the repeating pattern, and decreases at the tone presentation rate, during regularity. Neurons in AC, across the population, showed higher firing with more repetitions of the pattern and for shorter pattern lengths. Single-units within AC showed higher precision in their firing when responding to their best frequency during regularity. Neurons in AC and HPC both entrained to the pattern rate during presentation of the regular sequence when compared to the random sequence. Lastly, development of an optogenetic approach to inactivate AC in the ferret paves the way for future work to probe the causal involvement of these brain regions

Functional connectivity and dendritic integration of feedback in visual cortex

A fundamental question in neuroscience is how different brain regions communicate with each other. Sensory processing engages distributed circuits across many brain areas and involves information flow in the feedforward and feedback direction. While feedforward processing is conceptually well understood, feedback processing has remained mysterious. Cortico-cortical feedback axons are enriched in layer 1, where they form synapses with the apical dendrites of pyramidal neurons. The organization and dendritic integration of information conveyed by these axons, however, are unknown. This thesis describes my efforts to link the circuit-level and dendritic-level organization of cortico-cortical feedback in the mouse visual system. First, using cellular resolution all-optical interrogation across cortical areas, I characterized the functional connectivity between the lateromedial higher visual area (LM) and primary visual cortex (V1). Feedback influence had both facilitating and suppressive effects on visually-evoked activity in V1 neurons, and was spatially organized: retinotopically aligned feedback was relatively more suppressive, while retinotopically offset feedback was relatively more facilitating. Second, to examine how feedback inputs are integrated in apical dendrites, I optogenetically stimulated presynaptic neurons in LM while using 2-photon calcium imaging to map feedback-recipient spines in the apical tufts of layer 5 neurons in V1. Activation of a single feedback-providing input was sufficient to boost calcium signals and recruit branch-specific local events in the recipient dendrite, suggesting that feedback can engage dendritic nonlinearities directly. Finally, I measured the recruitment of apical dendrites during visual stimulus processing. Surround visual stimuli, which should recruit relatively more facilitating feedback, drove local calcium events in apical tuft branches. Moreover, global dendritic event size was not purely determined by somatic activity but modulated by visual stimuli and behavioural state, in a manner consistent with the spatial organization of feedback. In summary, these results point toward a possible involvement of active dendritic processing in the integration of feedback signals. Active dendrites could thus provide a biophysical substrate for the integration of essential top-down information streams, including contextual or predictive processing

Early, sustained and broadly-tuned discharge of fast-spiking interneurons in the premotor cortex during action planning

Preparatory neural activity in premotor areas is critical for planning and execution of voluntary movements. Previous studies in monkeys and mice have revealed how the discharges of pyramidal, excitatory neurons (PNs) encode a motor plan for an upcoming movement (Afshar et al., 2011; Chen et al., 2017; Li et al., 2015). However, the contribution of GABAergic interneurons, specifically fast-spiking interneurons (FSNs), to voluntary movements remains poorly understood. Putative premotor areas involved in action planning have been demonstrated in rodents. In particular, in mice, a premotor area controlling voluntary licking has been identified in the anterior-lateral motor cortex (ALM) (Komiyama et al., 2010). Also, ALM partially overlaps with the rostral forelimb area (RFA), the previously defined premotor region involved in the control of paw movement in rats and mice (Rouiller et al., 1993; Tennant et al., 2011). To understand the excitatory-inhibitory microcircuit involved in action planning, here I compare directly the response properties of PNs and FSNs during licking behaviour and forelimb retraction in the mouse. Recordings are carried out with both acute electrodes and chronic microelectrode arrays from both the two premotor areas, i.e. the ALM \u2013 responsible for licking \u2013, and RFA \u2013 involved in paw movement. Specifically, in a first set of experiments, I used head-restrained mice that spontaneously lick a reward delivered at random intervals from a drinking spout. Mice voluntary performed either single isolated or a burst of consecutive licks, which I categorized, a posteriori, in single (= 1 lick) and multiple licks ( 65 3 licks). During the task, I extracellularly recorded single units\u2019 activity from ALM, using acute in vivo electrophysiology. I identified putative PNs and FSNs, based on well-established features of their waveforms, and investigated their functional properties during the movement. Unexpectedly, I report that optogenetically-verified FSNs showed an earlier and more sustained activation than PNs. In particular, most of the neurons\u2019 activity anticipated the licking onset, consistently with an involvement of the ALM in movement planning. The majority of the neurons (~90%) increased their firing frequency in correspondence with the movement, but suppressive modulations were also observed in a subset of units. For both PNs and FSNs, I found significantly greater discharge during multiple than single licks and the peak discharge was significantly delayed for both subclasses during multiple licking events. However, FSNs modulated their activity about 100ms earlier than PNs. Furthermore, almost all FSNs showed a peak in their response before the beginning of the sequence of licks. Analysis of mean information content confirms that FSNs predict licking onset not only significantly better, but even earlier, than PNs. Chronic electrode arrays covering both the ALM and RFA were next used to simultaneously probe neural responses during (i) licking and (ii) forelimb pulling in a robotic device (Spalletti et al., 2017). I report that most of the FSNs respond with a stereotyped increase in their firing rates during both licking and pulling. In stark contrast, PNs show a variety of behaviours, dependent on movement type. At least for a minority of them, licking behaviour and forelimb retraction are represented as two different motor acts, reaching significant levels in the PNs. Accordingly, computational analysis shows that PNs carry more independent information than FSNs. Altogether, these data indicate that a global rise of GABAergic inhibition mediated by FSNs firing contributes to early action planning. Next, encouraged by the deeper understanding of the cortical microcircuits underlying movement planning in mice, I exploited this knowledge to explore more complex mechanisms, as action understanding. The neural circuits that integrate performed and observed actions have been found in the premotor cortex of monkeys and named as \u2018mirror neurons system\u2019 (di Pellegrino et al., 1992). Recently, the presence of mirror neurons have been demonstrated in rodents in the anterior cingulate cortex (Carrillo et al., 2019), but whether they could contribute to action understanding in the premotor cortex is still unclear. At behavioural level, the observation of actions can actually lead, in some cases, to the repetition of those same actions. This phenomenon has been named social facilitation, and the underlying motor program has been attributed to the mirror system (Ferrari et al., 2005). Here, I set up a behavioural task similar to the one exploited in monkeys to explore social facilitation in mice. I took advantage of licking behaviour to set up the social facilitation experiment. Therefore, head-restrained mice were allowed to lick water from a feeding needle. I found that mice can actually facilitated to lick more when another individual was engaged in the same action, supporting the hypothesis of a social facilitation in mouse. Altogether these results indicate that the observers\u2019 behaviour was actually influenced by the demonstrators\u2019 one, laying the groundwork for the study of mirror neurons in mice at cellular level

Fast-spiking parvalbumin^+ GABAergic interneurons: From cellular design to microcircuit function

The success story of fast-spiking, parvalbumin-positive (PV+) GABAergic interneurons (GABA, γ-aminobutyric acid) in the mammalian central nervous system is noteworthy. In 1995, the properties of these interneurons were completely unknown. Twenty years later, thanks to the massive use of subcellular patch-clamp techniques, simultaneous multiple-cell recording, optogenetics, in vivo measurements, and computational approaches, our knowledge about PV+ interneurons became more extensive than for several types of pyramidal neurons. These findings have implications beyond the “small world” of basic research on GABAergic cells. For example, the results provide a first proof of principle that neuroscientists might be able to close the gaps between the molecular, cellular, network, and behavioral levels, representing one of the main challenges at the present time. Furthermore, the results may form the basis for PV+ interneurons as therapeutic targets for brain disease in the future. However, much needs to be learned about the basic function of these interneurons before clinical neuroscientists will be able to use PV+ interneurons for therapeutic purposes

장소와 그 가치를 저장하는 배측과 중간 해마의 차별적 역할

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 뇌인지과학과, 2021.8. 강민수.오래전부터 해마는 자신의 경험, 즉 일화 사건의 기억에 필수적인 영역으로 알려져왔습니다. 이러한 일화 사건에는 특정 장소에서 겪은 감정적 경험들이 기억으로 저장됩니다. 이러한 일화 기억의 특성을 고려하면, 해마는 감정 정보를 처리할 가능성이 매우 높고, 실제로 중간 해마와 복측 해마는 편도체로부터 해부학적으로 직접 연결되어 있습니다. 또한 중간 해마는 배측 해마로부터 많은 공간 정보를 받아드린다고 알려져있습니다. 따라서 중간 해마는 장소의 위치와 그 장소에서 경험한 감정정보를 연합할 가능성이 높습니다. 하지만, 중간 해마의 이러한 장소-감정 연합 기억의 역할은 거의 알려지지 않았다. 그래서, 저는 중간 해매가 특정 공간에서 발생하는 사건의 가치를 저장하는데 중요하고, 배측 해마는 정확한 위치 정보를 표상하는데 중요하다는 가설을 세웠습니다. 이를 검증하기 위해 쥐의 배측과 중간 해마의 개별 뉴런을 동시에 리코딩하였으며, 선호도가 다른 먹이를 이용해 장소의 가치 정보를 변화시키는 실험을 진행했습니다. 이 학위 논문의 첫 파트에서는 쥐가 2차원 공간에서 자유롭게 돌아다닐 때의 배측부터 복측해마의 장소 세포가 어떻게 달라지는지 살펴보았습니다. 구체적으로, 중간해마보다 배측 해마에서 장소 선택적 활동이 더 강하게 나타났으며, 복측 해마에서는 장소 세포의 활동이 거의 관찰되지 않았습니다. 두번째 파트에서, 해마가 필요없는 간단한 과제에서 먹이의 가치가 바뀐 이후에, 배측과 복측 해마의 장소 세포의 공간 표상 변화를 살펴보았습니다. 주어진 공간에서 제공되던 맛있는 먹이가 맛없는 먹이로 바뀌고 나면, 중간 해마의 장소세포는 재빠르게 공간 표상을 재배열하게 됩니다. 하지만, 동일한 조작에서 배측 해마의 장소세포는 공간 표상을 일정하게 유지하였습니다. 마지막으로, 세번째 파트에서는 해마가 필요한 기억 과제에서 가치-의존적 공간 재배열을 추가적으로 알아보았습니다. T 모양의 미로에서 장소 선호 과제를 진행하는 동안, 중간 해마의 장소 세포는 맛있는 먹이가 나오는 공간을 집중적으로 표상하며, 이러한 집중된 표상은 맛있는 먹이의 위치가 바뀌어도 동일하게 관찰됩니다. 반면, 배측 해마 장소 세포의 공간 표상은 이러한 조작에 거의 영향을 받지 않습니다. 그리고 이 장소 선호 학습을 하는 동안, 배측 해마보다 중간 해마의 신경망 상태가 빠르게 변하는 모습을 보였습니다. 종합하자면, 위 결과들은 배측 해마와 복측 해마는 서로 다른 기능을 맡고 있다는 점을 보여줍니다. 즉, 배측 해마는 동물의 정확한 장소를 표상하는데 특화되어 있으며, 중간 해마는 장소와 그 가치 정보를 연합하는 역할을 맡고 있습니다. 이러한 발견은 중간 해마가 행동 선택과 밀접한 정보를 처리하며, 이러한 정보를 내측 전두엽을 통해 다른 뇌 영역과 소통하는 기능적으로 중요한 영역이라는 것을 시사합니다.It has long been postulated that the hippocampus is vital for memorizing autobiographical episodic events. Because an episodic event often entails memories for certain places associated with their emotional and motivational significance, it is promising that the hippocampus processes spatial information in conjunction with its associated valence. Among the hippocampal subregions (i.e., dorsal, intermediate, and ventral), the amygdala, which plays key roles in processing valence information, sends direct axonal projection to the intermediate and ventral hippocampus. Also, there are extensive recurrent collaterals and associational projections (presumably spatial information) from the dorsal hippocampus to the intermediate hippocampus. Thus, the intermediate hippocampus may integrate emotional/motivational information in association with locational information. However, it is largely unknown that how the intermediate hippocampus process value-associated spatial information processing. Therefore, I hypothesized that encoding the value of an event at a specific location takes priority in the intermediate hippocampus, compared to the dorsal hippocampus, whose priority resides in representing the precise location of an animal, presumably in the cognitive map. To test this hypothesis, I simultaneously recorded single units from the dorsal and intermediate hippocampus while rats performed a battery of tasks in which the level of motivational significance of a place was controlled by foods with different palatability. In this dissertation of Chapter 1, I examined the changes in spatial firing patterns along the dorsoventral axis while rats foraged in an open field maze. Specifically, spatially selective firing was more eminent in the dorsal than in the intermediate hippocampus, and spatial signals were hardly observed in the ventral hippocampus. In Chapter 2, after changes in reward value during non-mnemonic tasks, differential global remappings of place cells were found between the dorsal and intermediate hippocampus. When more-palatable reward (i.e., sunflower seeds) were replaced with less-palatable one (Cheerios) in a given location, place cells in the intermediate hippocampus remapped immediately. In contrast, place fields recorded from the dorsal hippocampus maintained their spatial representations stably in the same manipulation. In Chapter 3, value-dependent remappings were further investigated in hippocampal-dependent tasks. During the place-preference task in the T-maze, place fields obtained from the intermediate hippocampus accumulated near the arm associated with more-preferred rewards, and overrepresented patterns shifted toward opposite arm after the locations of more-preferred and less-preferred rewards were reversed. However, spatial representations of place cells in the dorsal hippocampus were rarely affected by such manipulation. And, during the acquisition of the place-preference task, the ensemble network state in the iHP changed faster than that in the dHP. Taken together, our results suggest that there are functional segregations between the dorsal and intermediate subregions of the hippocampus. That is, the dorsal hippocampus is specialized in representing the animal's precise locations in the environment, whereas the intermediate hippocampus takes part in the integration of spatial information and its motivational values. These findings imply that the intermediate hippocampus is a functionally significant hippocampal subregion through which critical action-related information (i.e., spatial information from the dorsal hippocampus and emotional/motivational information from the amygdala) is integrated and communicated to the rest of the brain via the medial prefrontal cortex.BACKGROUND AND HYPOTHESIS. 1 1.1 BACKGROUND 1 1.1.1 Episodic memory and hippocampus. 2 1.1.2 Introduction of the rodent hippocampal researches. 2 1.1.3 Single-cell recording from the rodent hippocampus 4 1.1.3.1 Basic firing properties of place cells 4 1.1.3.2 Spatial representation of place cells. 5 1.1.3.3 Non-spatial representation of place cells 6 1.1.3.4 Value representation in the hippocampus. 6 1.1.4 Difference in anatomical connectivities along the dorsoventral axis. 7 1.1.5 Difference in functions along the dorsoventral axis. 10 1.2 HYPOTHESIS 12 CHAPTER 1. 13 2.1 Introduction. 14 2.2 Methods. 15 2.2.1 Subjects. 15 2.2.2 Maze familiarization and pre-training 15 2.2.3 Surgical implantation of the hyperdrive. 15 2.2.4 Electrophysiological recording procedures 16 2.2.5 Histological verification of tetrode tracks 16 2.2.6 Unit isolation 16 2.2.7 Basic firing properties 17 2.2.8 Definition of place fields 17 2.2.9 Theta-modulation and burst index 18 2.3 Results. 19 2.3.1 Anatomical boundary between dorsal, intermediate and ventral hippocampus. 19 2.3.2 Comparison of basic firing properties between hippocampal subregions 20 2.3.3 Degree of spatially selective firing patterns sharply decreased at the border between dHP and iHP. 23 2.4 Discussion. 28 CHAPTER 2. 30 3.1 Introduction. 31 3.2 Methods. 32 3.2.1 Behavior paradigm. 32 3.2.1.1 Food preference test. 32 3.2.1.2 Spatial alternation task. 33 3.2.2 Post-surgical training and main recording 33 3.2.3 Constructing the population rate map. 34 3.2.4 Categorization of place field responses 34 3.2.5 Reward-type coding analysis. 34 3.2.6 Speed-correlated cells. 35 3.3 Results. 35 3.3.1 Rat's food preference for sunflower seeds and Froot Loops over Cheerios. 35 3.3.2 Place cells in iHP, but not dHP, encode changes in motivational values of place via global remapping. 36 3.3.3 Identity of reward type is coded in the iHP by rate remapping, but not in the dHP. 49 3.3.4 Neural activity of single cells of vHP in response to motivational value changes. 51 3.4.5 Immediate coding of the changes in motivational values in iHP, but not in dHP. 53 3.4 Discussion 60 CHAPTER 3. 64 4.1 Introduction 65 4.2 Methods 65 4.2.1 Behavior paradigm. 65 4.2.2 Principal component analysis for neural ensemble state 66 4.2.3 Synchronization of spiking activity. 67 4.3 Results. 68 4.3.1 Overrepresentation of the motivationally significant place by the place cells in iHP, but not in dHP 68 4.2.2 Rapid changes of the ensemble network changes in iHP, compared to those in dHP. 77 4.2.3 Place cells in the dHP and iHP co-fire more strongly during a mnemonic task than non-mnemonic tasks. 79 4.4 Discussion 82 GENERAL DISCUSSION. 87 5.1 Conclusion 88 5.2 Limitation 88 5.3 Implication and perspective. 89 5.4 Future research direction. 93 BIBLIOGRAPHY 94 ACKNOWLEDGMENT 111 국문초록 112박

Two-photon all-optical interrogation of mouse barrel cortex during sensory discrimination

The neocortex supports a rich repertoire of cognitive and behavioural functions, yet the rules, or neural ‘codes’, that determine how patterns of cortical activity drive perceptual processes remain enigmatic. Experimental neuroscientists study these codes through measuring and manipulating neuronal activity in awake behaving subjects, which allows links to be identified between patterns of neural activity and ongoing behaviour functions. In this thesis, I detail the application of novel optical techniques for simultaneously recording and manipulating neurons with cellular resolution to examine how tactile signals are processed in sparse neuronal ensembles in mouse somatosensory ‘barrel’ cortex. To do this, I designed a whisker-based perceptual decision-making task for head-fixed mice, that allows precise control over sensory input and interpretable readout of perceptual choice. Through several complementary experimental approaches, I show that task performance is exquisitely coupled to barrel cortical activity. Using two- photon calcium imaging to simultaneously record from populations of barrel cortex neurons, I demonstrate that different subpopulations of neurons in layer 2/3 (L2/3) show selectivity for contralateral and ipsilateral whisker input during behaviour. To directly test whether these stimulus-tuned groups of neurons differentially impact perceptual decision-making I performed patterned photostimulation experiments to selectively activate these functionally defined sets of neurons and assessed the resulting impact on behaviour and the local cortical network in layer 2/3. In contrast with the expected results, stimulation of sensory-coding neurons appeared to have little perceptual impact on task performance. However, activation of non- stimulus coding neurons did drive decision biases. These results challenge the conventional view that strongly sensory responsive neurons carry more perceptual weight than non-responsive sensory neurons during perceptual decision-making. Furthermore, patterned photostimulation revealed and imposed potent surround suppression in L2/3, which points to strong lateral inhibition playing a dominant role in shaping spatiotemporally sparse activity patterns. These results showcase the utility of combined patterned photostimulation methods and population calcium imaging for revealing and testing neural circuit function during sensorimotor behaviour and provide new perspectives on sensory coding in barrel cortex

Mapping neural responses onto innate and acquired behavior: from insect olfaction to realizing a bio-hybrid chemical recognition system

In many organisms, the sense of smell, driven by the olfactory system, serves as the primary sensory modality that guides a plethora of behaviors such as foraging for food, finding mates, and evading predators. Using an array of biological sensors, the olfactory system converts volatile chemical inputs from an organism’s environment into well-patterned neural responses that inform downstream motor neurons to drive appropriate behaviors (e.g., moving towards food or away from danger). For many external cues, the elicited neural responses are often determined by the genetic makeup of the organism, which assigns an innate preference, or valence, for these different stimuli. However, our environment is constantly in flux, and the same stimulus can be encountered in a variety of different contexts, such as following other cues or under different ambient conditions (e.g., humidity). This can modify the neural activation pattern ascribed to the stimulus and potentially alter the corresponding behavioral output. The objective of this dissertation is to understand how neural responses in the early olfactory system of locusts (Schisctocerca americana) are spatiotemporally structured to robustly represent innate valence in different scenarios to drive appropriate behaviors and how they can be altered through learning. To achieve this goal, we used a large panel of chemically diverse odorants and characterized the neural responses they elicited in the antennal lobe (at the level of ensembles of principal or projection neurons) as well as the innate appetitive behavioral response they produced. We found that neural responses generated both during (ON response) and after (OFF response) termination of the odorant contained information regarding its identity and could be used to predict the innate behavioral outcomes. Notably, predictions made using the ON and the OFF responses differed in the sets of neurons they used to generate the predictions, indicating that neural-behavioral transformations could be achieved in multiple ways. Furthermore, both these ON and OFF neural response classifiers outperformed attempts to predict behavior using chemical features of the stimuli (detected by NMR or IR spectra), indicating that the antennal lobe was transforming and encoding olfactory inputs to map them onto the innate valence associated with the sensory cue. We found that the organization of odor-evoked neural responses that readily map onto innate preferences may also constrain learned odor-reward associations. While odorants with an innate positive behavioral preference alone could support learning odor-reward associations, the conditioned responses were not odor-specific but appeared to generalize to other odorants that evoked similar neural responses. The timing of the behavioral responses could be varied by delivering rewards during epochs when the odorant would generate either the ON or the OFF neural responses. Overall, we found that the organization of ON and OFF neural responses in the antennal lobe clustered into manifolds or subspaces that could be explained using innate behavioral preferences and suitability for reinforcement learning. To understand the robustness of these results, we developed novel minimally invasive experimental methods to record locust neural responses while they actively sampled their surroundings. We found neural responses in this more naturalistic scenario to maintain their manifold organization, and classical conditioning enhanced the separation between neural responses evoked by innately appetitive and non-appetitive odorants. Our results also indicate that neural and behavioral responses in freely moving locusts were consistent with those observed earlier in highly compromised preparations. Finally, we exploited our newly-developed recording techniques to engineer an insect-based chemical sensor that could be used for a real-world application