Information processing in dissociated neuronal cultures of rat hippocampal neurons

One of the major aims of Systems Neuroscience is to understand how the nervous system transforms sensory inputs into appropriate motor reactions. In very simple cases sensory neurons are immediately coupled to motoneurons and the entire transformation becomes a simple reflex, in which a noxious signal is immediately transformed into an escape reaction. However, in the most complex behaviours, the nervous system seems to analyse in detail the sensory inputs and is performing some kind of information processing (IP). IP takes place at many different levels of the nervous system: from the peripheral nervous system, where sensory stimuli are detected and converted into electrical pulses, to the central nervous system, where features of sensory stimuli are extracted, perception takes place and actions and motions are coordinated. Moreover, understanding the basic computational properties of the nervous system, besides being at the core of Neuroscience, also arouses great interest even in the field of Neuroengineering and in the field of Computer Science. In fact, being able to decode the neural activity can lead to the development of a new generation of neuroprosthetic devices aimed, for example, at restoring motor functions in severely paralysed patients (Chapin, 2004). On the other side, the development of Artificial Neural Networks (ANNs) (Marr, 1982; Rumelhart & McClelland, 1988; Herz et al., 1981; Hopfield, 1982; Minsky & Papert, 1988) has already proved that the study of biological neural networks may lead to the development and to the design of new computing algorithms and devices. All nervous systems are based on the same elements, the neurons, which are computing devices which, compared to silicon components, are much slower and much less reliable. How are nervous systems of all living species able to survive being based on slow and poorly reliable components? This obvious and na\uefve question is equivalent to characterizing IP in a more quantitative way. In order to study IP and to capture the basic computational properties of the nervous system, two major questions seem to arise. Firstly, which is the fundamental unit of information processing: 2 single neurons or neuronal ensembles? Secondly, how is information encoded in the neuronal firing? These questions - in my view - summarize the problem of the neural code. The subject of my PhD research was to study information processing in dissociated neuronal cultures of rat hippocampal neurons. These cultures, with random connections, provide a more general view of neuronal networks and assemblies, not depending on the circuitry of a neuronal network in vivo, and allow a more detailed and careful experimental investigation. In order to record the activity of a large ensemble of neurons, these neurons were cultured on multielectrode arrays (MEAs) and multi-site stimulation was used to activate different neurons and pathways of the network. In this way, it was possible to vary the properties of the stimulus applied under a controlled extracellular environment. Given this experimental system, my investigation had two major approaches. On one side, I focused my studies on the problem of the neural code, where I studied in particular information processing at the single neuron level and at an ensemble level, investigating also putative neural coding mechanisms. On the other side, I tried to explore the possibility of using biological neurons as computing elements in a task commonly solved by conventional silicon devices: image processing and pattern recognition. The results reported in the first two chapters of my thesis have been published in two separate articles. The third chapter of my thesis represents an article in preparation

Excitatory postsynaptic potentials in rat neocortical neurons in vitro. III. Effects of a quinoxalinedione non-NMDA receptor antagonist

1. Intracellular microelectrodes were used to obtain recordings from neurons in layer II/III of rat frontal cortex. A bipolar electrode positioned in layer IV of the neocortex was used to evoke postsynaptic potentials. Graded series of stimulation were employed to selectively activate different classes of postsynaptic responses. The sensitivity of postsynaptic potentials and iontophoretically applied neurotransmitters to the non-N-methyl-D-asparate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was examined. 2. As reported previously, low-intensity electrical stimulation of cortical layer IV evoked short-latency early excitatory postsynaptic potentials (eEPSPs) in layer II/III neurons. CNQX reversibly antagonized eEPSPs in a dose-dependent manner. Stimulation at intensities just subthreshold for activation of inhibitory postsynaptic potentials (IPSPs) produced long-latency (10 to 40-ms) EPSPs (late EPSPs or 1EPSPs). CNQX was effective in blocking 1EPSPs. 3. With the use of stimulus intensities at or just below threshold for evoking an action potential, complex synaptic potentials consisting of EPSP-IPSP sequences were observed. Both early, Cl(-)-dependent and late, K(+)-dependent IPSPs were reduced by CNQX. This effect was reversible on washing. This disinhibition could lead to enhanced excitability in the presence of CNQX. 4. Iontophoretic application of quisqualate produced a membrane depolarization with superimposed action potentials, whereas NMDA depolarized the membrane potential and evoked bursts of action potentials. At concentrations up to 5 microM, CNQX selectively antagonized quisqualate responses. NMDA responses were reduced by 10 microM CNQX. D-Serine (0.5-2 mM), an agonist at the glycine regulatory site on the NMDA receptor, reversed the CNQX depression of NMDA responses

흔적 공포 조건화와 맥락 공포 분별의 시냅스 기전

학위논문(박사) -- 서울대학교대학원 : 의과대학 의과학과, 2023. 2. 이석호.Learning the association between aversive events and environmental stimuli is essential for survival. Fear conditioning, a basic form of associative learning, is one of the widely studied paradigms in behavioral psychology. Fear conditioning is divided into cued and contextual fear conditioning (CFC). Depending on the absence or presence of a time interval between conditioned stimulus (CS) and unconditioned stimulus (US), cued fear conditioning is further subdivided into delayed fear conditioning (DFC) and traced fear conditioning (TFC), respectively. Amygdala is a center for associative memory formation and expression of fear response in all forms of fear conditioning. Whereas the amygdala is sufficient for DFC, CFC and TFC requires contributions of other cortical regions such as the hippocampus, prefrontal cortex (PFC) and retrosplenial cortex. Thus, CFC and TFC provide invaluable opportunities for studying cortical functions. Most previous studies have focused on the neuronal correlates of fear conditioning, but it has not previously been investigated whether synapse specific dysfunction of plasticity has any effects on the behavior. In this thesis, I challenged this issue by investigating the behavioral consequences of dysfunction of plasticity at layer 2/3 (L2/3) to layer 5 (L5) pyramidal cell synapses in the prelimbic (PL) area of the medial prefrontal cortex (mPFC) and perforant pathway (PP) synapses in the hippocampal CA3 and in rodents. Sustained increased activity in PFC during trace interval in TFC is essential for acquisition of trace fear memory. However, the neurophysiological mechanisms underlying increased activity are poorly understood. Post-tetanic potentiation (PTP) was proposed as short-term plasticity that might mediate the generation of sustained increased activity during working memory. I examined the neurobiological mechanism of PTP in the PL, and tested whether PTP plays a role in sustained increased activity and TFC. Using optogenetic stimulation, I stimulated afferent fibers to L5 pyramidal neurons (PNs) in cell-type and layer-specific manner. I found that PTP was induced in the L5 corticopontine (Cpn) PNs at the synapses made by L2/3 PNs and L5 commissural (COM) PNs, but not in the L5 COM PNs. While PTP at both synapse types onto Cpn cells was inhibited by protein kinase C inhibitor, tetraphenylphosphonium (TPP), a mitochondrial Na/Ca exchanger blocker, suppressed PTP only at L2/3-to-Cpn synapses. Studying the effect of TPP infusion into the PL area on TFC, I found that TPP did not affect the trace memory formation, but reduced the maintenance of the fear memory during fear memory extinction test. In vivo recordings revealed that c.a. 20% of PL-PNs exhibited sustained increased activity after the cessation of CS. The TPP infusion abolished such post-CS sustained increased activity during both conditioning and extinction training. In vivo calcium imaging of COM and CPn neurons during the tone test revealed that L5 CPn and COM neurons showed different proportions of activity patterns. The largest proportion of CPn cells, but not COM cells, belonged to a delay cell type that exhibited sustained increased activity from CS onset to the expected US timing. These results imply that PTP at L2/3-Cpn synapses is required for post-CS sustained increased activity during trace fear conditioning, and plays a role in maintenance of trace fear memory. The hippocampus is important for consolidation and retrieval of contextual fear. The network process that underlies formation of distinct ensembles representing two similar contexts is called pattern separation and is one of the important functions of the hippocampus. However, the network mechanisms underlying pattern separation of neuronal ensembles in CA3 is largely unknown. Kv1.2 expression in rodent CA3 pyramidal cells (CA3-PCs) is polarized to distal apical dendrites, and its downregulation specifically enhances dendritic responses to PP synaptic inputs. It has been previously shown that haploinsufficiency of Kv1.2 (Kcna2+/-) in CA3-PCs, but not Kv1.1 (Kcna1+/-), lowers the threshold for long-term potentiation (LTP) at PP-CA3 synapses, and that the Kcna2+/- mice are normal in discrimination of distinct contexts but impaired in discrimination of similar but slightly distinct contexts. I examined pattern separation of neuronal ensembles in CA3 and dentate gyrus (DG) that represent two similar contexts using in situ hybridization of immediate early genes: Homer1a and Arc. The size and overlap of CA3 ensembles activated by the first visit to the similar contexts were not different between wildtype and Kcna2+/- mice, but these ensemble parameters diverged over training days between genotypes, suggesting that abnormal plastic changes at PP-CA3 synapses of Kcna2+/- mice is responsible for the impaired pattern separation. Unlike CA3, DG ensembles were not different between two genotype mice. The DG ensembles were already separated on the first day, and their overlap did not further evolve. Eventually, the Kcna2+/- mice exhibited larger CA3 ensemble size and overlap upon retrieval of two contexts, compared to wildtype or Kcna1+/- mice. These results suggest that sparse LTP at PP-CA3 synapse probably supervised by mossy fiber inputs is essential for gradual pattern separation in CA3.환경적 자극과 혐오스러운 사건의 상관관계를 학습하는 것은 생존에 필수적인 요소다. 공포 조건화 (fear conditioning)는 상관 학습의 기초적인 형태로 행동 심리학에서 많이 이용되는 패러다임 중 하나이다. 공포 조건화는 문맥 공포 조건화 (contextual fear conditioning; CFC)와 단서 공포 조건화로 나뉜다. 또한 단서 공포 조건화는 조건 자극 (conditioned stimulus; CS)과 무조건 자극 (unconditioned stimulus; US) 사이의 짧은 시간 간격의 유무를 바탕으로 각각 지연 공포 조건화 (delayed fear conditioning; DFC)과 흔적 공포 조건화 (trace fear conditioning; TFC)로 세분화된다. 편도체는 모든 종류의 공포 조건화에서 상관 기억의 형성과 공포 반응의 발현에 핵심적인 역할을 한다. DFC에서는 공포 기억의 학습 및 발현이 편도체만으로 충분한데 반해, TFC와 CFC에서는 해마, 전전두엽 그리고 팽대후피질 (retrosplenial cortex)과 같은 다른 피질 영역의 기여가 필요하다. 따라서 CFC와 TFC는 피질 영역의 기능을 연구하는데 큰 도움을 준다. 지금까지 대부분의 연구는 공포 조건화와 신경망 활성의 상관관계에 초점이 맞춰져 있었기에 시냅스 특이적 가소성의 기능장애가 행동에 미치는 영향은 연구는 미진하다. 나는 내측 전전두엽 (medial prefrontal cortex; PFC) 변연계전 (prelimbic; PL) 부위의 레이어 2/3 (L2/3)와 레이어 5 (L5)의 피라미드 세포 (pyramidal neuron; PN) 사이의 시냅스와 천공경로 (perforant pathway; PP)와 해마 CA3의 시냅스에서의 가소성 장애와 행동 양상의 관계에 대해 연구하였다. TFC의 흔적 기간 (trace interval) 동안 PFC 신경세포의 지속적으로 증가된 활성은 흔적 공포 기억의 습득에 필요하다. 그러나 PFC에서 나타나는 지속적으로 증가된 활성의 신경생리학적인 메커니즘의 이해도는 낮다. 단기가소성의 종류 중 하나 강축후 강화 (post-tetanic potentiation; PTP)는 작업 기억 동안 지속적으로 증가하는 활성을 중개할 것으로 예상된다. 따라서 나는 PL에서 발생하는 PTP의 신경생물학적 메커니즘과 PTP가 TFC와 지속적으로 증가된 활성에 영향을 미치는지에 대해서 연구했다. 광유전학을 통해 나는 L5 PN의 들신경섬유(afferent fiber)를 세포 타입 그리고 층 특이적으로 자극했다. 그 결과 L2/3 PN과 L5의 맞교차 (commissural; COM) PN이 L5 피질뇌교투사 (corticopontine; CPn) PN과 연결되는 시냅스에서 PTP가 유도되고 L5 COM과 연결되는 시냅스에서는 유도되지 않는 것을 발견했다. 두개의 시냅스에서 유도되는 PTP는 protein kinase C (PKC) 억제제로 모두 억제되었지만, 미토콘드리아 Na/Ca exchanger (mNCX) 차단제인 tetraphenylphosphonium (TPP)는 L2/3-CPn 시냅스에서 유도되는 PTP만을 억제했다. TPP를 PL 부위에 주입한 후 TFC를 수행한 결과, 흔적 공포 소거 실험에서 TPP는 흔적 공포 형성에는 영향을 미치지 않았지만 공포 기억을 짧게 유지시키는 것을 밝혔다. 또한, 생체 내 기록 실험에서 TFC동안 CS 이후에 지속적으로 증가한 활성을 가지는 PL-PNs이 약 20% 인 것을 밝혔다. TPP의 주입은 공포 조건화 실험과 공포 기억 소거 실험에서 CS 이후에 나타나는 지속적인 활성 증가를 억제했다. COM과 CPn 세포 특이적 생체내 칼슘 이미징 실험에서 두 세포는 다른 비율의 활성 양상을 나타냈다. COM과 다르게 CPn은 CS의 시작점에서 예상되는 US 시간까지 지속적으로 증가한 활성을 보이는 Delay 세포 타입의 비율이 훨씬 많았다. 이러한 결과들은 L2/3-CPn 시냅스에서 발생하는 PTP가 TFC에서 CS 이후에 지속적으로 증가한 활성에 필요하며, 흔적 공포 기억 유지에 중요하다는 것을 시사한다. 해마는 맥락 공포의 형성, 강화 그리고 검색에 중요하다. 두 개의 유사한 맥락을 나타내는 앙상블 형성의 기초가 되는 네트워크 프로세스를 패턴 분리 (pattern separation)라고 하며 해마의 중요한 기능 중 하나이다. 그러나 CA3에서 신경 앙상블의 패턴 분리의 기본이 되는 네트워크 메커니즘은 거의 알려져 있지 않다. CA3 피라미드 세포 (CA3-PN)에서 Kv1.2 발현은 말단 정점 수상돌기 (distal apical dendrite)로 집중되어 있고, 그것의 하향 조절 (downregulation)은 PP 시냅스 입력에 대한 수지상 (dendritic) 반응을 향상시킨다. CA3-PN에서 Kv1.2의 반수체 부족 개체 (Kcna2+/-)는 PP-CA3 시냅스에서 장기 강화 (Long-term potentiation; LTP)의 임계값 (threshold)을 낮춘다. Kcna2+/- 개체는 확실히 구별되는 상황을 식별하는 행동은 정상이지만 약간의 차이가 있는 유사한 상황을 식별하는 행동에는 장애를 보이는 것을 이전 연구에서 밝혔다. 나는 초기 발현 유전자 (Homer1a, Arc)와 절편상 하이브리드 형성법을 사용하여 두 가지 유사한 컨텍스트에서 나타나는 CA3 및 치아이랑 (Dentate gyrus; DG)의 신경 앙상블의 패턴 분리를 조사했다. 비슷한 문맥에 처음 노출되었을 때 활성화되는 CA3 앙상블의 크기와 교집합은 야생형 (wildtype; WT)과 Kcna2+/- 마우스 간에 차이가 없었다. 그러나 두 그룹간의 앙상블 크기와 교집합은 실험이 진행될수록 점차 차이를 보였다. 이는 Kcna2+/- 마우스의 PP-CA3 시냅스에서 비정상적인 가소성 변화가 나타나며 결과적으로 WT과 다르게 비정상적인 패턴 분리를 야기한다는 것을 제안한다. 이와 다르게 DG의 앙상블은 두 그룹간의 차이가 없었다. DG 앙상블은 실험 첫날부터 이미 분리되어 있었으며, 앙상블의 교집합은 실험이 진행되어도 변화하지 않았다. 결과적으로 Kcna2+/- 마우스는 WT과 Kcna1+/- 마우스에 비해서, CA3 앙상블의 크기와 두개의 문맥에 대한 앙상블의 교집합이 크다. 이러한 결과는 이끼 섬유 (mossy fiber)의 입력에 의해 지도되는 PP-CA3 시냅스의 희소한 LTP가 CA3의 점진적 패턴 분리에 필수적임을 시사한다.Chapter 1. Role of post-tetanic potentiation in persistent activity of medial prefrontal neuron during trace fear conditioning and retrieval 1 Introduction 2 Materials and Methods 14 Results 34 Discussion 112 Chapter 2. Gradual pattern separation in CA3 associated with contextual discrimination learning is impaired by Kv1.2 insufficiency 134 Introduction 135 Materials and Methods 140 Results 148 Discussion 172 References 180 Abstract in Korean 200박

MUSCARINIC MODULATION OF BASOLATERAL AMYGDALA

The basolateral amygdala (BL) receives a dense cholinergic innervation from the basal forebrain. Despite the importance of muscarinic acetylcholine receptors (mAChRs) in fear learning, consolidation, and extinction, there have been no studies that have systematically investigated the functional role of mAChRs in regulating emotional processing in the BL. To address this critical knowledge gap we combined brain slice whole-cell recording, optogenetics, and immunohistochemistry to determine how muscarine, acting on mAChRs, regulates neuronal oscillations, synaptic transmission and plasticity in the BL. Neurons in the BL oscillate rhythmically during emotional processing, which are thought to be important to integrate sensory inputs, allow binding of information from different brain areas and facilitate synaptic plasticity in target downstream structures. We found that muscarine induced theta frequency rhythmic inhibitory postsynaptic potentials (IPSPs) in BL pyramidal neuron (PN). These IPSPs synchronized PN firing at theta frequencies. Recordings from neurochemically-identified interneurons revealed that muscarine selectively depolarized parvalbumin (PV)-containing, fast firing, but not PV, regular firing or somatostatin (SOM)-containing interneurons. This depolarization was mediated by M3 mAChRs. Dual cell recordings from connected interneuron-PN pair indicated that action potentials in fast firing, but not regular firing interneurons were strongly correlated with large IPSCs in BL PNs. Furthermore, selective blockade of M3, but not M1 mAChRs suppressed the rhythmic IPSCs in BL PNs. These findings suggest that muscarine induces rhythmic IPSCs in PNs by selectively depolarizing PV, fast firing interneurons through M3 mAChRs. Furthermore, we found that rhythmic IPSCs were highly synchronized between PNs throughout the BL. The BL receives extensive glutamatergic inputs from multiple brain regions and recurrent collaterals as well. They are important for fear learning and extinction, which are tightly regulated by local GABAergic inhibition. We found that mAChRs activation suppressed external glutamatergic inputs in a frequency dependent and pathway specific manner but kept recurrent glutamatergic transmission intact. In addition, muscarine disinhibited BL PNs by attenuating feedforward and GABAergic inhibition. In agreement with these observations, long term potentiation (LTP) induction was facilitated in the BL by mAChRs activation. Taken together, we provided mechanisms for cholinergic induction of thetaoscillations and facilitation of LTP in the BL

The interaction between neuronal networks and gene networks

Periods of strong electrical activity can initiate neuronal plasticity leading to long-lasting changes of network properties. A key event in the modification of the synaptic connectivity after neuronal activity is the activation of new gene transcription. Moreover, calcium (Ca2+) influx is crucial for transducing synaptic activity into gene expression through the activation of many signalling pathways. In our work we are interested in studying changes in electrical activity and in gene expression profile at the network level. In particular, we want to understand the interplay between neuronal and gene networks to clarify how electrical activity can alter the gene expression profile and how gene expression profile can modify the electrical and functional properties of neuronal networks. Thus, we investigated the neuronal network at three different levels: gene transcription profile, electrical activity and Ca2+ dynamics. Blockage of GABAA receptor by pharmacological inhibitors such as gabazine or bicuculline triggers synchronous bursts of spikes initiating neuronal plasticity. We have used this model of chemically-induced neuronal plasticity to investigate the modifications that occur at different network levels in rat hippocampal cultures. By combining multielectrode extracellular recordings and calcium imaging with DNA microarrays, we were able to study the concomitant changes of the gene expression profile, network electrical activity and Ca2+ concentration. First, we have investigated the time course of the electrical activity and the molecular events triggered by gabazine treatment. The analysis of the electrical activity revealed three main phases during gabazine-induced neuronal plasticity: an early component of synchronization (E-Sync) that appeared immediately after the termination of the treatment persisted for 3 hours and was blocked by inhibitors of the MAPK/ERK pathway; a late component (L-Sync) -from 6 to 24 hours- that was blocked by inhibitors of the transcription. And, an intermediate phase, from 3 to 6 hours after the treatment, in which the evoke response was maximally potentiated. Moreover, gabazine exposure initiated significant changes of gene expression; the genomic analysis identified three clusters of genes that displayed a characteristic temporal profile. An early rise of transcription factors (Cluster 1), which were maximally up-regulated at 1.5 hours. More than 200 genes, many of which known to be involved in LTP were maximally up-regulated in the following 2-3 hours (Cluster 2) and then were down-regulated at 24 hours. Among these genes, we have found several genes coding for K+ channels and the HNC1 channels. Finally, genes involved in cellular homeostasis were up-regulated at longer time (Cluster 3). Therefore, this approach allows relating changes of electrical properties occurring during neuronal plasticity to specific molecular events. Second, we have investigated which sources of Ca2+ entry were involved in mediating the new gene transcription activated in response to bursting activity. Using Ca2+ imaging, a detailed characterization of Ca2+ contributions was performed to allow investigating which sources of Ca2+ entry could be relevant to induce gene transcription. At the same time, changes of gene expression were specifically investigated blocking NMDA receptors and L-, N- and P/Q-type VGCCs. Therefore, the analysis of the Ca2+ contribution and gene expression changes revealed that the NMDA receptors and the VGCCs specifically induced different groups of genes. Thus, the combination of genome-wide analysis, MEA technology and calcium imaging offers an attractive strategy to study the molecular events underlying long-term synaptic modification

Long-Term Activity-Dependent Plasticity of Action Potential Propagation Delay and Amplitude in Cortical Networks

Background: The precise temporal control of neuronal action potentials is essential for regulating many brain functions. From the viewpoint of a neuron, the specific timings of afferent input from the action potentials of its synaptic partners determines whether or not and when that neuron will fire its own action potential. Tuning such input would provide a powerful mechanism to adjust neuron function and in turn, that of the brain. However, axonal plasticity of action potential timing is counter to conventional notions of stable propagation and to the dominant theories of activity-dependent plasticity focusing on synaptic efficacies. Methodology/Principal Findings: Here we show the occurrence of activity-dependent plasticity of action potentia