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This chapter delves into the structure and function of the mammalian nervous system, with a particular focus on neurons and their role in signal transmission. It explores the intricate neural circuits of the hippocampus, highlighting the importance of local inhibitory circuits in maintaining excitation balance. The text discusses the generation of evoked potentials through electrical pulse stimulations and the effects of paired-pulse stimulations on neuronal responses. Additionally, it examines the responses of CA3 neurons to ventral hippocampal commissure (VHC) stimulations and the role of synaptic plasticity in learning and memory. The chapter concludes with a summary of the key findings and their implications for understanding the nervous system.
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Abstract
The animal experiments described in this book focus on the rat hippocampus. This chapter introduces hippocampal structures and neural circuits. It explains the generations of various evoked potentials in neuronal population responses to pulse stimulations, including orthodromically and antidromically evoked potentials. The chapter also explores the effects of feedforward and feedback inhibitory circuits through paired-pulse stimulations in the hippocampal CA1 and CA3 regions.
2.1 Overview of Nervous System
2.1.1 Neuron and Nervous System
The mammalian nervous system consists of two main parts: the central nervous system (CNS) and the peripheral nervous system (PNS). CNS includes the brain and spinal cord, while PNS includes cranial, spinal, and visceral nerves. The nervous system contains two main cell types: neurons and glial cells. Neurons are the basic structural and functional units that receive input signals, generate action potentials, and transmit them. Glial cells support neurons by providing protection, repair, and nutrition. They also form myelin sheaths and transport metabolic substances.
A neuron typically consists of three main structures: soma, dendrites, and axon (Fig. 2.1). The soma, varying in size and shape, serves as both the signal integration center and the metabolic and nutritional hub. It comprises membrane, nucleus, and cytoplasm. Cytoplasm contains both common organelles and specialized structures such as Nissl bodies and neurofibrils. Dendrites, typically shorter than axons, form tree-like structures that branch repeatedly and become progressively thinner. They receive input signals and propagate them to soma. Each neuron typically has one axon, ranging in length from a few micrometers to over a meter. Axons carry action potentials (also known as neural impulses) and transmit them to other neurons or effectors through synapses at axonal terminals. An axon maintains relatively uniform thickness throughout its length, beginning at the axon hillock where it emerges from the soma. The axon initial segment (AIS) is slightly thicker and lacks a myelin sheath. Axons with myelin sheaths are called myelinated nerve fibers, while those without are called unmyelinated nerve fibers. Axons typically end by splitting into multiple branches, each with swollen spines that form synapses with other neurons or muscle cells.
Fig. 2.1
Schematic diagram of neuronal structures and their functions
Neurons communicate with each other and with effector cells through specialized junctions called synapses. These synapses can be classified based on their locations, such as axon-dendrite, axon-soma, or axon-axon synapses. They can also be categorized into chemical and electrical synapses. Chemical synapses use neurotransmitters to transmit signals, converting electrical signals to chemical ones and back again. Electrical synapses, in contrast, conduct signals directly through localized currents.
Electrical synapses, also called gap junctions, are protein channels connecting the adjacent membranes of two neurons. These channels work like hollow rivets, allowing ions and small molecules to flow through. Unlike chemical synapses, electrical synapses have no distinct presynaptic and postsynaptic membranes. This allows them to transmit signals quickly in both directions with almost no delay. Though their exact functions are not fully understood, electrical synapses likely play a key role in promoting synchronous neuronal activity.
Chemical synapses, often simply called synapses, are the primary means of signal transmission in the nervous system. A typical synapse consists of three components: presynaptic membrane, synaptic gap, and postsynaptic membrane. The synaptic gap is about 20–40 nm wide, while both the presynaptic and postsynaptic membranes are approximately 7.5 nm—slightly thicker than typical neuronal membranes. Inside the presynaptic membrane, there exist numerous synaptic vesicles that store neurotransmitters. When neural impulses (action potentials) reach and depolarize the presynaptic membrane to threshold level, voltage-gated calcium ion (Ca2+) channels open, allowing extracellular Ca2+ to flow in. This influx causes a rapid increase in Ca2+ concentration in the axoplasm ([Ca2+]i) inside presynaptic membrane. The increased [Ca2+]i triggers synaptic vesicles to fuse with the presynaptic membrane, releasing neurotransmitters into the synaptic gap. The amount of released neurotransmitters is directly proportional to the [Ca2+]i level. Once in the synaptic gap, the neurotransmitters diffuse to the postsynaptic membrane, bind to specific receptors, and open specific ion channels. This process leads to either depolarization or hyperpolarization in the postsynaptic membrane, creating synaptic potentials.
Synaptic potentials have two types: excitatory postsynaptic potential (EPSP) and inhibitory postsynaptic potential (IPSP). These potentials arise from the opening of ligand-gated Na+, Cl−, and K+ channels, which depends on the types of neurotransmitters and their receptors. In the CNS, glutamate and aspartate are the main excitatory amino acid neurotransmitters. Their receptors include α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA). AMPA and KA receptors—together termed non-NMDA receptors—respond quickly to glutamate and mainly increase membrane permeability to Na+ and K+. In contrast, NMDA receptors respond slowly to glutamate and, when activated, become permeable to Na+, K+ and Ca2+. Unlike non-NMDA receptors, which are purely ligand-gated, NMDA receptors are both ligand-gated and voltage-gated. They contain binding sites for both glutamate and magnesium ions (Mg2+). Mg2+ ions block NMDA receptor channels in a voltage-dependent manner. When bound with glutamates, the post-synaptic membrane must also reach a specific level of depolarization before Mg2+ ions move away from their blocking sites and let the NMDA receptor channels open. The dual requirements explain why NMDA receptors respond slowly to glutamate. Most glutamate target neurons contain both NMDA and AMPA receptors, allowing the rapid membrane depolarization caused by AMPA receptors to trigger the opening of NMDA receptor channels.
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In the CNS, the main inhibitory amino acid neurotransmitters include γ-aminobutyric acid (GABA) and glycine (Gly). Postsynaptic membranes contain two types of GABA receptors: GABAA and GABAB. GABAA is an ionotropic receptor with Cl− channels that, when activated, increases Cl− influx. GABAB is a G-protein-coupled metabotropic receptor that, when activated, inhibits adenylate cyclase, activates K+ channels, and increases K+ efflux. Both Cl− influx and K+ efflux can hyperpolarize the postsynaptic membrane, generating IPSPs. Inhibitory synapses are prevalent in local neuronal circuits of the brain. They play a crucial role in controlling neuronal excitability, modulating information integration and action potential generation, regulating synaptic plasticity, and promoting synchronous neuronal activity (Cobb et al. 1995; Maccaferri and Dingledine 2002; Staff and Spruston 2003).
Synaptic transmission exhibits plasticity—the ability to undergo long-lasting changes in efficiency through repeated activations. Long-term potentiation (LTP) and long-term depression (LTD) are the two main forms of synaptic plasticity. They are considered as the foundations of high-level brain functions such as learning and memory. The mechanisms of LTP and LTD formations are outlined below.
1.
Long-Term Potentiation
LTP refers to an enhancement in EPSPs that occurs in postsynaptic neurons after presynaptic neurons are activated by a brief, high-frequency pulse train. This enhancement can persist for a long period. For example, applying 50–100 pulses at 100 Hz can produce LTP in the hippocampal CA1 pyramidal neurons, lasting for hours, days or even months. LTP generates when high-frequency stimulation increases EPSP through superimposition, which promotes the opening of NMDA receptor channels gated by both ligands and membrane potential. This results in increased Ca2+ influx and [Ca2+]i inside the postsynaptic membrane. The increased [Ca2+]i activates Ca2+-CaM dependent protein kinase IIs, which phosphorylate AMPA receptor-coupled channels and increase their conductance. It also causes more AMPA receptors to bind to the postsynaptic membrane, increasing receptor density. As a result, when presynaptic membrane releases neurotransmitters, there is increased opportunity and efficiency for neurotransmitters binding to AMPA receptors on the postsynaptic membrane. This enhances EPSP, resulting in LTP.
2.
Long-Term Depression
LTD refers to a decrease in synaptic transmission efficiency which can be also produced by repeated pulse stimulation but at a low frequency. For example, LTD can generate by applying 1–5 Hz pulses for a few minutes to the Schaffer collaterals—afferent fibers of the hippocampal CA1 region. While its generation follows a similar route as LTP, LTD produces the opposite outcome. The low-frequency pulses open only a small number of NMDA receptor channels, resulting in a slight increase in [Ca2+]i inside the postsynaptic membrane. This leads to dephosphorylation, rather than phosphorylation, of Ca2+-CaM dependent protein kinase II. The enzymes then dephosphorylate AMPA receptors, causing a decrease in AMPA receptor density on the postsynaptic membrane, thereby reducing EPSP. Both LTP and LTD generations involve NMDA receptor channels, Ca2+ influx, and changes in AMPA receptor efficiency on the postsynaptic membrane, though with different outcomes.
2.1.3 Information Integration in Neurons
1.
SynapticIntegration
Each neuron's dendrites contain numerous synapses—typically tens of thousands in total. These synapses are either excitatory or inhibitory, producing EPSPs or IPSPs, respectively. Spatiotemporal integration of these postsynaptic potentials can trigger an action potential (AP) when the membrane becomes sufficiently depolarized to reach the activation threshold. The AP typically starts at the AIS due to its high density of voltage-gated Na+ channels and small diameter, which requires fewer charges to achieve membrane depolarization during capacitance charging (Stuart et al. 1997). Once triggered, the AP propagates along the axon to its terminals to complete signal conduction. The AP generation depends on multiple factors, including EPSP magnitude, the spatiotemporal integration of EPSPs, and the activity of inhibitory synapses near soma (Bear et al. 2016).
First, larger EPSPs contribute more in triggering an AP. The postsynaptic membrane of each synapse contains many neurotransmitter-gated channels that activate in response to neurotransmitters released from the presynaptic membrane. The neurotransmitter release is controlled by Ca2+ influx into the presynaptic membrane, which varies with the strength and frequency of incoming APs. Because each released synaptic vesicle contains a fixed number of neurotransmitter molecules, the EPSP magnitude at postsynaptic membrane changes in discrete steps rather than continuously.
Second, the integration of multiple EPSPs over space and time can produce a large depolarization potential, increasing the likelihood to trigger an AP. This integration depends on the electrical properties of dendrites. Synaptic current gradually decays as it spreads toward the soma. The synapses’ contribution to soma depolarization decreases with increasing distance, governed by both the intracellular and membrane impedances of the dendrites. The intracellular impedance is determined by the dendrite diameter and the cytoplasm feature.
Finally, inhibitory synapses in peri-somatic region can exert bypass effects. These synapses, primarily of the axon-soma type, locate close to the soma. When they are activated during dendritic EPSPs, their IPSPs (produced by opening Cl− channels near the soma) can bypass the spreading currents from the EPSPs, significantly reducing the EPSPs' contribution to soma depolarization and preventing AP generation.
Moreover, the EPSP integration from a few excitatory synapses alone is usually insufficient to trigger an AP. A single EPSP typically produces only about 1 mV of soma depolarization, while its activation threshold is about 20–30 mV. This means that even without opposing IPSPs, a postsynaptic neuron needs 20–50 EPSPs occurring almost simultaneously to trigger an AP.
The traditional view of neuronal signal conduction is unidirectional: dendrite synapses receive input signals, which trigger the soma (or AIS) to generate an AP that then travels along the axon to terminal synapses, as shown in Fig. 2.1. This model casts dendrites and axons as simple conductors—dendrites for input and axons for output. The soma acts as an integrator, like a threshold calculator, combining dendrite signals to produce an output signal. However, research has challenged this basic model, revealing that dendrites also perform integrations and show complex hierarchical information processing. Additionally, APs can travel backward from soma to dendrites. These perspectives are outlined below.
2.
HierarchicalInformation Integration in Neurons
With advanced techniques like multi-electrode patch clamps and two-photon microscopy imaging, the structure and function of tiny dendrites have been revealed. Studies have shown that dendrites themselves can perform complex signal processing. When they receive inputs from numerous synapses, dendrites can carry out both linear and nonlinear signal processing, including integration, amplification, reduction, and detection of synchronous inputs (London and Häusser 2005; Sidiropoulou et al. 2006).
Synapses typically distribute on dendritic spines—tiny protrusions on dendrite branches. The pyramidal neurons in cerebral cortex and the Purkinje cells in cerebellar cortex have high densities of dendritic spines, with thousands per neuron. These spines not only greatly increase the surface area available for receiving input signals but also perform information integration (Spruston 2008).
Dendritic spines contain high densities of voltage-gated Na+ and Ca2+ channels and NMDA receptors, giving them active electrical properties. Like the ion channels on soma and axon that have a threshold mechanism for APs, conductance in spine channels can also change and result in varying gains to synaptic inputs. This mechanism enables dendritic spines to integrate input signals nonlinearly. Strong inputs can trigger localized dendritic spikes. These spikes, driven by voltage-gated ion channels, are regenerative and have distinct waveforms—occurring only when input signals reach a specific threshold. Unlike APs, these spikes have limited propagation and are confined to tiny dendritic branches, exhibiting independence and locality. However, they can help amplify input signals at neighboring dendritic spines.
As shown in Fig. 2.2, when dendritic spines receive dense inputs simultaneously, their nonlinear integrations—a threshold mechanism (denoted by S-shape symbols in the figure)—can enhance dendritic signals locally. In contrast, sparse inputs can decay quickly due to the filtering effect of spine membrane and spine narrow neck structure (Yuste and Urban 2004). The numerous dendritic spines on dendrites act as independent computational subunits (Poirazi et al. 2003). They first integrate their respective synaptic inputs by a threshold mechanism. Then, the outputs of these subunits are further integrated and threshold-computed on the dendritic trunk and soma to produce the final output of the entire neuron. This creates a two-level integration mechanism.
Fig. 2.2
Schematic diagram of the two-level integration model in pyramidal neurons. The S-shape symbols in the circles represent threshold computations. wi represents subunit weight
For a pyramidal neuron with extensive apical dendrites (as shown in Fig. 2.3), Häusser and Mel (2003) proposed a three-level integration model for its information processing. The three levels are: first, the integration of dendritic spines at the distal apical dendrites; second, the integration of fine branches at the proximal apical dendrites; and third, the output integration at soma based on the first two levels. Experimental data have supported this model, demonstrating areas that can produce secondary spikes at the main bifurcation of apical dendrites. The branches at distal apical dendrites function as independent integration units with their own spike generation mechanism—the first-level integration. When membrane potential exceeds a threshold, it triggers a Ca2+-dominated dendritic spike, producing significant dendrite depolarization that can substantially contribute to the somatic membrane potential. Additionally, a so-called “coupling zone” at the proximal apical dendrites forms the second-level integration, where input signals can modulate the interaction between somatic/axonal spikes and distal dendritic spikes.
Fig. 2.3
Schematic diagram of CA1 pyramidal neuron morphology
The multi-level integration model reveals the important role of apical dendrites in pyramidal neurons in brain regions such as the cerebral cortex and hippocampus. These neurons have extensive apical dendrites with numerous synaptic inputs located far from their soma. Due to passive potential attenuation in the dendritic membrane, it is challenging to explain how signals from these distant apical dendrites contribute to the somatic potentials without some compensatory mechanism. The persistence of these distal dendrites throughout biological evolution demonstrates their essential physiological functions.
Incoming signals from dendritic synapses do not always directly affect a neuron to generate action potentials. Rather, these signals undergo screening and processing before final integration at soma, where some are filtered out while others are enhanced. The dendrites' ability to integrate information depends on complex factors, including their morphological structures, the timing and location of synaptic inputs, the interaction between excitatory and inhibitory inputs, and the distributions of voltage-gated channels on dendritic membranes.
The above multi-level models only describe the unidirectional propagation of neural signals—the traditional flow from dendrites to soma and to axon. However, research has shown that action potentials can also travel backward from the soma to dendrites (Stuart et al. 1997). This reveals that a neuron's information processing is not simply “open-loop”, but involves feedback mechanisms. These mechanisms play important roles in dendritic function and synaptic plasticity (London and Häusser 2005). For instance, backpropagation of action potentials can activate slow voltage-gated ion channels on dendrites. The resulting channel currents can change the potentials at the soma and axon initial segment, triggering new action potentials. This soma-dendrite interaction can lead neurons to produce burst firing, a characteristic of certain principal neurons like hippocampal pyramidal neurons. Burst firing can enhance the reliability of neural information transmission (Buzsáki 2006; Koch 1999).
In summary, a neuron itself has sophisticated mechanisms for information processing. These include not only soma integration but also threshold processing through active properties in dendritic spines and main dendritic branches, as well as backpropagation of somatic action potentials. Each neuron acts as a collection of smaller units working together to integrate information. When a group of neurons connect through synapses to form neural networks (or neural circuits), they achieve even more complex information integration. The next section will examine integration at the neural circuit level and the responses of neuronal populations to external stimulations.
2.2 Hippocampal Structure and Evoked Potentials
The hippocampus, an essential part of the brain limbic system, is named for its resemblance to seahorse. The information processing in its neural circuits is closely related to brain functions such as learning and memory, as well as emotion and sleep. Though crucial for memory formation, the hippocampus does not permanently store memories. People with hippocampal damage can retain old memories but struggle to form new ones. In the 1950s, an epilepsy patient named Henry Molaison (H.M.) became an anterograde amnesiac after undergoing brain tissue removal involving hippocampus. This famous case, among others, confirms the vital role of hippocampus in learning and memory. Later, in the 1970s, Bliss and colleagues made a groundbreaking discovery—long-term potentiation (LTP) in hippocampal synaptic transmission (Bliss and Lømo 1973; Bliss and Gardner-Medwin 1973). This finding revealed that synapses have long-term plasticity—possibly the biological basis for brain learning and memory functions. In that same decade, the discovery of place cells in the hippocampus also indicated its crucial role in spatial cognition and memory (O'Keefe and Dostrovsky 1971). Additionally, the hippocampus is a common focus region of diseases such as epilepsy and Alzheimer's disease. Furthermore, its clear layered structure and well-defined neural circuits make it fascinating to researchers across various neuroscience fields, including neurophysiology, psychology, pathology, neural engineering, and computational neurobiology.
2.2.1 Hippocampal Structure and Major Excitatory Pathways
The hippocampus has a unique anatomical structure, with neuron types and fiber pathways fundamentally similar across various mammalian species (Andersen et al. 2007). As shown in Fig. 2.4A, the rat hippocampus lies at the bottom of lateral ventricle in the temporal lobe, forming part of the medial surface of cerebral hemisphere. It curves along its long axis into a crescent shape, extending first from anterior to posterolateral, then anteromedially. The hippocampi in the left and right cerebral hemispheres resemble a pair of sheep horns. The anterior portions of the two hippocampi connect through axonal fibers of the ventral hippocampal commissure (VHC), which pass beneath the corpus callosum. The hippocampal lateral surface forms the medial wall of lateral ventricle, while its inferomedial surface wraps around the brainstem. Its posterior edge contains the dentate gyrus, which connects to the entorhinal cortex through the subiculum.
Fig. 2.4
Anatomical location, sagittal section views, and major excitatory pathways of the rat hippocampus. A Schematic diagram showing the hippocampus location in the rat brain. B Sagittal section at 2.4 mm mediolateral to bregma from the rat brain stereotaxic atlas (Paxinos and Watson 2007). C A photo showing a sagittal section view of the rat hippocampus. D Schematic diagram of the “tri-synaptic circuit” showing excitatory pathways in the cornu ammonis (CA) and dentate gyrus (DG)
The principal neurons in the hippocampal region are arranged densely and orderly, forming distinct neural circuit structures. Figure 2.4B shows a sagittal section at 2.4 mm mediolateral to bregma from the rat brain atlas (Paxinos and Watson 2007), where stained somata appear blue-purple. In the hippocampal region, neuronal somata form two dark curves: one in the cornu ammonis (CA) and another in the dentate gyrus (DG). These curves stand out against surrounding areas due to the dense soma arrangement. The hippocampus well-defined laminar structure is visible to the naked eye even in unstained brain slices. Figure 2.4C shows a sagittal view of a rat brain, captured with a Canon camera (model EOS 6D) equipped with a macro lens (EF 100 mm f/2.8 USM). This photo displays the brain after paraformaldehyde immersion, sectioned at approximately 2.7 mm mediolateral to bregma. In the center of the photo, the hippocampus and its structure are visible, standing distinctly apart from surrounding brain tissues.
Figure 2.4D illustrates the major excitatory pathways in the two hippocampal regions—the CA and the DG. The CA forms a C-shape and is divided into four subregions (CA1, CA2, CA3 and CA4) based on differences in internal development and fiber formation. Pyramidal neurons (or pyramidal cells) are CA principal neurons. The DG, with granule cells as its principal neurons, curves into a V- or U-shape (depending on the septotemporal position) and nestles within the CA's C-shape, mostly surrounded by the CA.
The excitatory synaptic connections of hippocampus form a “tri-synaptic circuit”: (1) The perforant path (PP), formed by the axons of entorhinal cortex neurons, serves as the afferent fibers to the hippocampus and forms synapses with the dendrites of DG granule cells. (2) The axons of these granule cells form mossy fibers (MF), which connect to and form synapses with the dendrites of CA3 pyramidal neurons. (3) The axons of CA3 pyramidal neurons form the Schaffer collaterals that then make synapses with the dendrites of CA1 pyramidal neurons. Finally, the myelinated axons of CA1 pyramidal neurons form the hippocampal white matter, known as alveus—the efferent fiber of the hippocampus. The alveus creates a thin white sheet covering the dorsal surface of hippocampus, giving it a silvery appearance. When the overlying cerebral cortex is removed, the exposed hippocampus can be easily identified by this silvery color. Section 3.3 will describe an experiment approach with exposed hippocampus.
In addition to the ipsilateral excitatory connections, the axonal branches of rodent CA3 pyramidal neurons connect to neurons in the contralateral hippocampal region through VHC (Fig. 2.4D). Furthermore, inhibitory GABAergic interneurons exist throughout the hippocampal regions. They have short axons that form inhibitory synapses with nearby principal neurons and other interneurons, releasing GABA neurotransmitters to create local inhibitory effects. Although interneurons comprise only about 10% of the total neuron population—far fewer than principal neurons (i.e., CA pyramidal neurons and DG granule cells) (Andersen et al. 2007), they exhibit diverse morphologies, chemical properties and functions (Klausberger and Somogyi 2008; Kepecs and Fishell 2014; Pelkey et al. 2017). The axon terminals of each interneuron connect with multiple neurons to form inhibitory synapses. These synapses serve different functions depending on their locations: those on neuronal somata and axon initial segments can effectively regulate action potential generation, while those on dendrites can affect synaptic potential integrations. The effects of local inhibitory circuits can be tested using electrical pulse stimulations, as detailed in the subsequent sections.
2.2.2 Hippocampal Evoked Potentials by Pulse Stimulations
The rat hippocampus has a tightly packed cell body layer that contains only 3–5 layers of somata. The somata are densely arranged within a thin layer. The dendrites align directionally, forming a distinct layered structure. When the neurons are activated synchronously, this arrangement can result in unique potential waveforms in each layer (Richardson et al. 1987; Kloosterman et al. 2001; Vreugdenhil et al. 2005; Andersen et al. 2007). Figure 2.5A1 illustrates the layers of hippocampal CA1 pyramidal neurons. From dorsal to ventral (top to bottom in the figure), the layers are: stratum oriens (so), pyramidal cell layer (pcl), stratum radiatum (sr), and stratum lacunosum-moleculare (sl-m). The so layer, about 300 μm thick, contains the basal dendrites of pyramidal neurons, which form a thicket-like structure with numerous branches and no clear distinction between main and secondary trunks. The pcl layer contains the somata. The sr and sl-m layers contain the apical dendrites, together spanning about 600 μm. The apical dendrites of a CA1 pyramidal neuron are long, featuring distinct main and secondary trunks that extend broadly. The neuron has a cone-shape soma with basal dendrites growing from the cone base and apical dendrites from its apex. Its axon grows from the cone base and ascends to form the alveus. In the CA1 region, the somata of pyramidal neurons average about 15 μm in diameter—smaller than those in the CA3 region, which can reach 30 μm (Andersen et al. 2007).
Fig. 2.5
Lamellar structures and layer-specific evoked potentials in the rat hippocampal region. A Lamellar structures in the hippocampal CA1 region (A1) and DG region (A2). B When an electrical pulse is applied to the CA1 Schaffer collaterals (B1) or to the DG PP pathway (B2), the resulting excitatory inputs can induce OPS and fEPSP in the soma and dendritic layers, respectively. C Schematic diagram of evoked potential generations. Small circles with “+” and “−” represent the accumulated positive and negative charges in the extracellular space, forming current “source” and “sink” respectively. The left (C1) and right (C2) diagrams, separated by a thick dashed line, respectively show the ionic flows during excitatory synaptic activation on dendrites (C1) and action potential generation at the soma (C2)
As shown in Fig. 2.5A2, the DG lies beneath the CA1 region, beyond the hippocampal fissure (hf). The DG comprises three layers: the molecular layer (ml), granule cell layer (gcl), and polymorphic layer (pl). A DG granule cell has a round or oval soma, with its dendrites mainly in the ml layer.
When a pulse is applied to the Schaffer collaterals—the afferent pathway in the sr layer of CA1 region (Figs. 2.4D and 2.5A1), it can activate a bundle of axons. The resulting APs travel collectively towards the synapses at the apical dendrites of CA1 pyramidal neurons. Through synaptic transmissions, a group of these downstream pyramidal neurons can be activated to fire APs synchronously. Similarly, applying a pulse at the PP—the afferent pathway to the DG—can activate PP axons and then their synapses in the ml layer of the DG (Fig. 2.5A2). Through synaptic transmissions, a group of DG granule cells can generate APs synchronously.
As shown in Fig. 2.5B, unique potential waveforms from activated neuron populations can be recorded in the extracellular space. In the dendritic layer receiving excitatory synaptic inputs, the recorded signal consists mainly of the field excitatory postsynaptic potential (fEPSP)—a primarily negative potential generated by synapses. In the soma layer, the recorded signal consists mainly of the negative population spike (PS), which forms from APs (Andersen et al. 1971). When excitatory synaptic inputs produce a PS, it is called an orthodromic population spike (OPS) (Kloosterman et al. 2001). The fEPSP amplitude or slope can indicate the strength of excitatory synaptic transmission, while the OPS amplitude relates closely to the number of neurons firing action potentials and their discharge synchronicity (Theoret et al. 1984).
Figure 2.5C1 illustrates ion flows during the activation period of excitatory synapses on dendrites, while Fig. 2.5C2 illustrates the period of action potential generation at soma. The diagrams explain the formation of fEPSP and OPS. When receptor ion channels open in the postsynaptic membranes, positively charged ions flow into the membranes, leaving negative charges outside. This creates the negative fEPSP outside the dendrites. Through the current loops (indicated by dashed lines with arrows in the figure), this fEPSP appears as a positive potential outside the somata (indicated by hollow triangles in Fig. 2.5B). Similarly, the negative OPS forms outside the somata when positively charged Na+ ions flow into membranes during APs, leaving negative charges outside. The current loop causes the OPS to appear as a small positive spike superimposing on the negative fEPSP in the dendritic layer (indicated by small circles in Fig. 2.5B).
In summary, the distinct laminar structures in the hippocampal region provide unique biomarkers in evoked potentials. These potentials can serve two purposes: evaluating the excitability of neurons and their networks, and guiding proper electrode placement in electrophysiological experiments (see Sect. 3.2 for details).
2.3 Local Inhibitory Circuits in the Rat Hippocampal CA1 Region
The principal neurons in the hippocampal region—CA pyramidal neurons and DG granule cells—are responsible for information integration and transmission. These neurons connect both upstream and downstream neurons along projection pathways, while also forming local neural circuits with surrounding neurons, especially the interneurons in local inhibitory circuits. Complex regulation mechanisms within these neural networks maintain the excitation balance in principal neurons (Pelkey et al. 2017; Andersen et al. 2007; Mori et al. 2004; Sik et al. 1994; Kullmann 2011).
2.3.1 Effect of Inhibitory Circuits During Orthodromic Activations of CA1 Pyramidal Neurons
Figure 2.6A illustrates the local inhibitory circuits in the hippocampal CA1 region. Pink triangles represent pyramidal neurons, while blue ovals represent interneurons (IN). Through the Schaffer collaterals, axon terminals of upstream CA3 pyramidal neurons form excitatory synapses with both CA1 pyramidal neurons and interneurons (IN1 and IN2). The CA1 pyramidal neurons also form excitatory synapses with interneurons (IN2 and IN3), which in turn form inhibitory synapses back onto the pyramidal neurons. These connections create two types of inhibitions: feedforward (indicated by solid grey lines) and feedback (indicated by dashed grey lines).
Fig. 2.6
Local inhibitory circuits in the hippocampal CA1 region. A Schematic diagram showing feedforward and feedback inhibitory circuits. B Testing the effect of inhibitory circuits by paired-pulse stimulations at the Schaffer collaterals. The four signals were recorded respectively from the pcl layer (upper) and the sr layer (lower), with normal inhibitory circuits (B1) and blocked inhibitory synapses by PTX (B2). C Interneuron firing induced by two single pulses at intensities of 0.05 and 0.1 mA. D Spontaneous burst firing of pyramidal neurons, along with induced OPS and a “silent period” following a pulse stimulation. Red small arrows in figures B–D indicate truncated stimulus artifacts
When CA1 pyramidal neurons respond to excitatory inputs from the Schaffer collaterals, they undergo both feedforward and feedback inhibitions. Feedforward inhibition occurs when CA3 pyramidal neurons fire along the Schaffer collaterals to activate both CA1 pyramidal neurons and interneurons (such as IN1 and IN2). These interneurons in turn inhibit CA1 pyramidal neurons via inhibitory synapses. This process requires two synaptic transmissions. Feedback inhibition occurs when the firing of CA1 pyramidal neurons activate interneurons (such as IN2 and IN3), which then produce synaptic inhibitions back on the pyramidal neurons. This process requires three synaptic transmissions. The two types of inhibitions primarily target different sites: feedforward inhibition mainly acts on the dendrites of pyramidal neurons, while feedback inhibition acts mainly near the soma (Lipski 1981). Notably, an identical interneuron can participate in both inhibitory circuits simultaneously, like IN2 in Fig. 2.6A.
The activation of CA1 pyramidal neurons by the Schaffer collaterals requires only one synaptic transmission, meaning these neurons are activated before inhibitions occur. This raises a question: are the inhibitions from interneurons too late to be effective? Intracellular recordings of postsynaptic potentials in a pyramidal neuron have shown that the initial EPSP rapidly reverses to an IPSP within milliseconds (Mori et al. 2004), demonstrating that inhibition indeed follows excitation. Nevertheless, this inhibition affects subsequent incoming excitations rather than the current ones. The effect of inhibitory circuits can be detected by applying paired-pulse stimulation (PPS) to the Schaffer collaterals.
When two suprathreshold pulses were applied to the Schaffer collaterals at a 25-ms interval, the first pulse induced a large OPS1 in the CA1 pyramidal cell layer (pcl, Fig. 2.6B1). The second identical pulse hardly induced OPS2 due to the effect from inhibitory circuits. In the apical dendritic layer (sr), however, both pulses induced large fEPSPs (fEPSP1 and fEPSP2). GABAA receptors mediate the fast inhibitory potentials in the CA1 region (Leung et al. 2008; Pelkey et al. 2017). When an inhibitory synapse blocker, such as the GABAA receptor antagonist picrotoxin (PTX), was applied to weaken the inhibitions, both pulses induced large OPSs with continuous discharges forming multiple peaks (Fig. 2.6B2). These negative peaks demonstrate the inherent bursting behavior of pyramidal neurons. Under normal inhibition condition (Fig. 2.6B1), the induced OPS1 appeared as a single peak, because the effect of inhibitory circuits suppressed subsequent burst discharges.
Due to their lower activation threshold (Csicsvari et al., 1998; Buzsáki 2006), interneurons activate more quickly than pyramidal neurons when a pulse is applied to the Schaffer collaterals, as shown in Fig. 2.6C. The figure displays two single-pulse stimulations at different intensities, with the recordings aligned by their stimulus artifacts (indicated by a red arrow). Blue dots mark the firing of an interneuron. (Note: extracellular recording of an AP is called a unit spike, or simply spike; refer to Sect. 4.1 for details). The two low-intensity pulses (0.05 and 0.1 mA) activated the same interneuron, which fired at least twice in succession immediately following the pulses—its additional firing might be masked by the OPS from pyramidal neurons. If this activated interneuron participated in feedforward inhibition, its inhibition could rapidly act on pyramidal neurons.
Figure 2.6D shows spontaneous bursts from pyramidal neurons—series of APs with intervals of only a few milliseconds. The enlarged insets at the bottom use solid and hollow triangles to distinguish the bursts from two pyramidal neurons. When a single pulse was applied, it induced an OPS with one peak, followed by a “silent period” without any spontaneous neuronal firing. This silence resulted from inhibitory circuits. Particularly, feedforward inhibitions can occur immediately after the stimulation activates the pyramidal neurons (Pouille and Scanziani 2001), causing the induced OPS as a single peak.
Thus, a pulse at the Schaffer collaterals can induce two opposite effects on CA1 pyramidal neurons: excitation and inhibition (Fig. 2.6A). The inhibitory effect starts within milliseconds after pulse application and can last for hundreds of milliseconds. However, in a PPS, the evoked OPS1 and OPS2 can exhibit complex changes based on pulse intensities and inter-pulse intervals (IPI), rather than consistent OPS2 decrease or disappearance. Figure 2.7 shows the effect of varying pulse intensity while keeping a constant 50-ms IPI. The strength of incoming activation determines the number of postsynaptic neuron discharges, which can be evaluated by the OPS amplitude under certain conditions (Theoret et al. 1984). At a low pulse intensity (e.g., 0.05 mA, Fig. 2.7A1), the first pulse produced either no OPS1 or only a small one, indicating a subthreshold stimulation for most postsynaptic CA1 pyramidal neurons. However, the second pulse—at the same intensity—produced an OPS2 with significantly increased amplitude, showing paired-pulse facilitation (PPF).
Fig. 2.7
CA1 neuronal responses to orthodromic paired-pulse stimulations (PPS) at the Schaffer collaterals using a 50-ms IPI and varying pulse intensities. A Examples of induced OPSs at three different intensities. Red arrows indicate truncated stimulus artifacts. B Changes of OPS1 amplitudes against pulse intensity. C Changes of OPS amplitude ratios (OPS2/OPS1) against pulse intensity. n represents the number of experimental rats
With a low-intensity PPS at an IPI shorter than 100 ms, the two evoked OPSs typically exhibit PPF. This occurs because the first pulse activates only a small number of axons in the Schaffer collaterals, causing insufficient neurotransmitter release for EPSPs to reach activation threshold. The rapid second pulse supplements neurotransmitter release, thereby enhancing EPSPs (Debanne et al. 1996; Neher and Sakaba 2008; Regehr 2012). The integration of consecutive EPSPs can reach the threshold and trigger pyramidal neuron firing, producing a larger OPS2. Are inhibitory circuits activated during this period? Interneurons have a lower activation threshold than pyramidal neurons, so a low-intensity pulse that fails to activate pyramidal neurons may still activate some interneurons in the inhibitory circuits, causing mild inhibition. Nevertheless, when the first low-intensity pulse fails to activate sufficient pyramidal neurons (producing no or small OPS1), only the feedforward inhibitory circuits can be activated while most feedback inhibitory circuits remain inactive. Therefore, when the second pulse arrives, the increased EPSPs can overcome the weak inhibitions, resulting in enhanced firing rather than a decrease.
As pulse intensity increased, the OPS1 also increased and tended towards saturation at pulse intensities above 0.4 mA (Fig. 2.7B). However, the OPS2 decreased, causing a reduced amplitude ratio of OPS2/OPS1—a phenomenon known as paired-pulse depression (PPD, Fig. 2.7C). A large OPS1 indicated that the first pulse produced synchronous APs from a large population of pyramidal neurons, which can activate interneurons in the feedback inhibitory circuits to in turn suppress pyramidal neurons (Fig. 2.6A). The feedback inhibition acted together with feedforward inhibition. Thus, when the second pulse arrived, the inhibitory effect on pyramidal neurons overwhelmed the excitatory input, preventing OPS2 generation (Fig. 2.7A2 and A3).
Additionally, the change in OPS1 amplitude against pulse intensity shows an S-shape form (Fig. 2.7B), reflecting both the threshold and saturation mechanisms of post-synaptic neuron activations. Postsynaptic excitatory potentials must integrate to reach the firing threshold of postsynaptic neurons to produce APs (Spruston 2008). Around this threshold, small changes in pulse intensity can cause substantial changes in neuronal discharges. Therefore, the OPS1 amplitude changed rapidly in the 0.1–0.3 mA range. Increasing pulse intensity can expand the stimulated area and activate more axons in the Schaffer collaterals. However, once the excitatory inputs to downstream postsynaptic neurons become sufficient to produce APs, additional increase in pulse intensity has little effect. Moreover, during orthodromic activation by afferent axons, the broad distribution of axon branches and their terminals enables each axon to control multiple postsynaptic neurons, resulting in high activation efficiency (López-Aguado et al. 2002). This drives the OPS1 quickly to a saturation level, forming an S-shape input–output relationship (Wierenga and Wadman 2003).
What happens if more pulses are applied in succession? Can inhibitory circuits keep suppressing pyramidal neurons from firing indefinitely? The answer is no. Additional pulses can trigger other mechanisms to produce more complex processes. For instance, when a GABAergic inhibitory synapse of interneuron is activated continuously at high frequencies, it can switch from inhibitory to excitatory, exciting rather than suppressing pyramidal neurons (Staley et al., 1995). As detailed in Sect. 5.3, pyramidal neurons can also show altered responses under sustained pulse stimulations.
2.3.2 Effect of Inhibitory Circuits During Antidromic Activations of CA1 Pyramidal Neurons
Unlike orthodromic activation of CA1 neurons involving synaptic transmissions, antidromic activation occurs when pulses are applied to the alveus—the efferent fiber of the CA1 region (i.e., the axons of CA1 pyramidal neurons themselves). This activation can travel back towards the somata, generating APs that form an antidromic population spike (APS). Simultaneously, the axonal activation can also orthodromically excites interneurons in the feedback circuits through synapses in axonal terminals (Fig. 2.6A). This raises a question: do PPF and PPD also occur during antidromic activation?
As shown in Fig. 2.8, the IPI of PPSs was kept at 50 ms. As pulse intensity increased, the APSs induced by both pulses gradually increased due to greater neuronal activations, but without substantial amplitude difference between the two APSs. The mean amplitude ratios of APS2/APS1 were slightly above 1, indicating minor PPF but no PPD. This differs from the results of orthodromic PPSs shown in Fig. 2.7. However, this doesn't mean the local inhibitory circuits are inactive. Figure 2.9 shows that after blocking GABAA-mediated inhibitory synapses with PTX, the induced APSs appeared as multiple peaks rather than a single peak, similar to the same situation with orthodromic stimulation shown in Fig. 2.6B2. The multi-peak firing occurred due to the bursty feature of hippocampal pyramidal neurons. With normal inhibitory circuits, a single pulse of either orthodromic or antidromic stimulation can produce at most single-peak OPS or APS, because feedforward and feedback inhibitions can prevent subsequent burst firing.
Fig. 2.8
CA1 neuronal responses to antidromic paired-pulse stimulations at the alveus using a 50-ms IPI and varying pulse intensities. A Examples of induced APSs at three different intensities. B Changes of APS1 amplitude against pulse intensity. C Changes of APS amplitude ratio (APS2/APS1) against pulse intensity
Antidromic activation does not involve synaptic transmission and integration, making it hardly affected by inhibitory synapses (Lipski 1981). Therefore, under normal conditions, the second pulse of PPS can induce an APS as large as the first one. However, subsequent spikes in burst firing are suppressed because they depend on integration of excitatory activity in dendrites (see Sect. 2.1.3). Additionally, unlike Fig. 2.7B, the input–output curve in Fig. 2.8B shows no pronounced S-shape, indicating that no clear threshold and saturation appeared. Within this pulse intensity range of 0.05–0.6 mA, the APS1 amplitude increased linearly with pulse intensity as more neurons became activated. With further increases in pulse intensity, the APS can eventually reach saturation (Zheng et al., 2022).
2.3.3 Analyzing Feedforward and Feedback Inhibitions Using Paired-Pulse Stimulation
Orthodromic stimulation at the Schaffer collaterals can induce APs in downstream CA1 pyramidal neurons and activate both feedforward and feedback inhibitory circuits (Fig. 2.6A), generating two inhibition components. In contrast, antidromic stimulation at the alveus can activate only feedback inhibitory circuits, producing feedback inhibition. Using a combination of orthodromic and antidromic paired-pulse stimulation, we analyzed the feedforward and feedback inhibition components to address two questions (Feng et al. 2011): How do the two inhibitions change over time in regulating CA1 neuronal firing? What is the relationship between the time course of these synaptic inhibitions and the AP refractory period?
As shown in Fig. 2.10, we set the IPI between two pulses in a range of 0–400 ms, and applied both antidromic-orthodromic paired-pulse stimulation (AO) and purely orthodromic paired-pulse stimulation (OO) to examine the suppression of OPS2 evoked by the second pulse. In an AO, the OPS2 was affected only by feedback inhibition. In this case, the ratio OPSR = OPS2/OPS1 reflected the strength of the feedback inhibition alone. Note: OPS1 here was the OPS evoked by a separated single pulse, serving as a control (Fig. 2.10A). In an OO, the OPS2 was affected by both feedforward and feedback inhibitions. The OPSR now reflected the combined strength of both inhibitions (OPS1 here was the evoked potential of the first pulse). A smaller OPSR indicates a stronger inhibition. (1-OPSR) can indicate the inhibited portion. Under certain conditions, subtracting the inhibited portion of AO stimulation from that of OO stimulation can estimate the strength of feedforward inhibition.
Fig. 2.10
Ratios of evoked potentials for OO and AO paired-pulse stimulations with IPIs ranging from 0 to 400 ms and a fixed 0.3mA pulse intensity. A Examples of evoked potentials. B OPSR data of OO and AO stimulations changing against IPI. C Enlarged OPSR curves in an IPI range of 0–50 ms.
Figure 2.10B shows the OPSR data for OO and AO stimulations, respectively. When the IPI was shorter than 100 ms, the mean OPSR values were all below one, exhibiting obvious PPD. At IPI = 50 ms, the OPSR of OO was 0.43, with an inhibited portion of 0.57 = 1–0.43, indicating that the combined feedforward and feedback inhibitions suppressed over half of the OPS amplitude. At this IPI, the OPSR of AO was 0.71, with an inhibited portion of 0.29 = 1–0.71, indicating comparable proportions of the two inhibition types, i.e., (1–0.71) ≈ (1–0.43)/2. As IPI shortened, the proportion of feedback inhibition increased, reaching 70% at IPI = 25 ms (Fig. 2.10C). When IPI < 10 ms, the OPSR of OO remained at 0, no evoked OPS2. Meanwhile, the OPSR of AO reached its lowest point at IPI = 4 ms but increased again as IPI shortened further, reaching 0.36 at IPI = 0 when the two pulses of AO were applied simultaneously. Although the two pulses were applied at the same time, their evoked potentials occurred at different times due to the synaptic delay in orthodromic activation (Fig. 2.11). Additionally, the estimated 86% feedback inhibition at IPI = 10 ms may be inaccurate, as the zero OPSR of OO made it impossible to determine whether over-inhibition existed.
Fig. 2.11
Examples of evoked potentials in OA and AO paired-pulse stimulations at different short IPIs.
The plots in Fig. 2.10B describe the OPSR data changing against IPIs (intervals between pulses) rather than intervals between evoked potentials—inter-spike-interval (ISI). For OO stimulations, both pulses follow identical activation pathways, making their IPI equivalent to ISI. However, for AO stimulations, the antidromic activation pathway do not involve synaptic transmission. Also, length variations in activation pathways across rat experiments can result in additional differences in evoked potential latencies. Antidromic stimulation typically induced APS with a latency of 1–2 ms, while orthodromic stimulation induced OPS with a latency of 4–9 ms (Fig. 2.11A, B). With longer IPIs, these latency differences between evoked potentials become negligible. However, when using shorter IPIs to study quick effects of inhibitory circuits, the latency differences can be crucial and must be considered.
As shown in Fig. 2.11E, even when the IPI of AO was 0, a substantial ISI existed between APS and OPS. The ISI was measured by the interval between the two negative peaks. We defined ISI as positive when APS preceded OPS, and negative when OPS preceded APS. Since feedback inhibition in AO stimulation is produced by the evoked APS, ISI can reflect the time course of quick effect of this inhibition more accurately than IPI. To investigate how feedback inhibition changes as ISI approaches 0, in addition to AO stimulations, OA stimulations with short IPI (where the O pulse precedes the A pulse) were also employed (Fig. 2.11C–E). Notably, in OA stimulations with very short IPIs, the evoked APS may appear before OPS, as shown in Fig. 2.11D and E.
The subplots in Fig. 2.11 show the different suppression of the second evoked potentials during OA and AO stimulations with different short IPIs. Figure 2.11A, B respectively show the evoked potentials of single O and A stimulations, serving as baseline controls. (The amplitude of control OPS was used as the denominator for calculating the OPSR data shown in Fig. 2.12). Figure 2.11C–H show the changes of APS and OPS with ISI. When O pulse preceded A pulse by 10 ms (IPI = 10 ms), the initial OPS suppressed the subsequent APS partially (Fig. 2.11C). However, at IPI = 2 ms, although O pulse still preceded A pulse, APS occurred first and significantly suppressed the following OPS (Fig. 2.11D). Here, with ISI = 2 ms, the refractory period following APS also contributed to the OPS suppression. As IPI decreased to 0 (Fig. 2.11E), the ISI between APS and OPS increased, and the OPS rose substantially as it escaped the refractory period of APS. When ISI further increased (becoming AO stimulations), OPS decreased again (Fig. 2.11F, G). At ISI≈10 ms, OPS almost disappeared, indicating powerful feedback inhibition produced by APS (Fig. 2.11G). Only as ISI increased further did OPS gradually recover (Fig. 2.11H). These changes revealed a weak period of OPS suppression between the waning refractory period and the onset of feedback inhibition.
Fig. 2.12
Suppression of APS on OPS in AO paired-pulse stimulations with short ISIs.
Figure 2.12 displays the OPSR curves plotted against ISI (the interval of APS preceding OPS) in six rat experiments. Although with individual variations, the curves exhibit a refractory period inhibition at ISIs below 3 ms and a strong phase of feedback inhibition at ISIs between 7 and 17 ms.
The above experimental data indicate that the combined effect of feedforward and feedback inhibitions is strong within 50 ms and can almost completely suppress firing within 10 ms. During the 10–50 ms period, the proportion of feedback inhibition increases as time shortens. However, it decreases during the 3–7 ms period, suggesting that the activation period of feedback inhibition doesn't seamlessly connect with the AP refractory period. At this period, the supplement of feedforward inhibition becomes crucial.
When the inputs from Schaffer collaterals orthodromically activate the downstream CA1 neurons, the induction of feedforward inhibition requires two synaptic transmissions which take about 3 ms (Pouille and Scanziani 2001). Because the simultaneous induction of OPS requires only one synaptic transmission, feedforward inhibition can begin its effect about 2 ms after OPS, allowing overlap between the feedforward inhibition and the refractory period. Moreover, if the orthodromic stimulation directly activates nearby interneurons, it can produce single-synapse feedforward inhibition on CA1 pyramidal neurons more quickly. Such fast inhibition can overlap more with excitatory postsynaptic potentials induced by the same stimulus (Papatheodoropoulos and Kostopoulos 1998). The rapid and powerful effect of feedforward inhibitions plays a crucial role in enhancing the synchronicity of neuronal firing, raising the firing threshold of CA1 pyramidal neurons, and suppressing abnormal epileptiform discharges.
2.3.4 Responses of CA3 Neurons to Paired-Pulse Stimulations
The pyramidal neurons in the rat hippocampal CA3 region have two major axonal branches. One forms the Schaffer collaterals (thick solid lines in Fig. 2.13A), which project to the ipsilateral CA1 region. Another forms the commissural fibers (CF), which extend to the contralateral hemisphere and connect to pyramidal neurons in the contralateral CA3 and CA1 regions (thick dashed lines in Fig. 2.13A). Excitatory synaptic connections also exist between the ipsilateral CA3 pyramidal neurons. Thus, the projection range of rat CA3 pyramidal neurons is extensive. These neurons innervate both ipsilateral and contralateral hippocampal CA3 to CA1 regions. The CF, extending from bilateral CA3 regions to their contralateral sides, forms an intertwined fiber—VHC. Note: This commissural fiber projection is almost non-existent in primate brains (Andersen et al. 2007).
Fig. 2.13
Responses of rat CA3 neurons to VHC pulse stimulations with varying intensities. A Schematic diagram showing the VHC connections between left and right hippocampi and the commissural fibers (CF) within the hippocampi (green and blue dashed lines). B Schematic diagram showing the stimulation electrode at VHC and the recording electrode in the CA3 region. C Diagram showing the activations of CA3 neurons by a VHC stimulus. D Examples of CA3 population spikes evoked by VHC pulses with varying intensities. Red arrows indicate stimulus artifacts. E and F Changes in the population spike amplitude (E) and latency (F) against stimulus intensity (n = 10)
Based on the neuronal connections shown in Fig. 2.13B, C, when a pulse is applied to the VHC, the evoked potentials recorded in the CA3 region contain two components (Fig. 2.13D). The first is the antidromically-evoked population spike (APS) produced by the direct activation at the axons of ipsilateral CA3 neurons themselves. The second is the orthodromically-evoked population spike (OPS) produced by the activation of afferent fiber—the axons of contralateral CA3 neurons. A single pulse to the VHC can trigger both the evoked populations simultaneously. The APS appears first because its induction doesn't involve synaptic transmission, resulting in a shorter latency than the OPS. As shown in Fig. 2.13E, increasing pulse intensity from 0.1 to 0.35 mA caused growth in both APS and OPS. However, due to the refractory period—which prevents rapidly repeated firing—a large initial APS can suppress the subsequent OPS. Therefore, when pulse intensity increased beyond 0.25 mA, the OPS began to decrease rather than continue growing.
Additionally, fine-tuning the stimulation electrode position in the VHC fiber can alter the sizes of the two evoked potentials and their amplitude ratio under a same pulse intensity. Due to individual variations in rat experiments, this size change resulted in the considerable variances in statistical amplitude data as shown by the large error bars (one standard deviation) in Fig. 2.13E. For example, at the pulse intensity of 0.25 mA, the APS amplitude is 5.4 ± 2.5 mV, and the OPS amplitude is 5.4 ± 2.8 mV (n = 10). The variations in latencies of the two evoked spikes were much smaller (Fig. 2.13F). Within the pulse intensity range of 0.1 to 0.35 mA, the mean APS latency remained stable at around 1.7 ms, with a standard deviation of only ~ 0.2 ms. The mean OPS latency, however, decreased with increasing pulse intensity, from 4.1 ± 0.51 ms to 3.6 ± 0.50 ms (n = 10). This latency reduction can be caused by faster depolarization in postsynaptic neurons produced by increased excitatory inputs.
Like the hippocampal CA1 region, the CA3 region also contains GABAergic local inhibitory circuits. Applying PPS at the VHC can reveal the inhibitions through evoked potentials. Figure 2.14A shows the evoked potentials of PPSs with an intensity of 0.35 mA and IPIs ranging from 10 to 400 ms. The antidromic and orthodromic evoked potentials from the two pulses were denoted as APS1, OPS1 and APS2, OPS2, respectively. The figure shows superimposed evoked-potentials at six different IPIs, aligned to the first pulses. The first pulses consistently evoked large APS1 and OPS1 waveforms with constant amplitudes, while the second pulses produced varying evoked-potentials across different IPIs. At a 10-ms IPI, APS2 decreased and OPS2 disappeared, indicating strong inhibitions. As the IPI extended beyond 50 ms, OPS2 became pronounced and gradually increased. Similarly, APS2 returned to match APS1 when the IPI exceeded 25 ms.
Fig. 2.14
Responses of CA3 neurons to paired-pulse stimulations (PPS) at the VHC with varying IPIs and a fixed 0.35-mA intensity. A Examples of evoked-potentials by PPSs with IPIs of 10, 25, 50, 100, 200 and 400 ms. The six recordings are superimposed and aligned to the first pulses (indicated by the larger red arrow). The IPI values are shown above the second pulses (indicated by smaller red arrows). B Changes in the amplitude ratios of OPS2/OPS1 and APS2/APS1 against IPIs (n = 9 rats)
The amplitude ratio of OPS2/OPS1 showed that shorter IPIs (10-50 ms) resulted in PPD—where OPS2 was smaller than OPS1 (Fig. 2.14B), while longer IPIs of 100 and 200 ms produced PPF—where OPS2 exceeded OPS1 and even appeared with multiple peaks, as indicated by the hollow triangle on the evoked-potential at 100 ms IPI in Fig. 2.14A. At 400 ms IPI, the two OPSs approached equality. The amplitude ratio of APS2/APS1 showed that APS2 was smaller than APS1 only at very short IPI (10 ms). In this situation, the second pulse arrived within about 5 ms after the first pulse triggered APS1 and OPS1 consecutively. Since the neuronal membrane had not fully recovered from its refractory period, APS2 was suppressed. At 25 ms IPI, APS2 slightly exceeded APS1. As IPI increased further, the two APSs approached equality.
The evoked potentials varied with both IPI and pulse intensity. At a low intensity of 0.1 mA with 25 ms IPI, OPSs exhibited PPD. However, with a longer IPI of 50 ms, OPSs showed PPF. As pulse intensity increased to 0.25 mA, OPSs exhibited PPD at both 25 and 50 ms IPIs (Fig. 2.15A). Statistical data showed that for intensities from 0.1 to 0.35 mA, OPSs consistently exhibited PPD at the shorter 25 ms IPI (Fig. 2.15B). The PPD increased with pulse intensity; at intensities above 0.2 mA, the mean OPS2 amplitude dropped to only about 10% of OPS1. At 50 ms IPI (Fig. 2.15C), OPS2 was approximately 140% of OPS1 at 0.1 mA intensity, exhibiting PPF. As the intensity increased to 0.2 mA and above, this ratio decreased, with OPS2 about 80% of OPS1. Notably, APSs consistently exhibited a slight PPF with both IPIs (25 and 50 ms) at the intensity range of 0.1–0.35 mA, with APS2 being about 120% of APS1.
Fig. 2.15
Responses of CA3 neurons to PPSs at the VHC with varying intensities. A Examples of evoked-potentials at two IPIs with two intensities. B and C Amplitude ratios of OPS2/OPS1 and APS2/APS1 at 25 ms IPI (B) and 50 ms IPI (C) against intensities (n = 12 rats)
The PPD of OPS2 in hippocampal CA3 neurons can be primarily caused by the combined effect of feedforward and feedback inhibitions similar to those in the CA1 region shown in Fig. 2.6A. The PPF of OPS2 can result from increased neurotransmitter release and EPSP integrations from the two pulse activations. Additionally, excitatory connections among CA3 pyramidal neurons themselves may also contribute to PPF. Due to the opposing effects—PPD and PPF—the neuronal responses exhibit complex changes under different stimulation conditions. When assessing the states of neurons and local circuits, specific conditions must be carefully considered. For example, when evaluating inhibitory circuits based on PPD, OPS2 suppression should be determined using consistent IPI and pulse intensity, along with similar OPS1 amplitudes. Otherwise, even with identical IPI and intensity, weak neuronal excitability can lead to a smaller OPS1, resulting in weaker feedback inhibition. This may cause reduced suppression or even an increase in OPS2 (due to PPF dominance). In such case, concluding that inhibitory circuits are impaired would be incorrect.
As shown in Fig. 2.13D, the evoked-potentials in the CA3 region produced by single-pulse stimulations at VHC contain two peaks (APS and OPS) with a latency difference of about 2 ms. This firing pattern resembles the multiple-peak discharges evoked in the CA1 region when inhibitory circuits are impaired by PTX, as shown in Fig. 2.6B2 and Fig. 2.9 (bottom). To verify that these two-peak potentials in the CA3 region stem from antidromic and orthodromic activations of two axon types (Fig. 2.13C), I used a calcium ion chelator (ethylene glycol tetraacetic acid, EGTA) to reduce the Ca2+ concentration in the hippocampus extracellular fluid ([Ca2+]o). This intervention can block synaptic transmissions and eliminate orthodromic activation, allowing verification of the evoked-potential sources. The experimental method is detailed in Sect. 3.3.
Figure 2.16 shows the results of this EGTA experiment. PPSs (0.35 mA intensity, 25 ms IPI) were applied to VHC at 5-min intervals. During the 30-min baseline recording with normal 2 mM [Ca2+]o, the first pulses produced both large APS1 and OPS1. After switching to a Ca2+-free solution containing EGTA for 40 min, APS1 increased while OPS1 gradually decreased. Upon returning to the normal Ca2+ solution, OPS1 gradually recovered and APS1 returned to its baseline level (Fig. 2.16A). Throughout the 100-min recording, both APS1 and APS2 remained large, with stable APS2/APS1 amplitude ratios (Fig. 2.16B).
Fig. 2.16
Verification of orthodromic and antidromic evoked-potentials in the rat hippocampal CA3 region by VHC stimulation. A Examples of evoked-potentials (upper) and amplitude changes of APS1 and OPS1 produced by paired-pulse stimulations (0.35 mA intensity, 25-ms IPI) at VHC during the 100 min recording period—including baseline with normal solution (30 min), application of Ca2+-free EGTA solution (40 min), and return to normal solution (30 min). B Changes in the APS2/APS1 amplitude ratio throughout the recording period
During the last 20 min of EGTA application—corresponding to the 50–70 min period in Fig. 2.16—the mean OPS1 amplitude decreased significantly to 5.9% ± 4.1% (n = 6) of its baseline value. This indicated that most synaptic transmissions were blocked by lowering [Ca2+]o. In contrast, the mean APS1 amplitude remained at 109% ± 16% (n = 6) of its baseline value without substantial change. Additionally, the mean amplitude ratio of APS2/APS1 (evoked by PPSs at 25 ms IPI) decreased from 129% ± 7.4% at baseline to 103% ± 5.1% (n = 6).
These data confirmed the distinct properties of the two components evoked by VHC stimulations. Together with other data such as the latency of evoked-potentials and the PPD and PPF produced by PPSs, we can conclude that the dual-spike potentials were generated by orthodromic and antidromic activations of afferent and efferent axon fibers. The features of these evoked-potentials, including their amplitudes, provide valuable indicators for assessing the states of neurons and their local circuits.
2.4 Summary
Neurons are the fundamental building blocks of the nervous system. Each neuron consists of a cell body (or soma), dendrites, and an axon. It receives signals through dendrite synapses and processes them using complex integration mechanisms. Once the input integration reaches activation threshold, the neuron can fire an action potential (or nerve impulse) that can travel outward along the axon. Besides forming excitatory projection connections, neurons also create local circuits. Among them, local inhibitory circuits play a vital role in maintaining the balance of neuronal excitation.
The animal experiments described in this book focus on the rat hippocampus. In this brain region, principal neurons create distinct laminar structures through their dense and orderly arrangement. The structures allow for extracellular recordings of both population spikes (PS) and field excitatory postsynaptic potentials (fEPSP) when neurons respond to electrical pulse stimulations. These evoked potentials can reach millivolts in amplitude, facilitating investigations into neurons and their circuits, as well as the modulation effects of electrical stimulations.
This chapter introduces the structure and neural circuits of the rat hippocampus, including excitatory synaptic pathways and local inhibitory circuits. It examines the waveforms, meanings, and generation mechanisms of pulse-evoked potentials—including orthodromic potentials (produced by pulses applied to presynaptic input fibers) and antidromic potentials (produced by direct axon activations). The chapter also presents our findings on the effects of feedforward and feedback inhibitory circuits in the hippocampal CA1 region by using paired-pulse stimulations. Additionally, it explores how CA3 pyramidal neurons respond to VHC stimulations.
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