6th Lecture PDF - Synaptic Transmission & Neurophysiology

Key Concepts and Mechanisms: 1. Parallel Fiber Information and Error Signals: ○ Parallel fibers carry sensory information, such as a tone. ○ The error signal, possibly generated by climbing fibers, is crucial for modulating the response (e.g., air puff-induced blink)....

Key Concepts and Mechanisms: 1. Parallel Fiber Information and Error Signals: ○ Parallel fibers carry sensory information, such as a tone. ○ The error signal, possibly generated by climbing fibers, is crucial for modulating the response (e.g., air puff-induced blink). ○ This combination results in a complex spike—a burst of activity with multiple characteristics, reflecting the rich information being processed. 2. Axon Recordings and Spike Propagation: ○ Researchers recorded from Purkinje cells' axons, observing both simple and complex spikes. ○ Simple spikes propagate more consistently, while complex spikes show variability in their propagation. ○ Variability is due to the probability of certain spikes successfully traveling down the axon, influenced by several factors. 3. Complex Spike Characteristics: ○ Key features of spikes include amplitude, rate of rise, slope, and interspike intervals. ○ These characteristics are predictive of whether the spike will propagate down the axon. ○ Sodium channel dynamics, particularly inactivation, play a significant role in determining this propagation. 4. Sodium Channel Inactivation: ○ Sodium channels open to generate action potentials but inactivate soon after. ○ Inactivation prevents further spikes unless the membrane is sufficiently hyperpolarized, allowing the channels to reset. ○ Complex spikes often fail to propagate because the available sodium channels have been inactivated by preceding spikes. 5. Neuronal Efficiency and Synaptic Transmission: ○ Neurons need to transmit information rapidly and efficiently. ○ Synaptic transmission is designed to ensure the swift relay of signals over long distances, followed by efficient transmission across synapses. ○ Chemical synapses have evolved to modulate signals (e.g., amplifying or inhibiting them), which allows for flexible neuronal computation. 6. Electrical vs. Chemical Synaptic Transmission: ○ Electrical synapses use gap junctions to allow ions to flow directly between cells, offering fast and efficient signal transmission.  ○ Electrical synapses are less energy-intensive but offer limited flexibility in signal modulation (e.g., both hyperpolarizing and depolarizing signals are transmitted equally). ○ Chemical synapses, in contrast, are slower but allow for greater computational diversity (e.g., inhibition via chloride ions). 7. Calcium Influx and Neurotransmitter Release: ○ Chemical synapses rely on calcium influx to trigger the release of neurotransmitters. ○ Vesicles containing neurotransmitters fuse with the presynaptic membrane, releasing the neurotransmitter into the synaptic cleft, where it binds to receptors on the postsynaptic cell. 8. Efficiency and Modifiability: ○ Chemical synapses allow for greater modifiability than electrical synapses. ○ The efficiency of synaptic transmission is critical due to the high energy cost of neuronal activity. ATP usage must be minimized while maintaining high-speed transmission. Additional Insights: 1. Sodium Channel Availability: ○ Repeated spikes consume sodium channel availability, making it harder for subsequent spikes to propagate. ○ This limitation is seen in low spike amplitudes and slower rates of rise in action potentials. 2. Neural Networks and Computation: ○ Synapses not only relay information but also modify it, enabling neurons to perform complex computations. ○ The computational power of a neural network is related to its ability to adapt and modify synaptic strength. 3. Electrical Coupling in the Brain: ○ Some regions of the brain, such as the inferior olive, rely heavily on electrical coupling, especially among inhibitory neurons. ○ This type of transmission is advantageous for synchronizing neuronal activity in certain brain regions. Summary of Synaptic Transmission: Electrical Synapses: Fast, efficient, but limited in computational flexibility. Useful for synchronizing activity, especially in inhibitory circuits. Chemical Synapses: Slower but highly adaptable. They allow for complex computations and modulation of signals (e.g., excitation vs. inhibition). These notes emphasize the balance between speed, efficiency, and computational power in synaptic transmission, with a focus on how different types of synapses contribute to neuronal processing and information propagation Class Notes on Synaptic Transmission and Neurophysiology 1. Bernard Katz and Synaptic Transmission: ○ Katz recorded from muscles while stimulating motor axons in frog preparations. ○ Katz was initially interested in action potentials but shifted focus to synaptic transmission after being dissatisfied with certain experimental techniques like voltage clamping. ○ His research played a significant role in the understanding of synaptic transmission, particularly at the neuromuscular junction (NMJ). 2. Calcium and Magnesium Effects on Synaptic Response: ○ Katz observed that altering the concentrations of calcium (Ca²⁺) and magnesium (Mg²⁺) could significantly impact synaptic responses. ○ High magnesium reduces calcium currents presynaptically, thereby preventing postsynaptic muscle spiking, leading to varied synaptic responses. ○ In low calcium and high magnesium conditions, Katz noticed extremely small, variable postsynaptic responses and even failures, indicating a dependence on calcium for neurotransmission. 3. Spontaneous and Evoked Synaptic Events: ○ Katz’s experiments showed spontaneous synaptic events occurring without stimulation, further supporting the concept of spontaneous neurotransmitter release. ○ These small, spontaneous events were consistent in amplitude, around 0.24 millivolts, forming distinct peaks on histograms that Katz compiled. ○ When looking at stimulated synaptic responses, Katz observed variability in the amplitudes, which led to the idea that synaptic transmission occurs in discrete packets or "quanta" of neurotransmitters. 4. Quantal Hypothesis of Synaptic Transmission: ○ Katz proposed that synaptic transmission occurs in quantal units, where neurotransmitters are released in packets rather than continuously. ○ The characteristic size of these quanta could be observed in spontaneous synaptic events as well as evoked responses, where multiples of the base quantum size were seen.  ○ This hypothesis was a key development in understanding that neurotransmitters are released in fixed amounts, governed by the number of vesicles and their probability of release. 5. Mathematical Model of Synaptic Transmission: ○ Katz introduced a model where the average excitatory postsynaptic potential (EPSP) is determined by three factors: 1. Number of release sites (N) – how many active zones are available for vesicle release. 2. Probability of release (P) – the likelihood that a vesicle will release its contents. 3. Quantal size (Q) – the postsynaptic response to one vesicle’s worth of neurotransmitter (miniature EPSP). ○ This model could predict synaptic behavior and was used to calculate the probability of different outcomes (failures, single vesicle releases, multiple vesicle releases) using binomial expansion. 6. Application of Binomial and Poisson Distributions: ○ Binomial distribution was used to describe the independent probability of vesicle release at each active zone. ○ In cases where the number of release sites is large and the probability of release is small, the Poisson distribution can simplify the model. ○ While this method is classical, it’s not frequently used in modern neurophysiology as the conditions it describes (large N and small P) are rare in typical synapses. 7. Challenges with the Quantal Model: ○ Real-world synaptic transmission is more complex due to factors like non-uniform quantal sizes and varying probabilities of release across different synapses. ○ Despite these complications, the quantal model remains a useful tool for understanding synaptic transmission at the NMJ and in other simpler systems. 8. Ultra-Structural Evidence of Quantal Release: ○ Electron microscopy (EM) studies confirmed Katz’s quantal hypothesis by showing vesicles at the presynaptic terminal undergoing exocytosis and endocytosis. ○ The so-called "omega shapes" observed in EM images represent vesicles fusing with the membrane to release neurotransmitters. 9. Calcium’s Role in Vesicle Release: ○ Experiments showed that calcium influx is essential for synaptic vesicle release. Blocking calcium channels with cadmium eliminated both the calcium current and the postsynaptic response.  ○ At the squid giant synapse, controlled voltage steps allowed precise measurements of calcium currents and their effect on postsynaptic potentials (EPSPs). ○ By stepping the presynaptic membrane potential to the calcium reversal potential, the calcium current was eliminated, showing that without calcium influx, synaptic release does not occur. 10. Synaptic Vesicle Release in Specialized Synapses: Certain large synapses like the squid giant synapse and the calyx of Held allow for detailed study of both presynaptic and postsynaptic activity. These specialized synapses have provided key insights into the mechanisms of synaptic transmission and vesicle release. 11. Implications for Synaptic Plasticity: Katz’s findings laid the groundwork for later studies on synaptic plasticity, where changes in the probability of release or number of active zones can alter synaptic strength. Understanding these mechanisms is crucial for studying learning and memory, as synaptic plasticity underlies these processes. In summary, Katz’s work on synaptic transmission revealed the fundamental mechanisms of neurotransmitter release, demonstrating that it occurs in quantal units regulated by calcium-dependent processes. His mathematical modeling and experimental approaches continue to inform our understanding of synaptic physiology and plasticity. Here are detailed notes based on the transcript: 1. Voltage Manipulation in Experiments: ○ The cell is hyperpolarized to around -70 mV, which represents a typical resting membrane potential in neurons. This manipulation affects ion channel behavior. 2. Channel Dynamics and Driving Force: ○ Ion channels (specifically calcium channels in this context) respond to changes in voltage by opening or closing, but this process is not instantaneous. ○ When the voltage is drastically shifted, channels remain open briefly before closing, leading to what is called a "tail current." This occurs because the driving force for ion movement is high before the channels fully close. ○ Driving force: After stepping down to a hyperpolarized voltage (-70 mV), the driving force for calcium becomes significant, possibly around 200 mV, causing a large influx of calcium ions briefly until the channels close. 3. Tail Current and Calcium Dynamics: ○ The tail current is a result of the lag in calcium channel closure when the voltage changes rapidly. ○ Calcium channels exhibit delayed closure, which leads to a tail current and, consequently, delayed neurotransmitter release. ○ In normal action potentials, this behavior is uncommon but occurs in these experimental voltage conditions. 4. Calcium's Role in Action Potentials: ○ Calcium currents are much smaller compared to sodium and potassium currents in an action potential, which is why calcium is often not blocked when studying action potentials. 5. Synaptic Transmission Dynamics: ○ Synaptic transmission involves rapid calcium entry, vesicle fusion, and neurotransmitter release. ○ The delay between action potential initiation and postsynaptic response is very short (milliseconds to microseconds), especially at physiological temperatures (around 37°C for mammals). 6. Calcium's Influence on Vesicle Fusion: ○ The dependence of neurotransmitter release on calcium follows a fourth-power relationship, implying that four calcium ions typically bind to trigger vesicle fusion. ○ Synaptotagmin, a protein involved in this process, binds 4-5 calcium ions, facilitating vesicle fusion with the presynaptic membrane. 7. Calcium Localization and Microdomains: ○ Calcium channels are located very close to release sites (synaptic vesicles) to minimize the amount of calcium needed and the energy required for calcium clearance. ○ Different calcium chelators (e.g., EGTA and BAPTA) show that calcium signaling is highly localized. Fast chelators like BAPTA can prevent vesicle fusion by rapidly binding calcium, while slower chelators like EGTA cannot, highlighting the importance of calcium microdomains. 8. Vesicle Fusion and Membrane Capacitance: ○ Vesicle fusion during neurotransmitter release causes an increase in membrane surface area, which can be measured by changes in capacitance. ○ Larger capacitance jumps correlate with more vesicles fusing, and these jumps can be blocked by calcium channel inhibitors, confirming the role of calcium in vesicle fusion. 9. Experimental Calculations on Vesicle Fusion:  ○ A mathematical approach to estimating the number of vesicles fusing based on changes in capacitance and vesicle size yields estimates of about 1800 vesicles, though these numbers were debated in class. ○ The calculation is based on the specific capacitance of membranes (around 1 microfarad per square centimeter) and the surface area of individual vesicles (about 5000 square nanometers). 10. Fluorescent Techniques for Vesicle Imaging: ○ Techniques like FM dyes and genetically encoded indicators (e.g., synapto-pHluorin) are used to visualize vesicle fusion and neurotransmitter release in living cells. ○ These dyes exploit the acidic interior of vesicles and fluoresce upon vesicle fusion, allowing researchers to monitor synaptic activity. 11. Recent Advances in Fluorescent Techniques: ○ The Glusniffer is a fluorescent tool specifically designed to monitor glutamate release, offering a precise readout of synaptic transmission, though it is specific to glutamate and not other neurotransmitters like GABA. 12. Channel Recordings: ○ Calcium channels have been a central theme in physiological recordings, particularly with techniques like whole-cell patch-clamp recordings that allow for detailed studies of channel behavior during synaptic transmission. ○ These studies help elucidate the intricate dynamics of calcium entry, vesicle fusion, and neurotransmitter release, forming the basis of our understanding of synaptic physiology. These notes synthesize the key concepts discussed in the transcript, focusing on the physiological mechanisms underlying synaptic transmission, particularly the role of calcium. Class Transcript Notes on Patch-Clamp Recordings and Ion Channels Inside-Out vs. Outside-Out Recordings: Inside-Out Recording: Useful when studying how intracellular molecules affect channel activity. Allows direct application of substances to the intracellular side of the channel. Outside-Out Recording: More useful when studying how extracellular molecules or conditions affect channel function. Single-Channel Recordings and Noise Reduction: When isolating one or two channels in the pipette, the noise level is significantly reduced, allowing more precise measurements.  A small patch of membrane is captured, which minimizes interference and enables the observation of individual channel activity. Voltage and Channel States: A baseline current is established, and when voltage is applied, the channel alternates between two current states (open and closed). The fluctuating current is a signature of having one channel in the pipette. Channels do not open gradually; they flick between open and closed states rapidly. Analyzing Channel Activity: You can create histograms showing how much time the membrane spends in different current states (e.g., 0 pA vs. 6 pA). This helps quantify channel activity over time, revealing the probability of channel states. Channel States and Simplification: Channels have two primary states: open and closed. Though some intermediate states exist, they are less common. Recordings of single-channel activity are crucial for understanding biophysical properties of channels. These will be examined in future papers. Stochastic Nature of Channel Opening: Opening and closing of channels is a stochastic process, governed by probabilities. Even when voltage is applied, channels do not uniformly open or close. The applied voltage increases the likelihood of a channel being in the open state. In a pipette with two channels, the recordings will show discrete steps corresponding to the opening of one or both channels, reflecting their independent behavior. Patch-Clamp Technique: Patch-clamp allows precise control over the small membrane patch being measured, isolating a few channels to study their behavior individually. Researchers often pull the patch away from the cell to isolate a small number of channels (one or two), avoiding the complexities of measuring many channels simultaneously. Summing Channel Activity: Whole-cell currents, like calcium currents, are the sum of individual channel activities. When many channels are active simultaneously, their summed activity produces the type of current typically observed in experiments. Types of Calcium Channels: There is diversity in calcium channels, which were first defined based on their distinct conductance properties and response to voltage changes. ○ T-type Channels: Transient, flicker on and off rapidly.  ○ L-type Channels: Large single-channel conductance, contributing to sustained currents. ○ N-type Channels: Intermediate between T and L. The distinction between these types is less about memorizing specifics and more about understanding the variety of calcium channels and their different roles. High vs. Low Voltage Activated Calcium Channels: High-voltage activated channels open at around 0 to +20 mV and are critical for triggering neurotransmitter release in presynaptic terminals. Low-voltage activated channels open at more negative potentials (around -30 mV). PQ and N-type Channels: PQ-type Channels: Found primarily in presynaptic terminals. N-type Channels: Also found in certain neurons. L-type Channels: More prevalent at the neuromuscular junction. R-type Channels: Rare, less frequently encountered. Short-Term Synaptic Plasticity: Chemical synapses exhibit short-term plasticity, where the response to a second stimulus is often larger than the response to the first. This can result in a significant increase in response size (up to 200%), suggesting modifications in neurotransmitter release mechanisms. These notes cover the key concepts related to patch-clamp techniques, single-channel recordings, and the diversity of ion channels, particularly calcium channels. They also touch on synaptic plasticity, a critical concept in neuroscience. Spike Propagation and Synaptic Transmission 1. Spike Propagation Importance: ○ Pathway: Mossy fibers → Granule cells → Simple spikes (conditioned stimulus) ○ Inferior Olive Climbing Fibers: Trigger complex spikes (error signal, unconditioned stimulus). 2. Key Features of Neurons: ○ Speed: Millisecond-range signaling. ○ Distance: Signals can travel long distances (1 meter). ○ Precision: High precision in signaling due to action potentials. Synaptic Transmission 3. Challenges of Synaptic Transmission:  ○ Speed: Rapid transmission (no delay greater than 1 ms). ○ Efficiency: Must transmit large amounts of information using minimal energy. ○ Modifiability: Neuronal networks must adjust connection strength to enable learning and memory. ○ Computation: Neurons must process multiple computations efficiently. 4. Electrical vs. Chemical Transmission: ○ Electrical Synapses: Fast and efficient. Connexins form pores between neurons, allowing direct ion travel. Signals propagate bidirectionally but lack modifiability. ○ Chemical Synapses: More modifiable (e.g., metabotropic receptor activation). Can convert excitatory signals to inhibitory (inverts the sign of the signal). Uses more energy but provides computational flexibility. Quantal Hypothesis and Synaptic Release 5. Quantal Hypothesis: ○ Bernard Katz: Synaptic transmission occurs in quantal units, where neurotransmitter release is not continuous but occurs in discrete packets. ○ Recording at the Neuromuscular Junction (NMJ): End Plate Potentials (EPPs): Postsynaptic potentials in muscle cells. In low calcium, spontaneous events and small EPPs occur in consistent sizes, supporting the quantal release hypothesis. 6. Synaptic Response Equation: ○ EPSP=N×Pr×q\text{EPSP} = N \times Pr \times qEPSP=N×Pr×q NNN: Number of release sites (active zones). PrPrPr: Probability of release for one vesicle. qqq: Postsynaptic response to one vesicle. 7. Binomial and Poisson Distributions in Synaptic Release: ○ Binomial Expansion: Used to calculate probabilities of release at multiple sites. ○ Poisson Distribution: Applied when the number of release sites NNN is large and PrPrPr is small. Calcium's Role in Synaptic Transmission 8. Calcium Hypothesis: ○ Llinas' Experiment: Voltage-clamping the presynapse shows that calcium influx drives synaptic release. When the membrane potential reaches calcium’s reversal potential, no driving force exists, and release is blocked.  Upon repolarization, calcium tail currents (ion fluxes after voltage returns to rest) activate the postsynaptic response. 9. Calcium Cooperativity: ○ Dodge and Rahamimoff: Release depends on calcium binding to synaptotagmin. ○ Synaptotagmin: A key protein that binds 4-5 calcium ions to trigger vesicle fusion. 10. Calcium Microdomains: ○ EGTA vs. BAPTA: EGTA (slow calcium chelator) does not block release, while BAPTA (fast chelator) dramatically reduces release. This suggests that calcium enters very near the release sites, forming microdomains of high calcium concentration. Modern Approaches to Measuring Vesicle Release 11. Capacitance Measurements: ○ Vesicle Fusion: Each vesicle fusion adds membrane area, increasing capacitance. ○ von Gersdorff Experiment: Depolarizing the presynaptic terminal shows changes in capacitance corresponding to vesicle fusion. 12. FM Dyes: ○ Use: Fluorescent dyes that bind to membranes and report vesicle fusion. ○ pHluorin Dyes: Genetically encoded pH-sensitive dyes that fluoresce upon vesicle fusion, useful for tracking synaptic activity. Single-Channel Recordings 13. Patch-Clamp Technique: ○ Single-Channel Conductance: Measured by recording small patches of membrane to isolate individual ion channels. ○ Ion Channel Properties: Summing individual recordings reveals the overall current characteristics (e.g., N-type, L-type calcium channels). 14. Calcium Channel Diversity: ○ High Voltage Activated (HVA): Used for neurotransmitter release. ○ Low Voltage Activated (LVA): Associated with pacemaking and rhythmic activities. Summary of Key Concepts  Quantal Hypothesis: Neurotransmitters are released in discrete quanta (vesicles). Calcium's Role: Critical for vesicle fusion, driven by localized microdomains. Synaptic Plasticity: Chemical synapses are modifiable and central to learning processes. Single-Channel Recordings: Provide detailed insight into ion channel activity at the presynaptic terminal. These notes cover the main content and key diagrams from the slides, synthesizing the detailed explanations of spike propagation, synaptic transmission, and modern methods in neurophysiological research.