Axon Guidance and the Foundation of Neurophysiology

• Availability: The first problem set will be accessible today at 11:00 AM Pacific Standard Time (PST), immediately following the lecture.

• Location: Students can find the assignment on the Canvas course website under the "Assignments" tab. The folder will automatically open at the specified time.

• Deadline: The problem set is due this Thursday at midnight PST. Students residing outside of California must ensure they adjust their schedules accordingly.

• Format and Ethics: The assignment is a take-home exam. Students are permitted to use their personal notes and required readings. However, sharing information or collaborating with peers is strictly prohibited to ensure individual understanding is assessed.

• Support: Office hours are scheduled for Wednesday and Thursday for students seeking clarification on confusing concepts.

Structural Components of the Neuron

• The Cell Body (Soma): This is the central part of the neuron, which contains the nucleus (with DNA) and standard organelles common to most cells. It is the site of basic biological maintenance.

• Dendrites: These are tendrils extending from the cell body that serve as the input side of the neuron. They receive information from other neurons. A single neuron can have anywhere from one to thousands of dendrites.

• The Axon: This is the output element of the cell.

  • Polarity: As deduced by Santiago Ramón y Cajal, information flows in a single direction: from dendrites to the cell body and out through the axon.

  • Singularity: A neuron typically has only one axon emerging from the cell body. While it may branch hundreds of times, it remains a single output structure.

  • Length: Axons are significantly longer than dendrites. While dendrites are measured in micrometers, axons can reach lengths of several centimeters (orders of magnitude larger).

Foundation and Concepts of Axon Guidance

• Definition: Axon guidance is the process by which axons grow, navigate space, and identify specific targets to form functional circuits during the development of the nervous system.

• Non-Random Connectivity: Neurons do not connect randomly. The process is guided by a precise, though not yet fully decoded, set of chemical signals.

• Pioneer Cells and Axons:

  • Pioneer Cells: These are the first neurons to send out axons searching for targets.

  • Navigate the "Unknown": These axons move outward through the developing tissue to establish initial pathways.

• The Timing Element: Precision in axon guidance is temporal. Target cells are often eliminated or die if the connecting axon does not reach them within a specific developmental window.

Experimental Evidence: The Grasshopper Study (1970s)

• Model Organism: Researchers used the developing grasshopper because its eggs are transparent, easy to grow, and allow for detailed observation of limb buds.

• Mechanism of Navigation: Sensory neurons (e.g., T11) sprout axons that follow "guidepost cells." These specific cells along the route produce chemical signals that act as milestones for the axon.

• Ablation Experiments:

  • Definition: "Ablation" is the professional term used for destroying a cell.

  • Method: Researchers used finely guided lasers (developed in the 1960s) to "zap" or fry specific guidepost cells.

  • Result: When a guidepost cell was ablated, the growing axon would reach the previous milestone but then become lost, wandering in random directions because it could no longer "sniff out" the necessary signal.

The Growth Cone: The Navigational Engine

• Discovery: Identified in the 1890s, the growth cone is a specialized structure at the tip of a developing axon. Mature axons in the adult brain do not possess growth cones.

• Function: It is responsible for detection, steering, and movement.

• Cytoskeletal Structure:

  • Microtubules: These protein fibers provide structural support and are primarily concentrated in the center of the growth cone.

  • Actin: These fibers are located mostly at the edges (tendrils) and are responsible for the actual pushing, pulling, and gripping.

• Internal Labeling: Researchers use immunofluorescence to color-code these proteins artificially for study under microscopes.

• Growth Location Experiment (1980s): By using a weak laser to "bleach" (break) fluorescent markers on an axon, researchers created a dark spot. Observation showed the spot remained stationary while the axon lengthened at the tip, proving growth occurs at the growth cone, not the cell body.

Steering and Environment Interaction

• Steering Mechanism: Similar to a rider guiding a horse with reins, the growth cone turns by altering the stability of its cytoskeleton.

  • Destabilizing Chemicals: Breaking down actin/microtubules on one side causes the growth cone to turn in the opposite direction.

  • Stabilizing Chemicals: Making filaments stronger on one side causes the growth cone to steer toward that side due to increased "grip."

• Surface Adhesion: Axons must be able to grip a surface to grow. In experiments, axons only grew on "sticky" coated surfaces and avoided non-sticky ones. In the developing brain, axons navigate by grabbing onto sticky substances in the environment.

Four Categories of Guidance Cues

• 1. Long-range Attraction: Diffusible, soluble molecules that create gradients to attract axons from a distance.

• 2. Long-range Repulsion: Diffusible molecules that create gradients to push axons away from incorrect areas.

• 3. Contact Adhesion: Non-diffusible molecules that stay at their source. Axons must touch them to recognize the "attractive" signal (acting like the pavement of a road).

• 4. Contact Repulsion: Non-diffusible molecules that push axons away upon physical contact (acting like the yellow lines on the edge of a road to keep the axon in a "tunnel").

• Signal Transduction: Once a cue is detected by a receptor on the growth cone, a signal transduction pathway is triggered, relaying instructions to the cytoskeleton to change direction.

Principles of Neurophysiology and the Resting Potential

• The Action Potential: The fundamental unit of activity and the "language" of the nervous system. Every thought, movement, and feeling is coded as action potentials.

• Resting Membrane Potential:

  • Standard Voltage: All neurons are negatively charged inside relative to the outside.

  • Range: Typically between $-60\,mV$ and $-100\,mV$, with an average of $-70\,mV$.

• Ion Concentration Gradients:

  • Potassium ($K^+$): High concentration inside the cell.

  • Sodium ($Na^+$): High concentration outside the cell.

  • Chloride ($Cl^-$): High concentration outside the cell.

• The Sodium-Potassium Pump ($Na^+/K^+$ ATP-ase): This energy-intensive transporter forces sodium out and potassium in against their concentration gradients to maintain non-equilibrium.

• Origin of Negativity: The membrane is highly permeable to Potassium ($K^+$) due to numerous "leak channels," but has low permeability to Sodium and Chloride. As positively charged Potassium leaks out down its concentration gradient, the interior of the cell becomes negatively charged.

Mathematical Modeling of Membrane Potentials

• The Nernst Equation: This calculates the equilibrium potential for a single ion ($X$). $E_x = \frac{RT}{zF} \ln \frac{[X]_{\text{outside}}}{[X]_{\text{inside}}}$

  • $R$ is the Gas Constant.

  • $T$ is the Temperature (in Kelvin).

  • $z$ is the Valence of the ion.

  • $F$ is the Faraday Constant.

• The Goldman-Hodgkin-Katz (GHK) Equation: A more precise model that accounts for the relative permeabilities ($P$) of multiple ions ($Na^+$ , $K^+$ , and $Cl^-$). $V_m = \frac{RT}{F} \ln \left( \frac{P_K[K^+]_{out} + P_{Na}[Na^+]_{out} + P_{Cl}[Cl^-]_{in}}{P_K[K^+]_{in} + P_{Na}[Na^+]_{in} + P_{Cl}[Cl^-]_{out}} \right)$

• Electrochemical Gradient: The final membrane potential is a balance between the chemical gradient (concentration) and the electrical gradient (charge attraction/repulsion).