GABA Receptors and Neurotransmitter Function

Basic Properties of Neural Cells

• Mature nerve cells cannot divide or regenerate their cell processes, but humans are born with 10-100 billion cells with a daily loss of 10,000 cells throughout life, causing little reduction of the entire cell population.
• Each cell makes about 1,000 synaptic contacts resulting in 10-100 thousand billion total contacts.
• Nerve cells consist of a cell body (soma or pericaryon) from which two cell processes emanate.
• Dendrites are often highly branched and receive electrical signals from other neurons, and covered with dendritic spines.
• Axons (neurites) send out electrical signals and have uniform diameter along their entire length, varying considerably, and divide into collaterals forming right angles.
• Axon tips are branched leading to 1,000 nerve endings per axon.
• Synapses are structures at nerve endings that allow chemical or electrical signal transfer to target cells.
• Glial cells can divide throughout life.
• Neurons are larger than glial cells and possess a uniform set of cell processes.
• Four major subtypes of glial cells include:
  • Astrocytes
  • Oligodendrocytes
  • Microglia
  • Schwann cells
  • Ependymal cells.
• Neurons and glial cells depend on physiological dependence and functional cooperation.
• Staining of thin tissue sections with non-specific dyes (8 µm) is used for microscopic analysis of brain tissue to analyze morphology.
• A technique devised by Camillo Golgi and Ramon y Cajal stains a small subset of the neural cell population with silver salts allows for discrimination of neuronal cell types mainly on dendritic tree shape.

Neuron Categories

• Multipolar Neurons
  • The most predominant in the brain and spinal cord
  • Are inclusive of motor neurons and interneurons
  • Have single axon from one end of the cell body
  • Have several dendrites branching
• Bipolar Neurons
  • Associated with afferent impulses
  • Have a single axon projecting from one end of the oval cell body
  • Have lone dendritic tree extending from the other end
• Unipolar Neurons
  • Have a single axon projecting from spherical cell body
• Pseudounipolar neurons
  • Have one process bifurcating close to the cell body
  • Serves both the role of the axon and the role of the dendrite
• Dendritic trees vary between neuronal types, contributing to differential processing and computation of synaptic or sensory inputs
• Dendritic geometry shapes intrinsic firing patterns, and coincidence detection.

Intracellular Structures

• Neurons contain cytoplasm, a thick, gelatinous solution enclosed by the cell membrane, which is composed of water, salts, and proteins, and provide a cellular environment.
• The cytoplasm of neural cells contains a highly efficient scaffolding system, the cytoskeleton comprising protein fibers of different sizes, structures and chemical composition:
  • Microtubules
    • Long unbranched hollow tubes of approx. 20 nm diameter
    • Consist of tubulin proteins as alpha and beta heterodimers
    • Primarily consist of 13 protofilaments arranged in a hollow tube.
  • Intermediate filaments - Solid filaments of 10 nm diameter
    • Consist of three neuron-specific neurofilament proteins
    • Have flexible polymer arms that repel neighboring neurofilaments
    • Determine the radius of the axon
    • Glial filaments consist of the glial fibrillary acidic protein (GFAP) instead
    • Neuronal injuries lead to concentration increase of neurofilament light chain (NFL) in cerebrospinal fluid (CSF) and blood
    • Mikrofilaments/actin-filaments
      • Small solid filaments of 5 nm diameter
      • Consist of the globular monomeric protein actin
      • Support muscle contraction in muscle cells
• Cytoskeletal elements resist deformation, but reorganize in response to external forces or internal stimuli.
• In the nucleus, genetic material is stored in the form of deoxyribonucleic acid (DNA).
• Genes are converted into proteins by specific control mechanisms
• Gene activation is initiated by gene transcription (biosynthesis of messenger RNA), and regulated by transcription factors.
• Protein and lipid biosynthesis happen in the endoplasmic reticulum (ER).
• Protein biosynthesis (translation) requires ribosomes which associate with mRNA produced in the nucleus present on the ER surface.
• Proteins are sorted and packaged into transport vesicles in the Golgi-complex.
• Some vesicles fuse with the plasma membrane, while others deliver content to intracellular sites.
• Energy for cell survival is in the mitochondria.
• Complex physiological mechanisms energy production and storage involve the biochemical breakdown of glucose leading to adenosine-tri-phosphate(ATP) production.
• This process requires oxygen where nerve cells rely on a glucose and oxygen supply from the blood stream.

Axonal Transport

• Transport between the cell body and cell processes for nerve cells.
• Important for nerve endings remote from the soma.
• Two main modes:
  • Slow axonal transport
    • Velocity range of 1-6 mm per day
    • Transports proteins of the cytoskeleton
    • Supports nerve finer growth during development and regeneration
  • Fast axonal transport
    • Velocity range of 100-400 mm/day
    • Transports larger particles like vesicles and mitochondria - Anterograde transport from the cell body to the nerve ending - Retrograde transport from the nerve ending to the cell body occurs - Fast axonal transport is guided by microtubules brought to the desired site by vehicle proteins - Anterograde transport affected by kinesin protein
    • Retrograde transport relies on dynein protein
    • Kinesin and dynein are called motor proteins
    • Use adenosine-triphosphate (ATP as energy source for transport

Neuronal Plasma Membrane Basics

• The plasma membrane is a double layer mainly of phospholipid molecules.

• It acts as a barrier that prevents the contents of the cell from mixing with extra cellular space contents.

• Plasma proteins are half proteins and half lipids.

• Phospholipids are composed of two hydrophobic fatty acid chains, linked via glycerol to hydrophilic head groups.

• Phospholipids contain a hydrophilic portion and a hydrophobic portion.

• In an aqueous environment, phospholipids spread out in one molecular layer to allow headgroups to contact the water surface while the fatty acid tails oriented to the opposite side.

• In the plasma membrane, two layers of phospholipid molecules are joined together.

• The fatty acid groups of each layer face in the hydrophobic center of the membrane, and headgroups are aligned.

• The core of the plasma membrane forms a barrier against the passage of hydrophilic molecules.

• Proteins are the second major constituent of the neuronal plasma membrane.

• Proteins consist of amino acids with an amino group and an acidic group.

• Side chains affect the physico-chemical properties of the chain with only twenty different species of amino acids are sufficient to create the protein molecules.

• Proteins are chains of differing amino acids whose fundamental properties are defined by amino acid sequence known as primary structure of the protein.

• Proteins do not exist as a long-unfolded chain of amino acids (polypeptide chain) but adopt structures depending on the amino acid sequence.

• Types of regular secondary structures:

  • α-helix: Single polypeptide chain turns regularly to make a rigid cylinder
  • β-sheet: Polypeptide chain folds back and forth upon itself
  • Random coil: Irregular structure often forms the flexible parts of a protein

• Tertiary structure/conformation gives the protein its overall three-dimensional shape that is stabilized by chemical bonds between the variable residues of amino acids.

• Conformational changes with protein kinesin coincide with specific activity

• Protein polypeptides or subunits grouped together to form a functional unit, dimer (kinesin-two subunits), tetramers (four subunits).

Structural Characterization of Proteins

• The primary protein structure is obtained through direct methods
• Secondary protein structure CD-spectroscopy.
• Secondary structures are predicted from the amino acid sequence by computer
• Tertiary structure requires biochemical and biophysical techniques: isolating the protein of interest in high purity for crystallization.
• 3D structure of proteins can be solved using x-ray crystallography.
• X-ray beams are generated in synchrotron storage rings where electrons travel
• Parallel beams of x-rays diffracted by the protein crystal and recorded on a detector/x-ray film to calculate the atomic structure of a protein

Functions and Features of Enzymes

• Molecule modified by the enzyme is called the substrate, which an enzyme modifies
• Enzyme names end in "-ase"
• An enzyme modifying DNA is called DNAse, one that modifies ATP would be called ATPase
• Enzyme substrate specificity relies on unique structural matching
• Enzyme activity can be controlled by modifying its tertiary structure to matching or mismatching its binding pocket with substrate.
• Enzymes split substrate into fragments of various sizes that are used for physiological processes
• Kinases are enzymes controls activity others proteins, by attaching or removing phosphate residues
• If the substrate is a protein, they are called protein kinases.

Membrane Proteins

• Membrane proteins are important in the function of nerve cells
• Membrane proteins control physiological interactions and signalling with other cells
• The constituents of proteins (amino acids) differ widely in side chain properties.
• Proteins can be fixed on membrane surface as so-called peripheral membrane proteins but also be deeply embedded into the hydrophobic core as so-called integral membrane proteins.
• Of the twenty amino acids used for protein biosynthesis, several have highly hydrophobic side chains
• Polypeptide chains into the core stretch into the protein stretches comprising at least 20 hydrophobic residues, then the membrane-spanning segments are identified. The number of transmembrane segments helps to classify protein families

Bioelectricity

• Electricity requires the separation of charges
• Technical electricity is based on the movement of electrons and hence works extremely fast
• Living cells electrical signalling involves the movement of ions
• Ions involved include:Anions (chloride) and Cations (sodium/potassium/calcium) enveloped by water.
• Membrane potentials are made up of the unequal distribution of charges on membrane sides since ions cannot pass through.
• Neuronal plasma membrane collects charges which is similar to a capacitator
• Neuronal plasma membrane possesses a relatively high capacitance (C=area/distance).
• The squid has proven as a biological model due to the squid giant axon which is greater than 0.5 mm in diameter.
• Squid axon size allowed recording of membrane potentials
• Hodgkin and Huxley derived basic electrical signalling information in nerve cells in the mid-20th century.
• The ion concentration of the squid axonal cytoplasm differs from extracellular fluids
• Sodium concentration (Na+) is eight-fold higher in extracellular
• Potassium (K+) occurs in twenty-fold higher concentration compared to extracellular
• Chloride is enriched in the extracellular fluid, similar as sodium to maintain steep gradients of ionic concentrations
• Active transport mechanisms balance the potassium and sodium diffusion
• The sodium-potassium pump is fuelled by ATP (two thirds of the total nerve cell energy)
• The transport process has unequal exchange of sodium and potassium: three sodium out of the cell, two potassium ions into the cell.
• Splitting of bound ATP leads to a conformational change in the intracellular part of the sodium-potassium pump.
• Sodium is occluded in a binding pocket close to membrane
• Removing ADP opens the binding pocket and releases sodium into a hydrophilic tunnel between the transmembrane segments of the pump protein
• Removal of phosphate residue splits from ATP evokes a conformational change that occludes potassium ions in a binding pocket at the extracellular face
• Unbalanced exchange-sodium to potassium ions and selective permeability make up the membrane potential.

The Resting Membrane Potential

• Plasma membrane is selectively leaky to potassium ions, potassium diffuses following previously built concentration gradient.
• Efflux of cations creates an electrical potential across the membrane
• When forces equilibrium (chemical ion gradient and counteracting electrical potential)- stable membrane potential (resting potential attained).
• The resting potential is determined by outward diffusion of potassium ions.

The Nernst equation

• Formula can calculate electrical potentials generated by diffusion of ions.
• The Goldman, Hodgkin and Katz extends on the Nernst equation

The Action Potential

• In principle it can be done two ways: injecting current increases the potential. difference leading to hyperpolarization, or reversing the polarity before injection leads to decrease called depolarization.
• Membrane potential follows and returns if the the pulse only causes small depolarization.
• Around -40 mV depolarization critical strength/threshold is reached, observing strong overshooting superimposed on passive response for milliseconds.
• Depolarization goes beyond 0 mV leading to a brief positivity, or action potential.
• If the stimulus strength is further increased, the action potential will retain size as information about stimulus intensity must be encoded by a frequency code mechanism.

Refractory Period

• If pairs of stimuli are applied minimal interval to normal response are determined
• Axons are refractory to stimulation with a absolute and a relative refractory period.

Ionic mechanisms for action potential generation

• Action potential coincides with sudden change with sodium permeability resulting in sodium influx
• Experiments show with less extracellular sodium, a delayed action potential occurs which became smaller with less concentration.

Voltage Clamp Technique

• Technique has electronic feedback system that holds the membrane potential constant
• Voltage Clamp: Two electrodes are applied, with one attached to voltage and other attached to current-passing amplifier.
• During normal action potential, membrane voltage changes, but is compensated by current injection controlled by feedback amplifier
• Compensating current equates to the cellular membrane flowing during action potential.
• Hodgkin and Huxley are able to record with the Superconducting Quantum Interference Device (SQUID)
• After setting potential to 10mv they found Biphasic Current which had both an Inwards and Outwards flow after a few milliseconds.
• By ion substitution experiments it was found that inward flow was generated by Sodium with the outward flow due to Potassium.
• Best properties of Sodium was its spontaneous reaction a few milliseconds after its initial rise.

Pharmacology of Ionic Currents

• Selectively block either the potassium or sodium
• The poison of puffer fish Tetrodotoxin (TTX) is used to block the action potential by sitting within the channel.
• Potassium can flow freely while sodium currents are stopped by a selective effect
• Tetraethylammonium (TEA) can block potassium and allow Sodium Isolation

Protein Isolation

• Toxins used for isolation and help finding protein structure
• The electric eel is most used due to sodium concentration as the starting material enriched with voltage-gated sodium channels.
• Labeled toxin is used to find high molecular weight protein that helps form the pore
• (Alpha Subunit) has 2 accessory subunits with aid of labeled Toxin the cloning and sequencing become available through gene technology
• The Amino Acid Sequence of alpha subunits have Transmembrane Segments
• The Channel has 4 Large Domains and includes 6 Segments
• The S4 Has Unusual Properties with several positive charges
• S5 and S6 have short hairpin

Single Channel Analysis

• Use the patch clamp technique developed by Erwin Neher and Bert Sakmann
• Surface of the Patch Clamp is very smooth
• Patch Pipette is put against attached cell
• Whole cell: the membrane breaks and merges both solutions, to measure the entire membrane solution
• Other techniques: Pull apart the patch pipette and measure small cell, Inside out or Right-side-out
• The Open-Probability increases with the amount of membrane Depolarization
• Measure pico Siemens and the Ampere against 1 Volt measured by Pico-Siemens.

Axonal Signal Propagation

• Action Potential moves down the axon
• Similar system performed by technical cable systems

Cable Properties

• Active Physiological mechanisms that are suppressed and most achieved by local cooling
• Similar to electrical cables, Electronical potentials along the axon are affected by Resistance
• Electronical Potentials travel micrometers due to high internal Axial Resistance

Active Signals

• Action Signals need to travel Long Distances
• Voltage Channels need to have threshold and be reached
• Sodium Channels behind Stimulation must be Refractory as the signal is only one directional
• Continuous Impulse Conduction achieved up to 25 mm per second
• Velocity is linked to surface root of diameter

The Autonomic Nervous System

• Vertebrates have a unique system which insulate using the Myelin
• Schwann cells produce singular myelin but oligodendrocytes creates to more to attach
• The myelin squeezes to make a compact multi layer called Myelin-Specific Adhesion Proteins
• MBP attach to the cytoplasmic portion against the inter cellular membrane
• Protelipid, contains multiple transmembrane and binds itself
• Mpb common with schwann while PPL us unique
• Po has single transmembrane segment to stable itself within the extracellular
• Multiple Sclerosis is the most demyelinating disease caused with MBP specific proteins, Molecular Mimicry leads to infected organisms

Functions of Glial Cells

• Astrocytes -Have many cell processes and appear like star
• Possess cytoskeleton protein (GFAP)
• Branches connect with blood vessels, and provides nutrients for cell
• Remove Neurotransmitters that are dangerous to the cell, and regulates activity of nerves
• Microglia - Small Ramified cells that occur throughout the centra nervous system
• Transfer into macrophage like cells able to clear materials that are damaged
• Capacity to exert neurotoxic for their own
• M2 Provide protect to the Neuron
• Ependymal cell- that is line the surface of the brain that secretes the liquid

Ionic mechanisms for action potential generation

• Action potential coincides with sudden change with sodium permeability resulting in sodium influx
• Experiments show with less extracellular sodium, a delayed action potential occurs which became smaller with less concentration.

Voltage Clamp Technique

• Technique has electronic feedback system that holds the membrane potential constant
• Voltage Clamp: Two electrodes are applied, with one attached to voltage and other attached to current-passing amplifier.
• During normal action potential, membrane voltage changes, but is compensated by current injection controlled by feedback amplifier
• Compensating current equates to the cellular membrane flowing during action potential.
• Hodgkin and Huxley are able to record with the Superconducting Quantum Interference Device (SQUID)
• After setting potential to 10mv they found Biphasic Current which had both an Inwards and Outwards flow after a few milliseconds.
• By ion substitution experiments it was found that inward flow was generated by Sodium with the outward flow due to Potassium.
• Best properties of Sodium was its spontaneous reaction a few milliseconds after its initial rise.

Pharmacology of Ionic Currents

• Selectively block either the potassium or sodium
• The poison of puffer fish Tetrodotoxin (TTX) is used to block the action potential by sitting within the channel.
• Potassium can flow freely while sodium currents are stopped by a selective effect
• Tetraethylammonium (TEA) can block potassium and allow Sodium Isolation

Protein Isolation

• Toxins used for isolation and help finding protein structure
• The electric eel is most used due to sodium concentration as the starting material enriched with voltage-gated sodium channels.
• Labeled toxin is used to find high molecular weight protein that helps form the pore
• (Alpha Subunit) has 2 accessory subunits with aid of labeled Toxin the cloning and sequencing become available through gene technology
• The Amino Acid Sequence of alpha subunits have Transmembrane Segments
• The Channel has 4 Large Domains and includes 6 Segments
• The S4 Has Unusual Properties with several positive charges
• S5 and S6 have short hairpin

Electical Synapses

• Electrical Synapses do no not use Nutriotransmittes, it flows through the synaptic gaps cell to cell
• Dont have Synpatic Vesicle and reduced area to have passage, the pre existing have to connect
• Connectors from 6 Subunits which allow uncharged passage
• This is Quicker then chemical channels, and allows synchronization of electrical signalling

###Synpatic Intergration

• Nerves are generated, specifically by the Initial Exon Segment, through the Acumlation
• Passive Spread across membrane so no overfiring happens, its the local depolarization
• Synaptic Potentials arrive but do not generate due to fast Succesion instead(temportal Summation)
• Many signals generate, the hillock and synpatic potential

Trafficking Receptors

• estimated half-life for the turn over
• dynamic process with chemical transmission and postsynaptic density, for glutamate and calcium, in electric ZO1,

Synaptic Plasticity

• Adapt to change to respond by increasing and decreasing for given simiutaltuon
• High Freq simiutaltuon (PTP) with lasts short term, by Calcium Acummaltuon and Tetanic Reaction
• APYISIA, they can bridge an animal through animal cellular processes
• Sensory Fibers that axonic synapses use, serotonin to attach which activates GProtein Recpetors
• The G prootein then is released the A with all subs
• This turns to protein kinase A which phosphoyslates channel leads to increased rate.

Studying Mutant Flies

• The fruit fly, a mutation can be conveniently generated as a model
• Learning mutants, mutations in the task lead to encodings for cAMP and increased or decreased levels.
• Genes can affect protein and gene expression with the transcription factors
• Lead short or long for new cells even

Long Term Potentiation

• Hippocampus, in the fore brain is associated with memory and its removal
• Internal cone tons with in the the same plane to cut
• Electro recorded is achieved
• CA3 connects to CA1 and so on
• 1973 and Lomo, showed high freq generates for ESP
• NMDA, receptors which requires high frequency

Post Synaptic

• Calcium-kinas2

Presynpatic

• Calcium

Architecture Spine

• Central nerds are surrounded the vertebral side with is segmental and connected
• Central Grey matter neurons surround the nerves Spinal roots have GANGlia Detection happens thorough touch, pain and temperature Ventral Horn, leads back to muscle called FLEX are. Flex is fast and cant be controlled such as Knee jerk!

Autonomic

• Muscle attach is stretch and activates sensory within muscle to activate.
• Antagonsit will inhibt
• Local Process done by Segment, Ascending and Descending
• Cord injuries can be caused by Motorsides from cycle accidents, and injury creates paralysis of touch and feeling. Also the inverse tempers the other side.

Rhymic Movement Control

• Relives brain of movement by walking and running to regularized and allow higher movement