BPK 306 Midterm 1

What are neural networks composed of? What do neurons contain?

Neural networks are composed of neurons connected at synapses.

Neurons have a cell body (soma), varying number of dendrites (receive input) and a single (usually) axon that extends from the cell body (carries excitatory output in the form of action potentials)

What are glial cells? How do they differ from neurons?

Other neural cells (glial cells) include: astrocytes and oligodendria in CNS and Schwann cells and satellite cells in PNS

Glie (neruoglia) differ from neurons in several ways: do not form synapses, have only one type of projection, are able to divide, and are less electrically excitable

What are astrocytes? What are oligodendria?

Both contained in CNS

Astrocytes - "astroglia" - star-shaped glia cells in the brain and spinal cord

Oligodendria - characterized by variable numbers of sheetlike processes that are each wrapped around individual axons to form the myelin sheath of nerve fibers in the central nervous system (compared with Schwann cells in the peripheral nervous system)

- forms myelin in the central nervous system

- more numerous in white matter than in gray matter.

What are Schwann cells and Satellite cells?

Both contained in the PNS

Schwann Cells - in the PNS, form the myelin sheath around neuronal axons

Satellite Cells - glial cells that cover the surface of nerve cell bodies in sensory, sympathetic and parasympathetic ganglia

- similar role to astrocytes in the CNS

What does the CNS and PNS consist of? What do afferent and efferent pathways control? What do autonomic efferents control?

CNS: Brain and Spinal Cord

PNS: Sensory, somatic, and autonomic

Afferent pathways of the PNS deliver sensory input to the CNS

Somatic efferent pathways control skeletal/striated muscle

Autonomic efferents control smooth muscle

How does the brain and spinal cord INPUT signals?

How does the brain and spinal cord OUTPUT signals?

Somatic senses, special senses, and visceral sense send signals along afferents which input information to the brain and spinal cord.

Output is sent along efferent pathways to: 1) Somatic 2) autonomic pathways

1) Somatic pathways innervated skeletal muscle

2) Autonomic pathways innervate sympathetic and parasympathetic pathways which innervate the cardiac muscle, smooth muscle, glands, and enteric nervous system (gastrointestinal tract)

What does the PNS include? What does it interfaces with?

PNS includes all peripheral nerves exiting the spinal cord (and cranium) as well sensory receptors (muscle, skin and special senses).

PNS interfaces with the CNS in specialized regions of the spinal cord (ex. dorsal and ventral horns)

Explain the anatomy of the spinal cord and which pathways/tract are present.

1) White matter (outside) and grey matter (inside)

2) Different nerve tracts carry input to of from the brain

3) The Dorsal column medial lamniscus carries sensory input on fine touch, vibration and proprioception to the brain

4) The Spinothalamic pathway carries sensory input on temperature, crude touch and pain to the brain

5) Corticospinal tracts carry motor signals from the brain to proximal (medial corticospinal) and distal muscles (lateral corticospinal) to control voluntary (and some involuntary) movement

What are the functions of the CNS?

- Gather and integrate information from PNS

- Process and perceive information from PNS

- Organize reflex and autonomic responses

- Planning and executing voluntary movements

- Higher functions like cognition, learning and memory

What are the major regions of the brain? How is the spinal cord divided?

Major regions:

- cerebrum, cerebellum, pons, medulla, brain stem

Spinal cord is divided into regions named for the vertebra at which their nerve roots enter or leave

What regions are devoted to the special senses?

Special senses have devoted regions: visual cortex, auditory cortex, olfactory cortex, gustatory cortex

What are the functional areas of the cerebral cortex?

The cerebral cortex contains sensory areas for perception, motor areas that direct movement, and association areas that integrate information

Frontal Lobe - coordinates information from other association areas, controls some behaviours

- Skeletal muscle movement - primary motor cortex, motor association area (premotor cortex)

- Prefrontal association area

Parietal Lobe - Sensory information from skin, musculoskeletal system, viscera, and taste buds

- Primary somatic sensory cortex, sensory association area

Occipital Lobe - Vision

- Visual cortex, visual association area

Temporal Lobe - Hearing

- auditory cortex, auditory association area

Gustatory cortex - taste

Olfactory cortex - smell

How do neural pathways create perception?

Neural pathways extend from sensory areas to association areas, which integrate stimuli into perception

- We perceive different wavelengths of light as colours, pressure waves as sound and chemicals binding to chemoreceptors as taste

What are Brodmann Areas?

Cerebral cortex has ubiquitous 6 layers architecture:

- Superficial layers have connections with other cortical areas

- Intermediate layers receive input from subcortical areas

- Deep areas project to subcortical areas

Cell types or 'cytoarchitecture' is different between layers

Cell layer composition and thickness varies across areas of the brain and can be used to divide cortex into Brodmann areas

- defined by cytoarchitecture, conform well to functional neuroanatomy as defined by: animal neurophysiology, human functional neuroimaging, lesion studies, brain injury in patients

Explain the coordinated network of Brodmann Areas.

Cortical columns make up the basic 'processing module' for the cerebral cortex

The cellular makeup, together with their input and output connections, define the function and activity cortical columns

Coordinated network activity gives rise to behaviour, cognition, and perception

What are functional and structural brain networks?

Functional

- neurophysiological activity is correlated between functionally related networks

- this is how complex functions arise from distributed brain networks

Structural

- complex white matter connectivity between brain areas determined inputs and outputs

- Structural network connectivity constrains communication in the brain, but this 'functional connectivity' changes depending on mental state: awake? attending to visual space? Remembering events last week?

How is a biological membrane like a capacitor? What ions contribute?

Bi-lipid Membrane - hydrophilic and hydrophobic sides creating barrier preventing flow of ions in and out

- can pull charge across its membrane

- Capacitor holds different concentrations of ions and charges (bi-lipid membrane)

- has variable resistance to flow

3 main ionic contributors: Na, K, Cl

- Na and K most critical

- capacitance of nerves and brain can change due to changes in Na and K

- Variable resistance because of channels which can be inactive, open, or deactivated to allows ions to flow through

What are the 4 elements of the Hodgkin-Huxley model (HH)?

-Mathematical model that describes how action potentials in neurons are initiated and propagated.

1) Capacitor: plates correspond to inner and outer faces of the membrane

- can hold different concentrations and charges of ions across the membrane

2) Variable Resistance - (the inverse of conductance,g): corresponds to gate ion channels shown with a switch

3) Electromotive forces: separation of charged ions across the cell membrane

- electrical charge going to try and make ion move

- looks at the difference between ICF and ECF

- Normal conditions: -70mV

4) Based on the work of Dr. Alan Hodgkin and Dr. Andrew huxley, who shared the Nobel prize in 1963 for this (and other) work

What is Selective Permeability?

Biological membranes are permeable only to molecules with certain properties

- even if open, not easy for certain ions to pass (ex. Na channel doesnt pass Ca very well)

What are the properties of ion channels? What are the types? What causes ions to diffuse?

Channels have subunits stuck into the membrane when stimulated cause confromational changes which change the structural orientation of the pore causing it to become more permeable to certain ions.

- Each subunit composed of multiple transmembrane protein domains

- Ion channels can be selective or non-selective; usually gated

- crucial: ability to open and close

Gated channels open in reponse to:

- changes in membrane potential: voltage-gated (open at certain voltages)

- binding of a chemical or hormone: ligand gated (anything bind to receptor and opens channel)

- mechanically-gated or stretch-gated channels (can be mechanically gated (touch), physically/mechanically stress)

Ions the diffuse down their gradient

- concentration and electrical gradients

What are endogenous and exogenous ligands?

Endogenous Ligands - something inside the body part of natural biology, can cause channel open (ex. NA, epinephrine, Glutamate)

Exogenous Ligands - psychoactive drugs (ex. ecstasy, cocaine)

- Chemicals taken into the body that can cross blood brain barrier then act like ligand and cause opening or closing

- Will have psychoactive properties

Explain the structure of ion channels. What are they made up of? What does each type of channel contain unique to its function?

Subunits made up of single polypeptide chains containing transmembrane proteins surround the ion channel

Ion channels are transmembrane proteins with a central pore

- some are multimers of homomeric or heteromeric subunits (ex. Kv, HCN)

- some are monomers with repeating transmembrane units

- Nav (above) and Cav pores are formed by monomers with four repeating 6 Transmembran\e spanning regions

- Some channels are regulated by intracellular proteins, second messengers or ions

- Transmembrane protein subunits arranged around a central pore

- Selectivity filter that regulated which ions can permeate the pore

- Gate that can be opened or closed (some have more than one gate)

- Voltage-gated channels have a voltage sensor

- Ligand-gated channels have a ligand binding site (extracellular or intracellular)

- some channels have binding sites for intracellular proteins or second messenger molecules

- conformational change causes opening to permeable ions

- high-resolution structure (~1 A) of a growing number of ion channels have been generated using X-ray crystallography

What are two substances that can have negative effects on channels?

TTX - very effective Na channel blocker. Brain/Nervous System cannot fire action potentials. Loose ability to move, think, regulate heart/respiration

ScTX - scorpion venom, blocks Na channel

Explain the multiple states of voltage gated ion channels.

3 conformational states:

1) Closed - resting state - most of the time

2) Open - active state - allow the movement of ion

3) Inactive - inactive state until channel has been "reset"

(deactivated - refractory state, can't re-open channel/activate)

- other more complicated states and sub-states

- change in gate status involve protein conformational changes

Why is it important for Cav to inactivate?

Ca2+is toxic, too much can lead to excitotoxicity (to much excitability can cause neurons to dies)

- Ca inactivation prevents this

Explain the gating of a voltage gated Na channel.

Depending on membrane potential, Na channel either closed, opened, or inactivated

- Na closed at resting potential (~-70mV)

Na channels are essential for AP generation

- involves two gates: activation and inactivation

- Depolarization (becoming more positive) to threshold opens the activation gate

- Inactivation gate then closes, halting ion flow

- Inactivation gate cannot be removed until the membrane repolarizes (becomes more negative again)

- certain amount of depolarization opens Na channels, cause cascade of AP

What is the resting membrane potential?

Ions are distributed unequally across the cell membrane: ex. Na+ is higher outside the cell and K+ is higher inside the cell (resting condition)

- this differential distribution results in a resting membrane potential. All cells have an RMP

- This sets the RMP to about -70mV in a typical neuron

What is the Ionic composition of a typical cell?

Na+ (mEq/L) ECF: 135-147, ICF: 10-15

K+ (mEq/L) ECF: 3.5-5.0, ICF: 120-150

Cl- (mEq/L) ECF: 95-105, ICF: 20-30 (Cl important for maintaining RMP, less critical for AP, doesn't really contribute to mechanism)

HCO3- (mEq/L) ECF: 22-28, ICF: 12-16

Ca2+ (mmol/L) ECF: 2.1-2.8 (total), 1.1-1.4 (ionized), ICF: ~ 10^-7 (ionized)

Pi (mmol/L) ECF: 1.0-1.4 (total), 0.5-0.7 (ionized), ICF: 0.5-0.7 (ionized)

What are the driving forces that move ions?

Depends in permeability - driven by chemical diffusion and electrical differences

- cell permeability changes with the opening and closing of ions channels

- The direction an ion will move is dictated by chemical and electrical gradients (capacitance prevents movement- capacitance is changed by channels)

- The 'driving force' is the difference between the membrane charge and the equilibrium potential for a given ion (Vm-Eion)

- Vm is the membrane potential and Eion is dependent on the concentration gradient for that particular ion

- This Vm-Eion represents the net electrochemical driving force acting on the ion

- For ever ion, these is some membrane potential at which it has no net movement - equilibrium potential (Eion)

How do driving forces change with differences between Vm and Eion?

For a positive ion:

When Vm-Ex is greater than 0, the ion flows outward

- Vm= -80mV. Electrical driving force weak, concentration greater

When Vm-Ex is less than 0, the ion flows inward

- Vm= -120mV, strong electrical push in, medium concentration push out, electrical charge stronger cause ion move in

When Vm-Ex is equal to 0, ion flow stops

- 4mM K+ (out), 140mM K+ (in) Vm = Ek = -94mV (balanced)

What is the Nernst Equation? What is the equilibrium potential influenced by?

Predicts the equilibrium potential for a single ion

Eion = 61/zlog([ion]out/[ion]in)

Eion is influenced by

- concentration gradient

- membrane permeability

- "61" is 2.303 RT/F

- R = gas constant = 8.3145 J/K/mol

- F = Faraday constant = 96485 C/mol

- T = absolute temperature in K

- Z - ionic charge

What is the Goldman-Hodgkin-katz equation? What are the factors?

GHK predicts membrane potential using multiple ions

- Vm us determined by combined contributions of concentration gradients and membrane permeabilities of each ion (if concentrations are known, can determine Vm)

Vm = 61*log {(Pk[K+]out/Pk[K=]in) + (Pna[Na+]out/Pna[Na+]in) + (Pcl[Cl-]out/Pcl[Cl-]in)}

Pion is permeability

- [X] is concentration of the ion

- 61 is 2.303 RT/F

Who is the hottest guy ever?

Pavlo Zerebecky is definitely the hottest

How do passive potentials relate to action potentials?

Passive potentials are the results of inputs (integration of excitatory and inhibitory impulses) that either make an AP more or less likely

What is Ohm's Law? What is its relevance to membrane potential?

Voltage = Current x Resistance -> V=IR or V=I/g or I=gV

- A stimulus (change in current) will cause a resulting change in the membrane potential (voltage)

- Stimulus (current) can be the result of input from another neuron (at synapses), it can be spontaneous (ex. in pacemaker cells), OR it can be introduced to the system using electrophysiological techniques

- axons of one cell synapse onto dendrites of another cell

- The stimulus may cause a passive (graded) potential (current flowing down electrochemical gradient), or it may cause an active (action) potential (rely on neurochemical cascade to transmit information

- Propagation can be predicted by classical cable theory

How does distance affect a passive potential?

The strength of the passive potential decreases as distance from the stimulus increases

What needs to be met for an Action Potential to fire?

Threshold for firing

- once reached you get the initiation of an Action Potential causing neuron firing and a electrochemical cascade moving down the axon

What are the 3 electrical properties of the passive potential?

3 passive properties are important in neurons:

1) Membrane resistance

2) Membrane strength

3) Intracellular longitudinal (axial) resistance along axons and dendrites

- dropping off as get further away from point of stimulation

- These properties determine how far a passive (graded) potential generated in a dendrite will travel, and whether a passive potential will result in an action potential at the axon hillock

- Myelination is also important

- Propagation of passive potentials does not involve ion channels

Briefly explain how an input of current can cause an Action Potential.

An input of current at a dendrite propagates down the dendrite and through the cell body. If it causes enough of a potential change (threshold = -55mV) at the axon hillock (part of cell where axon attaches to the soma) will result in all or nothing Action Potential

How do membrane resistance and capacitance effect the voltage response?

Membrane resistance and Membrane Capacitance determine the shape and magnitude of the voltage response

- Membrane resistance also predicts how likely a small applied current is to generate an appreciable voltage response

- current change is instantaneous but voltage change is not

What can electrotonic potentials otherwise be referred to as?

Electrotonic potentials = passive response = graded potential = subthreshold response

What are the effects of inhibitory and excitatory stimulation of neurons?

Inhibitory causes hyperpolarization making the membrane potential more negative

- causing the neuron to be less likely to fire (pulling it further away from thresholed)

Excitatory stimulus causes depolarization making the membrane potential less negative

- excited cell more likely to meet threshold (-55mV) and fire action potential

- AP's only occur when the threshold is reached for opening of voltage gated Na+ channels

How does membrane capacitance effect voltage and current change?

Capacitance - ability to hold a charge across a membrane

- Voltage change lags due to membrane capacitance

- Current change is instantaneous

- In order for a membrane depolarization to occur, injected charges need to attract or pull away charges from the membrane - the higher the capacitance of the membrane the longer this will take

Explain the length constant of Passive Potentials.

Decay of passive responses occurs in all neurons ('fall off' of voltage)

- smaller diameter fibers decay over a shorther distance (have a smaller length constant, gamma)

- gamme is defined as the distance from the site of current injection where the voltage response is 1/e of its original amplitude

- related to membrane resistance and axial resistance

- the higher the ratio of membrane resistance to axial resistance, the lower the loss of current and the greater gamma is

gamma = sqrt (Rm/Ra)

- the longer, the harder it is to propagate

How does conduction relate to length and diameter?

Conductance is inversely proportional to the length of the conductor

- directly proportional to the cross-sectional area of the conductor

Higher conductance = thicker cells, shorter cells

Lower conductance = longer cells, thinner cells

- make conduction faster by making cells bigger

What is a subthreshold graded potential?

In the context of a neuron: a neuron is receiving input (stimulus, current) from the axon (blue) of another neuron

- the current diffuses passively, and diminishes

- A graded potential starts above threshold at its initiation point but decreases in strength as it travels through the cell body. At the trigger zone (axon hillock), it is below threshold and therefore does not initiate an action potential

What happens when stimuli are inhibitory and excitatory?

Inhibitory impulse coming in at the same time as excitatory can cancel out the stimulation.

- Impulses are additive whether they are strong enough for AP or not

How are electrical signals in neurons measured?

Electrical signals in neurons and other electrically-excitable cells can be measured using electrophysiology techniques.

- changes in Vm are measured using an internal electrode plus an extracellular reference electrode

How does active propagation occur?

Axon (long tube) - threshold cause Na channels to open and cause chain reaction to open more Na downstream

- much faster then passive propagation

Explain the 9 stages of Action Potentials.

1) Resting membrane potential - Na+ channels closed

2) Depolarizing stimulus - diffusion of passive propagation to axon hillock moving it to threshold

- depolarizing stimulus may be spontaneous (ex. due to the activity of pacemaker channels), received as input from neighbouring cells (ex. during synaptic transmission), or provided experimentally

3) Membrane depolarizes to threshold. Voltage gated Na+ channels open and Na+ enters cell. Voltage gated K+ channels begin to open slowly

- at about -55mV, voltage gated Na+ channels start to open rapidly depolarizing the membrane

- pore opens becomes very selective permeable to Na

4) Rapid Na+ entry depolarizes cell

- Voltage gated Na+ channels are maximally open and Na+ continues flux in the inward direction depolarizing the membrane. At about -20mV voltage gated K+ channels start to open

5) Na+ channels close (inactivate) and slower K+ channels open (+30mV)

- Although Vm has reversed (-70mV versus +30mV) the [gradients] of Na+, K+, and Cl- remain nearly identical to what they were before the AP. Large changes in Vm require movement of only a relatively small number of ions (1 out of every 10^6 K+ for a +100mV shift)

- voltage gated K= open (more slowly)

- Na channels inactivate at peak, then close at RMP

6) K+ moves from cell to extracellular fluid

- [K+] higher inside, K+ moves out. mV returns to more negative value

- K+ channels are maximally open and K+ fluxes in the outward direction repolarizing the cell. Nav inactivation gates are closed, preventing any further movement of Na+

7) K+ channels remain open and additional K+ leaves cell, hyperpolarizing it

- K+ channels remain open and undershoot the resting potential. This is known as the 'after-hyperpolarization'. As Vm becomes more negative, K+ channels close as Vm returns to Ek

- hyperpolarization because K+ remain open, makes it much harder for cell to fure during this period (contributes to absolute refractory period)

8) Voltage-gated K+ channels close, less K+ leaks out of the cell

- K+ channels close, slowing K+ leak. Retention of K+ and slow Na+ leak into the cell brings Vm back to the resting membrane potential of -70mV

9) Cell returns to resting membane ion permeability and resting membrane potential

- Na/K ATPase maintains intracellular and extracellular concentrations

In what direction does AP propagation occur?

AP propagation is unidirectional

- conduction of APs is unidirectional - once they are triggered they only move in one diretion and they dont go backwards

- Starts at axon hillock and goes to synapse

- can do the opposite experimentally, but its unidirectional

Explain the properties of Passive Responses.

Passive Potentials:

- propagation does not involve ion channels

- stimulus is current (spontaneous, from another neuron, or injected) and results in a voltage change but no AP

- The shape of a passive membrane voltage response is rounded due to the membrane properties of capacitance and resistance

- charge leaks out of the membrane and is not replenished

- voltage change will fall away exponentially with distance from the stimulation site

- the distance at which the potential is 1/e of its original value is the length constant (gamma)

- no channel opening as move down the cell, ion and current flow

Explain the properties of Active Responses.

Action Potentials: (all or nothing, need threshold)

- involve ion channels

- initiate when threshold membrane potential is reached

- at threshold, voltage-gated Na+ channels open, resulting in a large depolarization

- Different types of voltage-gated ion channels play a role in AP propagation

- In neurons, APs are initiated at the axon hillock, whereas passive potentials produced in dendrites are summed

What is Electrophysiology? What is an Electrophysiologist?

Electrophysiology - The branch of physiology that pertains to movement of ions in biological tissues and their flow across cellular membranes, as well as the electrical recording techniques that enable the measurement of these ionic movments

- techniques of how we know RMP, etc.

An Electrophysiologist studies the electrical and biophysical properties of organs, tissues, or cells.

- Hospital definition - neurologist that looks mainly at EEGs to diagnose

What are the type of questions an Electrophysiologist might ask? (Good exam type questions)

- Which ions are important for action potential generation?

- What channels are important for action potential generation (or another type of current)?

- Which ions are the channel of interest permeable to?

- What are the biophysical characteristics of the channel?

- What are the channel kinetics?

- Which protein structures are responsible for gating? Or for gating kinetics?

- Which protein structures are responsible for voltage sensing? Ligand binding? Selectivity of the channel?

- Which ions or second messengers can regulate channel activity?

- What chemicals can block the channel?

What are the components of an Electrophysiology Rig? How does it work?

- Intracellular electrode applies current or voltage commands

- Extracellular electrode is a reference

- Membrane potential Amplifiers increase small voltage (mV) or current (pA) changes so they can be detected by low impedance electronics

- Signal generators initiate the voltage or current injections. Can change voltage and current and measure what happens

- Feedback amplifiers ensure the supplied command matches the requested command

Components:

- Oscilloscope - display to see if the cell if firing

- Computer

- Digitizer and amplifier

- Stage and Headstage - where you put the preparation

- Micromanipulators - used to move electrode with accuracy of how small the axons are

- Inverted light microscope - light it above

- Faraday Cage - electrical shielding (ex. from cell phone signals)

- Air table - makes stable surface (block floor vibrations)

What is a voltage clamp?

- Applied at the signal generator of the electrophysiology rig?

The experimenter specifies the voltage and measures the resulting current. Useful if you want to:

- study single channels or one specific type of channel (ex. properties of channels under different voltages)

- investigate which ions the channel is permeable to or the speed of opening and closing

- Test if a particular chemical or substance alters any of the biophysical properties of the channel

What is Current Clamp?

- Applied at the signal generator of the electrophysiology rig?

The set up is the same as the voltage clamp, but the experimenter injects current, and measures the resulting changes in voltage (usually action potentials). Useful if you want to:

- Study excitable cells like neurons

- Find out which ions are important for action potentials

- Test if a particular drug blocks action potentials

What is Patch Clamp Electrophysiology?

- Developed by Dr. Erwin Neyer and Dr. Bert Sakmann in 1978

- They were the first to record currents from single channels and shared the 1991 nobel prize

- Involves forming a tight (high-resistance) seal between a glass micropipette containing an electrode and the plasma membrane of a cell

- Allows experimenter to record movement of ions or changes through single channels or a population of channels

- Allows experimenter to control the extracellular and intracellular composition

- There are several different patch-clamp conformations

- micropipette that can be placed on the surface of a cell. Use suction to seal pipette to surface of cell.

- Can be so accurate that can use to look at one channel

- Can measure current flow, electrical measurements using electrodes

How can you record Ion Channel Activity using patch clamp?

- Can do a patch clamp with voltage clamp - experimenter can specify the membrane voltage, and measure current through the channels in the membrane (voltage clamp)

Current can be measured: through individual channels (single channel current), through numerous channels (macroscopic or summed channel current), depends on the properties of the channel under study

- Same amount of voltage applied, but applying pipette to different amounts of channels

- single channel generates less current then aggregate of many channels opening at once

- smaller pipette = single channel

- larger pipette = many channels

Compare the advantages and disadvantages of recording in vivo and in vitro.

In vivo advantages:

- Real-time recordings in live animals

- Highest physiological relevance

- in the animal, freely behaving, recording from the neurons

- very much like natural situation, strongly representing how neuron behave in real life situation

Disadvantages:

- technically difficult

- little to no control of intracellular or extracellular fluids

In vitro advantages:

- Isolated tissues or cells are easier to work with

- can control ECF and sometimes ICF

- more experimental control - can manipulate and experiment

- in a dish/preparation, recording from that environment

Disadvantages:

- May be less physiologically relevant

- highly unrealistic

- dont know if the answer is unrealistic to how it would behave in real life

What is the limitation of recording in vivo and in vitro?

Always stuck somewhere from having full experimental control and having full knowledge of the environment and realistic situation that we care about (ex. disease in human)

- next step to in vivo would be testing on humans

What is Heterologous Expression?

Expression of gene/protein in organism not normally expressing it

- expressing gene in an animal where its not supposed to exist, then cause expression of that gene

Advantages:

- higher expression levels result in larger currents

- control over solution composition

- ability to modify channel structure using molecular biology techniques

- high control of experimental desing

Disadvantages:

- less physiological relevance

- some aspects of channel regulation may not exist in heterologous system

- extremely unrealistic situation, might act different in frog egg then in human

- Can upregulate channels to levels that dont exist and change normal combinations of channels to see results

Can be recorded from HEK293 cells and Xenopus oocytes (frog cells)

- HEk cells are derived from embryonic human kidney cells. Used to transfect gene into frog egg to express gene

What are I-V curves?

Used to graph the current-voltage relationships

- Ohm's law; V=IR

- In patch clamp, the experimenter specifies a voltage and measures the current

- for a single ion, plotting I as a function of V (an I-V curve) will be linear

- usually more than one ion is moving at the same time making I-V relationships more complicated

- gamma (conductance) is defined by the slope of the I-V curve

What are other types of electrophysiological date that can be measured?

Channel kinetics (how fast do they open and close) - what is actually moving in the channel and how fast/slow.

- ex. if got 4 subunits with multiple transmembrane domains, what is the actual conformational change at that level causing opening or closing

Which structures are responsible for kinetics

Which ion or second messengers can regulate channel activity

In general, what is the body water content of the human body?

60-70% of the human body is water (university aged students - female 60, male 70)

- decreases with age

What are the different body fluid compartments and how are they calculated? What determines volume and Composition of each compartment?

Total Body water (TBW) - 0.6 x body weight

- made up of:

Extracellular fluid (ECF) and Intracellular Fluid (ICF)

- separated by cell membrane

ECF - 0.2 x body weight -> ~1/3 TBW - made up of interstitial and plasma (plasma in ECF because of plasma proteins. Rest of the blood made up of cells part of ICF)

- Interstitial fluid and plasma separated by capillary wall

Interstitial fluid - 3/4 of ECF - (variable volume of TBW)

- made up of bone ~2L, Dense connective tissue (ligaments, tendons) ~3L

Plasma - 1/4 of ECF

ICF - 0.4 x body weight - ~2/3 TBW - contain transcellular water (TCW) ~1L (high turnover)

- TCW - gut secretions (9L/day), aqueous humor in eye, synovial fluid in joints

Solutes

What are the major solutes and their concentrations in the ECF and ICF? (check calculations)

Na+ - ECF: 142 mMol/L, ICF: 10 mMol/L

K+ - ECF: 4 mMol/L, ICF: 140mMol/L

Ca2+ - ECF: 1.2 mMol/L, ICF: 0.00005 mMol/L

HCO3- - ECF: 28 mMol/L, ICF: 10mMol/L

Phosphates - ECF: 4 mMol/L, ICF: 75 mMol/L

Glucose - ECF: 5 mMol/L, ICF: 0 to 1.1 mMol/L

Amino Acids - ECF: 2.7 mMol/L, ICF: 1.8 mMol/L

ECF protein concentration represents concentration of whole ECF not plasma, plasma protein concentration much higher

What are the differences between sex and age for fluid volumes? What are the solute levels for young adults?

Males - higher intracellular and extracellular fluid volumes

Females - higher solid mass (everything not aqueous) volume - females have a larger SM than males because carry more fat

Age:

- Solid mass volumes increase with age (higher proportion of fat in elderly)

- Extracellular fluid volumes decrease with age

- Intracellular fluid volumes increase to highest point in adulthood then decreases in elderly

Solute levels for young adults:

K+ - 88% ICF, 9% bone, 3 % ECF

Na+ - 7% ICF, 43% bone, 50 % ECF

Ca2+ - 99.9% bone, 0.1% ECF

Mg2+ - 59.9% ICF, 40% bone, 0.1% ECF

Pi - 11.9& ICF, 88% bone, 0.1% ECF

Cl- - 5% ICF, 95% ECF

What are the values used to describe body fluid compartments? Why is the implicit assumption male? Wy is the explicit assumption a BW of 70kg?

TBW - 42L, ECF - 14L, ICF - 28L, TCW ~1L, Interstitial fluid 10.5L, Plasma 3.5L

Implicit assumption is male cause the data was generated by the US military during WWII, for treatment of wounded soldiers

Explicit assumption of BW = 70kg because this represented the average body mass of recruits in the army in the mid 1940s

What are the 3 important concepts for fluid balance? What are effective and ineffective solutes?

1) Hydrostatic & osmotic pressures (move water [pressure])

- hydrostatic generated by heart and gravity

- osmotic generated by concentration differences

2) Selective Permeability of barrier between compartments (ex. Na impermeable, Urea highly permeable)

3) Osmolarity vs tonicity ("effective solutes")

- and the major ECF & ICF solutes are...

Effective solutes - exerts osmotic force due to impermeability

Ineffective Solutes - solute that is permeable and does not generate osmotic force

How is Osmotic Pressure calculated?

1 Osmole = 1 mole of osmotically active particles

Boyle's Law: P x V = nRT

- also applies to liquids where it is called van't hoff's law

Re-arranged:

- P = nRT/V & n/V = (C)oncentration

- P = RT x dC (concentration difference across barrier)

So osmolarity = 290 mOsmol/L generates P = 5.6 meters Hg across barrier

- much higher pressure then blood pressure

- But pressure has to push against something

What are the between the: ECF & ICF, Plasma & ISF, and ICF & TCW? What are they permeable to?

ECF & ICF: Cell membrane

- Permeable to water and to some small solutes (ex urea, CO2, O2)

- urea - ineffective solute at that barrier - doesn't generate any osmotic force at that barrier

- all solutes are generally effective at cell membrane

- regulated permeability of other small solutes

Plasma & ISF (Interstitial Fluid): endothelium (also vasculature, capillaries, excluding blood brain barrier)

- freely permeable to water and small solutes

- restrictive range of solutes effective at endothelium

- Restricts passage of proteins (oncotic pressure - osmotic pressure exerted by proteins at capillary wall [colloid osmotic pressure])

ICF & TCW - epithelium (tissue layer made up of polar cells that line a lumen)

- may be permeable to water & small solutes

- solutes largely transported from one side to other

What is the difference between Osmolarity and Tonicity?

Osmolarity

- total concentration of all solutes (including ions) in a solution

- does not depend on whether the solutes can cross cell membranes or not

- absolute scale that describes the concentration of osmolytes in a single solution, Osmoles/L (osmolarity) or Osm/kg H2O (osmolarity)

- absolute number

- osmotically active particles - particles that dissolve

- urea contributes to osmolarity (dissolved solutes)

Tonicity

- related to the osmotic GRADIENT across the barrier that separates two compartments

- ONLY solutes that cannot cross the membrane contribute to this effect - they are called EFFECTIVE solutes at this barrier

- relative comparison of one solution to another in which the first solution is hypertonic, hypotonic or isotonic to the second solution

- isotonic - the same, no pressure gradient

- is hypotonic one side, fluid move to hypertonic

What are the steps to determining fluid shifts between compartments?

- Determine between which compartments the fluid shift is happening

- Determine the permeability of the membrane between both compartments

- Need to know the initial conditions: Total volume, volumes in each compartment, content of each compartment, osmolarity of each compartment (same in both)

- Determine by inspection - conceptually determine the effect of the manipulation

- Calculate initial conditions:

Total solute content = Osmolarity x TBW

ECF content = ECF osmolarity x compartment volume

ICF content = ICF osmolarity x compartment volume

Determine the manipulation - ex. if add water both compartments affect, adding solute may only affect one side (depending on permeability of membrane)

- Determine new TBW

- Determine new osmolarity = total solute content (initial) / new TBW

- Determine new ECF volume = ECF content (initial) / new osmolarity

- Determine new ICF volume = ICF content (initial) / new osmolarity

NOTE - must keep in mind volume, solute concentrations and solute contents

What are the effect of adding water to fluid shifts and adding solutes?

Adding water: new volume added to TBW (initial)

Adding solute: new concentration added to total solute content (initial) then divided by new TBW

What are the barriers of movement between plasma and Interstitial fluid? What are the pressures? What equation governs this movement?

Movement between plasma and ISF (lateral movement across capillary wall)

- What are the barriers between plasma and ISF - endothelium

- plasma and ISF are fluids which move by pressure

4 pressures: hydrostatic inside capillary, hydrostatic in interstitial, oncotic in capillary (osmotic pressure generated by plasma proteins), oncotic pressure in interstitial

- hydrostatic pressure within capillary falls along length

- Starling equation governs fluid movement across capillary wall (another formulation of Ohm's law)

What is the Starling equation? What are its components?

Jv = Kfc x [(Pcap - Pisf) - @ x (nplasma - nisf)]

Jv - volume flow, flux (flow) across membrane

Kfc - filtration coefficient. Conductance made of permeability of endothelial barrier and surface area available for movement across capillary wall

@ - sigma (fudge factor) - if 1, impermeable to plasma proteins (full effect of oncotic pressure). If smaller then 1, leaks proteins. Want as close to 1 as can get

P - hydrostatic pressure

n (pie) - abbreviation for oncotic pressure

Where is data for fluid moved taken from and why?

Data from bat wings because:

- two dimensional tissue (could transluminate with light)

- can get rid of hair easily with nair

- no surgery on animal needed

Explain the normal conditions of fluid movement from plasma to ISF.

PA - Hydrostatic pressure in arterial capillary ~ 32mmHg

Pv - Hydrostatic pressure in venous capillary ~ 8mmHg

np - plasma colloid oncotic pressure - constant at 25mmHg

- if tissue very impermeable, could mean very little fluid movement into and out of wall. Oncotic pressure will change very little along length of capillary

- significant hydrostatic pressure drop along length of capillary

- When hydrostatic pressure greater then oncotic pressure - net pressure gradient outwardly directed driving fluid out of the capillary

- When oncotic pressure greater then hydrostatic pressure - net pressure gradient inwardly directed, tend to suck fluid from interstitial into capillary

- filtration and absorption generlly equal - may be slight more absorption

Explain the pathological situation of hypoproteinemia on fluid movement.

Hypoproteinemia - low plasma protein concentration causing low plasma oncotic pressure

- hydrostatic static hasnt changed from normal conditions (Pa ~32mmHg, Pv ~8mmHg)

- Oncotic pressure dropped from 25 to 20mmHg

- much larger region/net pressure gradient moving fluid outward (much more filtration then absorption)

- classic way to get edema

- ex. hepatitis - liver not producing enough protein -> conc. drops -> lower oncotic pressure

- edema in lower extremities

- ex. Nephrotic syndrome - glomeruli loses sensitivity -> lose lots proteins in the urine

Explain the pathological situation of elevated venous pressure on fluid movement.

Elevated venous Pressure - can be caused by obstruction in the veins (thrombus, phlebitis), right side heart failure

- hydrostatic pressure rises - PA ~37mmHg, Pv ~17mmHg

- downstream obstruction cause upstream pressure rise

- oncotic pressure at normal conditions - 25mmHg

- large gradient for outward fluid movement (much more filtration then absorption)

- ex. elephantiasis

Explain the 2 equations used to determine diffusion. What governs diffusion of neutral solutes?

J=-DA(dC/dX) - diffusion equation

J- flux of solute

D - diffusion coefficient (constant)

A - surface area across which transport occurs (available for diffusion)

dC - concentration gradient along

dX - distance along which transport occurs

- (given for direction)

Diffusion Coefficient determined by:

D = -kT/6nrN

k = boltzmann's constant -> k/6n = constant

T = ambient (absolute) temperature

r = radius of diffusing molecule

N = solvent viscosity

n (pie)

Flux (J) varies directly with: temperature, surface area, concentration gradient

Flux decreases as distance transported, molecular radius, or solvent viscosity increase

How do solutes move across membranes?

- Passive (diffusive) movement of solutes can only be downhill (2nd law of thermodynamics), just need to look at concentration gradients

- uphill transport requires free energy input (usually hydrolysis ATP)

- therefore separation of a solute into two compartments requires free energy input - Na-K-ATPase (consumes half calories we consume, generates membrane potential MP used to generate AP. Use MP store and transmit information in Nervous System and muscle)

- Water and most relevant solutes are not soluble in lipids (hydrophilis)

- they require specific permeation pathways to pass through the cell membrane (ex. aquaporins - regulated water permeability)

- transport of Na or glucose through membrane very slow unless have transport pathways inserted into membrane

What is the difference in downhill, passive transport with a transporter and without?

Without - termed black membrane (lipid only?) - very slow, no kinetics (linear), no saturation, diffusion slightly increases as concentration increases

With - Facilitated Diffusion - has transporter, has kinetics, has Vmax (max rate of transport), has concentration gradient where half of the max. -> facilitates transport

- allows transport to occur at much lower concentration gradients facilitating reaction of moving solute from one side to the other

What are the different types of channels involved in transport kinetics?

Pore (non-gated channel) - natural substance. Not useful for storing and maintaining cell volume

- Pores are conduits that are always open

Channel (gated pore) - has permeability, max transfer rates and kinetics

- conduits that are gated by a door

Carrier - 2 gates, passive transport, solute driven by concentration gradient

- opening of different gates depends on if the solute bound to interior channel, rectifying transport channel, preferentially in one direction

Na-K-ATPase - hydrolysis of 1 ATP, transports 3 Na out and 2 K in

- creates NaK gradient between ECF and ICF

- very complex cycle

Explain how a carrier channel work.s

Carriers are conduits that are gated by two "doors" that are never open at the same time

1) the carrier is open to the outside

2) X enters from outside and binds at a binding site

3)The outer gate closes and X becomes occluded, still attached to its binding site

4) The inner gate opens with X still bound

5) X exits and enters the inside of the cell

6) The outer gate closes, occluding an empty binding site. This cycle can also flow in reverse order

Explain how the Na-K-ATPase works. What can inhibit it?

1) The E1 form (binding sites inside) of the pump with ATP bound, is empty and exposed to intracellular space

2) Three Na+ ions within the cell then bind to sites on E1

3) The ATP molecule is hydrolyzed, phosphorylating the alpha subunit. This phosphorylation of E1 results in a conformational change, which occludes Na+ from both the cytosolic and extracellular space

4) The conformation of the pump spontanesouly changes to E2, exposing the bound Na+ to the extracellular space, allowing it to exit the pump

5) The E2-P form of pump is empty and exposed to the extracellular space

6) Two K+ ions outside the cell bind to sites on E2

7) Phosphate leaves E2 which changes its conformation once again and occludes K+

Ouabain (plant product binds to enzyme and inhibits attachment) binds to E2-P changing the conformation of the pump and blocking the pore. The pump is nonfunctional

Give exmaples of Primary and secondary active transport, and passive transport.

Primary Active Transport - Na-K-ATPase

Secondary Active - NCX - 3 Na in, 1 Ca out - antiporter

- regulate intracellular Ca2+

Passive transport - Na channel, K channel, Glucose transporter (uniporter)

- most cells have same membrane properties everywhere

Are epithelial cells polar? What are they connected by?

Epithelial cells are polar - apical and basal surfaces have different properties

- apical faces lumen, basal faces interstitial side. Both have different transport properties

What are the difference between primary, secondary, and tertiary transport?

Primary - anything the uses ATP directly

- Basal surface - Na-K-ATPase (generates big sodium gradient, everything else runs off it) pumps Na into interstitial, and K into cell

- Apical surface - H+-ATPase - pumps H+ into lumen

Secondary

- Basal surface - Na-K-ATPase

- Apical surface - SGLT (sodium linked glucose transporter), move glucose uphill using potential energy in sodium

- Na-H exchanger (NHG) - exchagne Na for H, moving H out (lumen) in exchange for Na moving in, driven by potential energy in Na gradient

Tertiary

-Basal - Na-K-ATPase

- Apical - NHG

- CL-HCO3- exchanger (anion exchanger) - used in proximal tubule to move chloride

- NHG pump H out, create acidic environment in lumen, pump HCO3- out to neutralize

- Cl inward gradient, HCO3- and H+ recombine to form CO2 and H20

Overall, what are the functions of the renal system? How does it accomplish this?

Waste Excretion - Urea (catabolized ammonium), uric acid, bilirubin, hormone metabolites, toxins

Na+, K+ content - primary regulated route of excretion of both

- also input level regulation of Na+ (governs ECF volume)

Osmolarity regulation - whether excreting dilute or concentrated urine

Acid-base balance - metabolic acid from catabolism of protein and nucleic acids

Hormones: Angiotensin II, Erythropoietin, 1,25 dihydroxy D3 (vitamin D)

Accomplished by

- 3 processes - filtration, reabsorption, secretion

- Axial specialization (along nephron)- each segment receives product from previous segment so anatomical relationships are important

- Solute handling that requires coordinated function of multiple segments (and sometimes multiple organs)

How does the excretion of the kidney react to a sudden intake of sodium?

Lags as kidney adjusts to new intake, reflected in small changes in fluid balance.

- Things dont happen instantaneously. Pathways have to be turned up/down - protein synthesis and removal, trafficking, etc. Nor is physiological regulation perfect

Explain the toilet analogy for an Action Potential.

1) A tolilet flush is similar to an action potential

2) All-or-none property. Pushing the toilet lever harder does not produce a bigger flush. Pushing a neuron past threshold does not increase the size of the action potential

3) Refractory Phase - Until the tank is full, the toilet will not flush again. Until the Na+ channels recover, the neuron cannot produce another action potential

- once axon fires, cant fire again for certain period of time

- Na+ channels inactivate

4) Direction - Like water in a properly operating toilet, an action potential always travels in one direction only

- initiated at axon hillock, travel to presynaptic terminal

How does synaptic input cause a passive potential? What properties are important?

Neurotransmitter release from pre-synaptic terminal of one neuron onto dendrites of another neuron creates input signal (passive potential)

- direction of propagation is from the site of stimulation towards the cell body and axon hillock

- length and diameter of dendrite are important factors in the propagation of passive potentials

How does the structure of dendrites affect passive potentials?

- Dendrites are generally long and thin, but do vary in their thickness (diameter)

- part of cell physiology that plays integration of function

- Dendrites generally have HIGH resistivity

- Voltage decreases as potentials travel from the input site towards cell body

- Since V=IR, membranes with higher membrane resistance have larger voltage changes with a stimulus

What are EPSPs and IPSPs?

- Push the RMP to more positive or negative states. Allow influx of ions

EPSP (Excitatory Postsynaptic Potential):

- Transient depolarization of pastsynpatic neuron due to increased conductance of the postsynpatic membrane to Na+/K+ in response to neurotransmitter binding

- At RMP, driving force for Na+ to enter cell is higher than for K+ to leave, resulting in depolarization

- going to make a neuron more likely generate AP

- allows positive ions influx (ex. Na+, K+)

IPSP (Inhibitory Postsynaptic Potential):

- Transient hyperpolarization of postsynaptic neuron due to increased Cl- conductance of postsynptic membrane in response to neurotransmitter binding

- Cl- enters cell at RMP causing hyperpolarization

- make neuron less likely to fire

- allow in negative ion (ex. Cl), make membrane more negative

How does summation at the Axon Hillock occur?

Neuron - basic information processing module of the nervous system

- Takes multiple inputs, integrates them, gives outputs

- integration (summation) and firing depend on EPSP and IPSP -> affect whether AP fire or not

-Recording from the cell body of a neuron

-Synaptic integration occurs at the axon hillock, which has the highest density of Nav channels and therefore the lowest threshold for spike initiation. Input is additive both spatially and temporally

Spatial summation - summation in space (same location)

- more EPSP summing, reach threshold, sum together and fire AP

Temporal Summation - summation in time

- ex. dont get AP because EPSP are transient, only gonna move RMP for certain amount of time then drift back to normal

- if EPSP at different times, don't summate

What causes the depolarizing stimulus during a neuronal action potential?

- EPSP involving influx of positive ions

What factors determine axonal conduction?

- relatively simple function, all or none

- Axons can be unmyelinated or myelinated

- Myelin - wrapping of lipid based glial cells around axon making propagation faster

- Nodes of Ranvier are densely populated with Nav channels

- Nodes of ranvier - breaks in myelin

- Produces 'saltatory' conduction, in which an AP jumps from node to node

- The larger the axon, the faster the conduction velocity

- Conduction is fastest in large, myelinated axons