Bio

Question 1

Defining the nervous system

Answer 1

The nervous system is made up of a network cells specialized to sense information (inputs)
from the “outside” world (light, chemicals, temperature, gravity, touch) senory neurons
from the “inside” world (internal states, signals from other cells)
These cells (neurons) can propagate information, along axons and dendrites, in the form of electrical impulses
Graded potentials
Action potentials
Neurons convey information to each other via chemical and electrical synapses
The nervous system generates outputs that integrate sensory information to elicit behavioral and/or physiological responses
e.g., muscle contraction and movement
e.g., heart rate, digestion, temperature
e.g., feeding, courtship, locomotion

Question 2

Information flow in the nervous system

Answer 2

Let’s use visual processing in the retina to review how information
flows through neural circuits:

Receptor cells in the retina have special proteins (photoreceptors) that detect light to trigger a change in voltage across the cell membrane

Here, light is converted into an electrical signal in the receptor cells

Electrical signals travels to synapses, where they trigger the release of chemicals (neurotransmitters) that bind neurotransmitter receptors on post-synaptic Bipolar cells

chemical synapses convert electrical signals into chemical signals

The neurotransmitter receptors on Bipolar cells produce graded electrical responses
Stronger light → more NT secretion by receptor cell
More NT → stronger change in membrane voltage
(depolarization) of the Bipolar cell we are back to an
electrical signal Graded responses reach Bipolar cell nerve terminals which synapse onto Ganglion cells
Neurotransmitters are secreted again
Bind to NT receptors on Ganglion cells to once again trigger graded electrical responses

Question 3

Graded vs action potential

Answer 3

When graded depolarization is strong enough, excitable neurons (like Ganglion cells) generate action potentials (APs)
All or none electrical responses that travel very fast along nerve fibers (e.g., axons)

Graded potentials “fizzle out” over time
Ions/charges that give rise to graded potentials are quickly depleted
Graded potentials can be excitatory (depolarizing) or inhibitory (hyperpolarizing)
Excitatory Post-Synaptic Potentials = EPSPs

Inhibitory Synaptic Potentials = IPSPs

Action potentials arise when graded potentials activate voltage-gated sodium (NaV) and potassium (KV) channels
Unlike graded potentials, APs don’t dissipate
All-or-none electrical impulses that can travel up to 120 m/s along axons!

NaV channels drive membrane depolarization, while KV channels drive repolarization/hyperpolarization
Some key action potential functions in neurons:
Transmit signals along axons

Trigger pre-synaptic Ca2+ influx at nerve terminals, through voltage-gated calcium channels, causing the regulated release of neurotransmitters (exocytosis)

Question 4

Graded vs. action potentials in other cells

Answer 4

Key function in muscle:
Driving contraction
Key function in endocrine cells:
Driving secretion of hormones (exocytosis)

Question 5

Challenges associated with understanding the nervous system

Answer 5

As biologists, we often think of biological systems in genetic and chemical terms:
i.e., reactions and interactions involving DNA, RNA, proteins, membrane lipids, etc.
However, the movement of electrical signals through neural structures requires
understanding some core principles in physics:
i.e., current, voltage, resistance/conductance, capacitance, etc.
In this course we aim to integrate these two ways of thinking about biological systems, to better understand how our own nervous system operates
And by extension, how disease states emerge at the intersect between molecular biology and electrophysiology

Question 6

Early inroads into understanding the nervous system

Answer 6

We didn’t always know that neurons are cells…
The cell theory (1838):
Made possible by technological advances in microscopy- used light microscope
Based on combined observations by numerous scientists
All living organisms are composed of cells
The cell is the basic unit of structure and organization in organisms
All cells come from pre-existing cells
Didn’t see discrete cells in neuronal structure- challenge had to be overcome to see that neurons are made of cells

Question 7

The reticular theory of the nervous system

Answer 7

The reticular theory of the nervous system ignored the cell theory (1861):
i.e., The nervous system is made up of a single contiguous network (not separate cells!)
Camillo Golgi and J. von Gerlach
Ironically, Golgi invented “la reazione nera”:
i.e., “the black reaction”
a.k.a. the Golgi stain
Apply potassium dichromate and silver nitrate to fixed neurons
Randomly labels a subset of neurons in their entirety, permitting single cell tracing
Ignores cell theory- nervous system made of single network
Golgi stain- applies chemicals to fixed(dead) neurons- a small subset of neurons turn black- can trace the stucture
Hard to distinguish distinct neurons, they are overlapping, do it enough can depict specific shapes
Golgi technique shows how neurons are made of single cells

Question 8

Santiago Ramón y Cajal…

Answer 8

Visionary histologist who studied a broad range of nervous system tissues
One of the first to see the great potential of the Golgi stain
Did seminal work that disproved the reticular theory in favour of the neuron theory (along with others)
i.e., The nervous system is made up of separate cells

Used golgi stain to find neurological structure of neurons
provided evidence that neurons are made of single cells

Question 9

neuron theory/doctrine

Answer 9

The “” is a broad synthesis of principles about nerneuron theory/doctrinevous system organization, put forth by Heinrich Waldeyer
However, it was Ramón y Cajal and his colleagues who generated the data that was used for formulating the neuron doctrine…

Question 10

the neuron doctrine:
Neurons (cells) are the functional units of the nervous system

Answer 10

Tenets of the neuron doctrine:
Neurons (cells) are the functional units of the nervous system
Nerve fibers project from the soma of single neurons
The nucleus is the nutritive center of the neuron
Have varying structures
All have dendrites, axonal projection

Question 11

Tenets of the neuron doctrine
Law of dynamic polarization

Answer 11

Nerve cells have a single axon that serves
as an effector
Dendrites and cell body serve as receptor surfaces of the neuron
Directionallity to nerve propogation, passes information in one way
Dendrites= receiver
Information travels from dendrites to
axons

Question 12

Tenets of the neuron doctrine
Neurons communicate via regions of cell-cell contact (synapses).

Answer 12

Neurons communicate via regions of cell-cell contact (synapses).
Charles Scott Sherrington later put forward the concept of synaptic transmission.
Dale’s law: single neurons utilize a single type of neurotransmitter (e.g., glutamate, glycine, GABA, etc.).
Came from discovery of electrical synapses

Question 13

issues with the neuron doctrine:

Answer 13

Extensive gap junctions (electrical synapses) in the CNS encroach upon the reticular theory.
Axons can act as dendrites, while dendrites can act as axons (reciprocal synapses).
Signals can travel against “polarity” (e.g., from soma/axon to dendrites).
Some neurons can secrete more than one neurotransmitter type.
Can have so many gap junctions that the cytoplasm of the cells interact and affect one another= matches reticular theory
Can have back propogation

Question 14

understanding how the nervous system operates

Answer 14

Thing to note: our first major inroads towards understanding how the nervous system operates came from detailed studies of neuron morphology, projection, and (synaptic) connectivity.
Also, technological advances paved the way for these important discoveries (i.e., microscopy and staining techniques).

Question 15

Ramón y Cajal’s influence:

Answer 15

Understanding nervous system structure & connectivity =
understanding information flow in the nervous system. If you can map synaptic transmissions, should understand how this is done
This paradigm in thinking remains prominent….
Many researchers have devoted their careers towards establishing new or improved neural imaging and tracing techniques.
This work has been extremely fruitful towards understanding
nervous system function.
However, there is an increasing appreciation that neural “connectomics” is not enough.
analogy: if you map the entire system of roadways in a city, would that be enough to understand all the traffic patterns that flow through it at different times of the day?

Question 16

Advances in neural imaging: Florescent dyes

Answer 16

Fluorescent dyes have electrons that absorb light and emit fluorescence as they drop back down to lower energy orbitals
Unlike the Golgi stain, dyes can be used to label live neurons
Inject into neuron → diffuses or is transported throughout
Some dyes can travel through gap junctions- to track synaptic transmission
Flourescent dyes- absorb light and emit different colours of light
Can inject this into living neuron and label neuron
Some dyes can label sub-cellular structures
MitoTracker (mitochondria)
LysoTracker (lysosomes)
Hoechst and DAPI (nucleus)
You can conjugate some dyes to other molecules
e.g., toxins such as phallacidin – binds to actin and prevents it from depolymerising (labels F-actin), causing cell to die
Bovine pulmonary endothelial cells: DAPI (blue), MitoTracker (red), and BODIPY FL Phallacidin (green)

Question 17

Advances in neural imaging: Protein and mRNA localization

Answer 17

Genes can be used to label and identify neurons
“Marker” genes are only expressed in cells of interest
Detecting mRNAs in cells/tissues = in situ hybridization
Labeled antisense RNA that complements a target mRNA is used as a probe
mRNAs are mostly located in the soma… you don’t get to see
where the translated proteins end up
Detecting proteins in situ = immuno-labelling
Protein “epitopes” are used to generate antibodies
Antibodies bind target proteins and can be detected
Marker genes- genes that the molecule expresses, makes it that specific neuron
Ex glutamate and glu1
Ex gaba and gab1
Make synthetic mrna molecule- make complimentary mrna- will bind specifically to mrna in cell, apply probe and the complimentary cells will bound with the cells ecxpressing that gene and use dye \

Protein in situ- make antibodies, take binding region and replicate it then insert it into another organism, the immune system will make antibodies for this antidote
Conjugate antibodies with a label \

Question 18

Advances in neural imaging: Fluorescent proteins

Answer 18

Like fluorescent dyes, fluorescent proteins absorb light and emit fluorescence, genetically encoded
Green fluorescent protein (GFP) was cloned from the jellyfish
Aequorea victoria
Can genetically express GFP in specific neurons
Use cell-specific promoters to drive expression in neurons of interest
Labels entire neuron, like fluorescent dyes
Can create fusion proteins
i.e., “stick” the GFP protein to another protein of interest
Visualize where the protein of interest spends its time inside/outside of the cell
Take the gene and use promoter of interest
Gfp doesn’t have preference for cell
If bind it to another protein, will be binded to those proteins
Researchers have since mutated GFP (and monomeric red fluorescent protein/mRFP) to generate many different colors
Brainbow: A “technicolor” Golgi stain
Developed by Jeff Lichtman and his team at Harvard
Each ball is a neuron, express proteins in different wavelengths

Question 19

central dogma of cell biology

Answer 19

DNA in the eukaryotic cell nucleus contains various genes.
Genes that encode proteins are transcribed by RNA polymerase enzyme to generate messenger RNAs (mRNA).
Need promoters in the DNA sequence that recruit transcription factors and other machinery required for transcription.
Transcribed RNA polymers need to be processed to make mature mRNA.
mRNA is shuttled to the cytoplasm, where ribosomes drive
translation of the RNA sequences into corresponding amino acid
protein sequences.
Proteins are the general machinery of the cell (enzymes, transcription factors, ion channels etc.).
Green fluorescent protein (GFP) and its derivatives (XFPs where X
denotes different colors such as yellow: YFP) are derived from genes in cells via the central dogma.

Question 20

Brainbow

Answer 20

Brainbow: A “technicolor” Golgi stain
Developed by Jeff Lichtman and his team at Harvard
Each ball is a neuron, express proteins in different wavelengths
Brainbow in vivo:
Approach: randomly integrate XFP genes into the mouse genome, each capable of randomly recombining to express either: CFP, YFP, or RFP
Neurons will randomly express a different combination of fluorescent proteins, producing a unique fluorescence profile
Akin to a technicolor Golgi stain!
In synthetic gene have promoter region, bind promoter to 3 genes
Mouse will make rna to complete gene
Added 2enxymes- to snip stop codon. Bringing the gene beside promoter
Mutually exclusive events- which ever gets to gene first wins, get product one or two not both

Question 21

Key limitation:
Fluorescent protein

Answer 21

Neuron projections in the CNS are tightly packed… Brainbow tracing is ineffective at high magnification where synaptic connectivity takes place

Question 22

. Advances in neuronal imaging: Tracing neural circuits

Answer 22

Several tools/technologies have been developed that can be used to label synaptically-connected neurons
Static tracers are transported within the cell in either retrograde or anterograde direction, but are restricted to that cell
e.g., Cholera toxin B (retrograde), Phaseolus vulgaris-leucoagglutinin (anterograde)
e.g., several modified adeno-associated viruses (AAVs) can label single neurons in either
retrograde or anterograde directions (promote expression of a detectable gene product
Retrograde- moves backwards
Anterograde- forward
Monosynaptic tracers can jump across a single synapse
Polysynaptic tracers can jump across a series of synapses
Generally, trans-synaptic tracers are derived from modified viruses
Modified rabies and pseudorabies viruses can label neural circuits in an
retrograde manner
Modified herpes simplex viruses can label neural circuits in an anterograde
manner

Question 23

Advances in neuronal imaging:
Serial EM reconstruction and connectomics

Answer 23

Serial sections are made from fixed nervous system tissue
Sections are imaged with scanning electron microscopy
Computer algorithms identify separate cells and create a 3D reconstruction

Question 24

Advances in neuronal imaging:
Serial EM reconstruction and connect

Answer 24

Full neural connectome for C. elegans
Achieved by Nobel laureate Sydney Brenner and his team (1986)
Only 302 neurons… should be simple to understand!!
Useful to guide research, but does not explain nervous system function
Take-home: need to understand the patterns in neuronal signaling (i.e., “the traffic”) that takes place along of the connectivity map
(“the roadways”)

Question 25

The study of bioelectricity: from Galvani to optical recording

Answer 25

Mapping the neural ”connectome” of an animal provides a
framework for understanding it
Reveals potential avenues for information flow
C. elegans, a simple nematode worm with only 302 neurons, had its connectome fully mapped over 30 years ago
Researchers are still trying to understand the neural bases of C. elegans
behavior
Ultimately, we need to understand both the connectivity, and how electrical and chemical information flows along these neural circuits
Chemical (neurotransmitters at synapses, neuromodulators)
Electrical (graded potentials, action potentials, electrical synapses- gap junction, electrical single pass

Question 26

Luigi Galvani is credited with the discovery of bioelectricity (1700s)

Answer 26

We’ve known about our “electrical” nature for a very long time…
Luigi Galvani is credited with the discovery of bioelectricity (1700s)
Applied electric current to dissected frog legs → caused them to twitch!
The process was dubbed “galvanization” by fellow scientist and sometimes competitor,
Alessandro Volta

Question 27

, Giovanni Aldini (London, 1803): experiment

Answer 27

Famous public experiment by Galvani’s
nephew, Giovanni Aldini (London, 1803):
Used body of executed prisoner
Minutes after execution, Aldini performed galvanization on the body, shocking his audience…
On the first application of the process to the face, the jaws of the deceased criminal began to quiver, and the adjoining muscles were horribly contorted, and one eye was actually opened. In the subsequent part of the process the right hand was raised and clenched, and the legs and thighs were set in The use of equipment to study bioelectricity, as pioneered by Galvani and others, is now referred to as electrophysiology0 use tools to simulate biological processes using electricity

Question 28

Julius Bernstein (1902)

Answer 28

A crucial step forward was the transition from merely stimulating nerve/muscle preparations with electricity, to recording electrical activity emanating from those preparations
Developed the “differential rheotome”
Sampled membrane voltage in microsecond scale
Can record AP
1st recording of a nerve action potential!
1st recording of resting potential @ ~-60 mV
Had a nerve and a stimulation electrode and a recording electrode
This thing recording triggers an action potential, causing it to propogate and go to the recorder, taking snapshots at different points of time and pointing it together

Question 29

Hodgkin, Huxley, Cole and the voltage-clamp technique

Answer 29

Later, we’ll look at fundamental advances in electrophysiological recording techniques that helped unlock the secrets of the action potential…
Hodgkin, Huxley, Cole and the voltage-clamp technique

Question 30

Erwin Neher, Bert Sakmann and the patch-clamp technique

Answer 30

Erwin Neher and Bert Sakmann are renowned for their groundbreaking work in neurophysiology, particularly for developing the patch-clamp technique. In 1991, they were jointly awarded the Nobel Prize in Physiology or Medicine for their discoveries concerning the function of single ion channels in cells.

The patch-clamp technique, developed in the late 1970s and early 1980s, revolutionized the study of ion channels. Ion channels are essential for a variety of biological processes, including nerve signal transmission and muscle contraction. Before the invention of this technique, it was difficult to study the electrical currents associated with the opening and closing of these channels.

The patch-clamp method allows scientists to isolate and record the electrical activity of single ion channels in cell membranes. By attaching a fine-tipped glass pipette to the cell membrane, the technique measures the minute ionic currents that pass through individual channels, providing unprecedented insight into their behavior.

This technology became a powerful tool for understanding the fundamental mechanisms of cellular activity and has applications in neurobiology, pharmacology, and even in medical research related to diseases like epilepsy and cardiac arrhythmias.

Neher and Sakmann’s contributions to neuroscience laid the foundation for modern electrophysiology, significantly advancing the study of cellular communication and the workings of the nervous system.

Question 31

There are 3 general paradigms for electrophysiological:

Answer 31

Extracellular recording (single or multiple
electrodes)
Record voltage/ion fluxes along the outside surface of a cell
Intracellular sharp electrodes (single or double)
Record voltage/ion fluxes across the cell membrane
Whole-cell patch electrodes, intracellular
Record voltage/ion fluxes across the cell
membrane
Large access into cell let’s you change the intracellular saline,
has wide recording electrode

Question 32

optical recording

Answer 32

And now we also have optical recording…
Example → protein engineers combined:
GFP
The Ca2+ sensor protein calmodulin (CaM)-detects calcium in cytoplasm, helps cell knoe when influx of calcium
The M13 alpha helix of the muscle protein myosin light chain kinase
The engineered protein (GCaMP) increases fluorescence when Ca2+ levels rise
APs cause Ca2+ influx → Ca2+ activates of CaM → CaM binds the M13 helix → this pulls on the GFP protein changing its structure → increased GFP fluoresce

Question 33

Other promising optical technologies…

Answer 33

Other promising optical technologies…
Voltage-sensitive fluorescent dyes
Genetically encoded fluorescent voltage indicators
Gcamp- limited in time, doesn’t let you see action potential fully

Question 34

Why might optical techniques be advantageous over classical electrophysiological approaches

Answer 34

look at multiple ones at once

Question 35

What might be some advantages of classical electrophysiological approaches?

Answer 35

different to beat temporal resoloution

Question 36

What is electricity?

Answer 36

What is electricity?
Movement of charged particles through a conductor/resistor
Referred to as current (symbol I)
Units for current are amperes (A)

What are the charged particles that move through metal wires?
Electrons
What are the charged particles that move through aqueous solutions?
Cations (e.g., Na+, K+, Ca2+)
Anions (e.g., Cl-, PO43-, HCO3-, proteins-, nucleotides-)
Charges are counted/measured in coulombs (C)
-1C = 6.2415×1018 electrons/anions
• +1C = 6.2415×1018 cations
Current (I) = number of coulombs moving through a conductor per second
• +1 ampere of current is equivalent 6.2415×1018 positively-charged particles moving through a
conductor per second
The unit for current can be broken down as follows: 1 A = 1 C/s or -1 A = -1 C/s Current= movement of charged particles
The conductors are metal wires
Aqueous solution alone- not good conductor, add sodium- makes it better, charges particles

Question 37

Ohm’s law

Answer 37

So, electricity is the movement of charged particles (current) through a conductor…
The energy for charge movement is the voltage…
Ohm’s law defines the relationships between current, voltage, and conductance
𝑰 = 𝑽 ∗ 𝑮
I is the current in amperes (symbol A ; coulombs per second)
V is the electrical potential energy in volts (joules per coulomb)
G is the conductance of the conductor in siemens (symbol S)

Question 38

A conductor is also a resistor…

Answer 38

G= 1/R
A good conductor makes a bad resistor
A good resistor makes a bad conductor
Ohm’s law becomes…
I is the current in amperes (symbol A ; coulombs per second)
V is the electrical potential energy in volts (joules per coulomb)
R is the resistance in ohms (symbol Ω)
Every conductor has a resistance- reciprocal of conductance
Cell membrane has high resistance but the ion channels give conductance pathway for the ions

Currents that pass these ions channels follow law of physics- can use same equatiosn

Question 39

A circuit diagram of the excitable cell

Answer 39

Cell membrane is a capacitor
Resistor= ion channel
Battery= ion gradients that supply energy for biological events
Ground- electrophysoloical reporting- ground is the earth, connect it to ground to dump electrons there and avoide electric surge or can take electrons from the earth

Question 40

voltage (energy) source

Answer 40

In your cell phone, the battery contains stored electrical energy (voltage)
In excitable cells, membrane voltage originates from ion concentration gradients across the cell membrane (i.e., Na+, K+, Cl-: more on this later)
This is the energy that underlies all electrical signaling in neurons and other excitable cells!
Voltage (mV)
In membrane- have negative charges outside and positive charges inside- causing asymmetrical charges across membrane, causes movement of ions
Biological systems have energy stored as the unequal distribution of charges

Question 41

conductor/resistor

Answer 41

Applying a voltage across a conductor/resistor will produce a current
In cells, ion channels are conductors/resistors across the cell membrane
Membrane voltage will produce current through open ion channels
Some ion channels conduct cations (Na+, K+, Ca2+), and others conduct anions (Cl-, HCO3 )
The cytoplasm is also a conductor
e.g., from dendrites → soma → axons
Resistor = ion channels, pathways for conducting current
Apply voltage across resistor- pushes charges aross the channel producing a current
In a wire- we only thing of electrons- single charge that is causing\
In cell- have negative and positive ions, ions channels thus discriminate between certain ions
Axon firing ap- conducts current into cell= cytoplasm= conductor

Question 42

: capacitor

Answer 42

Understanding capacitors is key for understanding bioelectricity, because the cell membrane is a capacitor
Definition: a capacitor consists of two conductive plates (i.e., cytoplasmic and extracellular salines) separated by an insulator (the cell membrane)
The insulator must be thin so that charges can “sense” each other across the plates
Is an insulator(prevents charges from leaving) beacsue of hydrophic regions
The cytoplasm and extracellular salines= conductive plates
And cell membrane= insulator
This is a capacitor
Opposite charges accumulate along opposite plates when a voltage is applied
Ions do not travel directly through the membrane (unless through ion channels)
Still, a current results from the repulsion/attraction of ions across the membrane
Applied charges accumulate until the energy in the capacitor matches the applied voltage
Thus, capacitors can hold an applied voltage (i.e., act as a voltage source)
However, they cannot generate voltages (only borrow)
Connect voltage to capacitor, voltage pushes charge onto leaflet of capacitor, that attaches opposite charges on the other side, have current even though not physically passing charge throygh circuit
Accumulation of voltage on capacitor- eventually reaches voltage of battery, causes circuit to stop, cant charge it anymore
Charge capacitor until match voltage of battery
Battery provide energy
Capacitor take energy byt cant make it can also give off source of voltage
Voltage in cell membrane= capacitor not battery

Question 43

The ability of a capacitor to hold charges is defined by:

Answer 43

Key concept: capacitor
The ability of a capacitor to hold charges is defined by:
C = q/V
Where C is the capacitance, q is the charge in coulombs, and V is the electrical potential energy in volts
Unit: coulombs/volt or farads (F)
A system with large capacitance (C) can store a lot of charge per unit volt
C is determined by the physical properties of the insulator, the conductive plates, and the surface area of the capacitor
Cell membrane is so thin- can detect charges
Capacitance connected to area- large cell= large capacitance

Question 44

Bringing these elements together into a circuit diagram of the excitable cell:

Answer 44

Bringing these elements together into a circuit diagram of the excitable cell:
In cells, ion gradients are the batteries (V) that move charges onto the cell membrane (a capacitor or C), leading to charging on the membrane voltage (Vm).
Batteries conduct charges through ion channels that provide pathways (i.e., resistance/conductance) for charging Vm.
Capacitor= membrane voltage, action potentials, resting potential

Conductors= ion channels or cytoplasm(AP propograting)
Voltage= battery source= ion gradient
The various applied voltages (V), or batteries, each made up of a different ion gradient, are defined by The Nernst equation
Potassium battery, charges mebrane capacitor by conductiong charges through potassium channels
Membrane- capacitor place for multiple batteries and respective ion channels
Bewtter conductir= easy reduce voltage

Question 45

Voltage change equation

Answer 45

Dv/dt= icap(capacitance current)/C\

Doubling icap, doubles the change in voltage
Double capacitance, half voltage
Accumulates charge- based of size of membrane and how fast you are applying charge

Add leak channels- two places charge can go, some leave and some change the voltage- eventually becomes asymptotic, once pressure builds up, charge leaves via leaks, wont change voltage

Question 46

Resting membrane potential (RMP)

Answer 46

There are three types of “potentials” to consider:
RMP is the membrane voltage when the neuron/cell is at rest
Action and graded potentials are fluctuations from RMP caused by
the opening of ion channels
Excitation equates to transient depolarization from RMP
Inhibition equates to transient hyperpolarization from RMP
Neurons, muscle and glial cells have more polarized RMPs
i.e., -30 to -90 mV
Non-excitable cells (e.g., epithelial and red blood cells) have less polarized RMPs
i.e., -8 to -30 mV

Question 47

In all living cells (including bacteria), ion pumps & exchangers ensure that

Answer 47

K+ is more abundant inside than outside
Na+ is more abundant outside than inside
Ca2+ is very low inside (toxic: precipitates proteins and organic/inorganic anions!!)
In neurons, [Cl-] gradient can vary during development (more on this later)

Question 48

Resting membrane potential (RMP) key concepts

Answer 48

Key concepts…
Like electrons in a wire, anions and cations can be moved through a conductor (i.e., solution, ion channels) by electrical potential energy (voltage)
However, ions in solution are also displaced by random kinetic energy, leading to diffusion along their concentration gradients
Thus, the total energy for moving a given ion through an aqueous cellular environment comes from:
Voltage (e.g., the membrane voltage or Vm)
That particular ion’s concentration gradient (e.g., across the cell membrane)

Question 49

Resting membrane potential (RMP) thought experiment:

Answer 49

The ion shown (purple) is positively charged
Its concentration is higher inside compared to outside
The membrane voltage is negative inside compared to outside (a.k.a. positive outside)
Q: In terms of kinetic energy (diffusion), in which direction will the ions want to travel?
Q: In terms of voltage, in which direction will the ions want to travel?
You can imagine these two forms of energy working
with each other, or in some cases, against…
How would you manipulate the voltage to enhance the flow of ions to the outside?
How would you manipulate the voltage to reverse the flow of ions to the inside?
How would you manipulate the voltage to make the net
flow of ions in this system equal to zero?
The balance between an ion’s gradient and the membrane voltage determines its direction of flow
Let’s say there is no voltage at first…
In what direction would ions flow?
What would happen to the membrane voltage as charges flow? (remember they are positive charges)
If pumps/exchangers maintain the concentration gradient constant, without themselves affecting voltage, at which point would the flow of ions stop?
So, an ion that can diffuse across the membrane (through ion channels) can travel along its concentration gradient, driving the membrane voltage to a point where ion flow stops (like a battery!)

Question 50

Nernst equation

Answer 50

The Nernst equation calculates the voltage that a given ion gradient can produce:
Tells us the membrane voltage for a given ion concentration gradient where movement of ions stops
i.e., the equilibrium potential or Eion (a voltage that exactly opposes the diffusion energy of an ion gradient)
R - thermodynamic gas constant (8.31… Joules x mol-1 K-1)
T - absolute temperature (in degrees Kelvin (K) = °C + 273.15)
z - valence of the ion (e.g., K+ = +1, Ca2+ = +2, Cl- = -1)
F - Faraday constant (9.65 x 104 C x mol-1)
For a monovalent cation at 20oC, RT/zF ~ 25 mV

𝐸𝐾 = −74.9 mV
Voltage at which K+ ceases to flow along its concentration gradient
.k.a. If the K+ gradient is maintained by pumps/exchangers, K+ ions will continue to flow until RMP = EK

Question 51

Resting membrane potential (RMP) Revisiting the cell circuit diagram:

Answer 51

Revisiting the cell circuit diagram:
Where does membrane voltage take place?
Cell membrane = capacitor
What charges the membrane voltage/capacitor?
Different ion gradients acting as “batteries”
Which “batteries” are better able to control Vm?
Those that can flow more quickly across the cell membrane Membrane voltage- on cell membrane
Capacitors- take on voltage while batteries create a voltage

What charges the membrane- ion gradients
Potassium- pushes outside cell
NA- pushes in cell
Squigly line= resistors- leak channels
Potassium- mostr negative voltage possible, na= most positive voltage possible

Potassium leak channels- very abundant

Question 52

look more deeply into how different ionic
batteries “compete” to control Vm and set RMP

Answer 52

So, we’ve noted that ion gradients can influence Vm
Now, lets look more deeply into how different ionic
batteries “compete” to control Vm and set RMP
Ions with higher conductances are better at “pulling” Vm towards their equilibrium potentials
The RMP of most neurons and muscle cells is close to EK
Why? cells are generally more permeable to K+ at rest compared to other ions, due to an abundance of K+ leak channels
Na+ leak channels allow Na+ ions to fight against K+ to produce
more “depolarized” RMPs Ion gradients= source of energy
Ions with greater conductance- have greater control over the voltage

Question 53

Julius Bernstein’s hypothesis:

Answer 53

Potassium greater inside than outside, sodium greater outside than inside
Determine voltage via Nernst equation If the K+ gradient is maintained by pumps/exchangers, K+ ions will continue to flow until RMP = EK

Na+ gradient is maintained by
pumps/exchangers, Na+ ions will continue to flow until RM1P7= ENa 1902, Julius Bernstein hypothesized that RMP depends only on [K+]out vs. [K+]in, and hence EK
i.e., only the K+ gradient sets RMP
So, Na+ (ENa) and other ions don’t matter???

Question 54

John Zachary (J.Z.) Young developed the famous squid giant axon preparation

Answer 54

John Zachary (J.Z.) Young developed the famous squid giant axon preparation
A giant axon that innervates muscles in the squid mantle
Used for escape swimming
Mantel contracts when get the axon- axon sends string impulses to cause contraction
Doesn’t have blood in the ganglion
Galvinization- electric impulse to cause contraction
Thereafter, neurobiologists started using the squid giant axon preparation for electrophysiological experiments The axon was so large, one could easily insert a recording electrode into it along its length
Intracellular recording permits measurement of
voltage across the axon’s cell membrane Axon is so wide, can put electride in axon and connect to amplifier, and amplifier on other side is connected to ground, we can record the voltage

Question 55

Hodgkin and Horowicz used squid axon to test Bernstein’s hypothesis.

Answer 55

Hodgkin and Horowicz used this prep to test Bernstein’s hypothesis…
If Bernstein was right, changing [K+]out while keeping [K+]in constant would change EK, and RMP/membrane voltage (Vm) should follow EK
Modify outside K concentration to get different voltages

Ex more outside
If it is reliant on K concentration, change the extracellular conc and calculate Nernst value and resting voltage should equal EK value

Instead, here’s what they saw…
Everything seemed fine at high [K+]out concentrations…
But at near-physiological [K+]out, things started to go in an unexpected direction!
When concentration outside is rlly high, 500,200- voltage was very close to the EK
When the concentration outside- in physiological conditions- they found that the EK wasn’t determining factor
Why?
as EK pulls Vm away from ENa, Na+ ions are further from equilibrium, flowing faster into the cell and counteringk current

EK- tends to have stronger control of membrane voltage bc of more abundant leak channels
Change potassium conc and EK gets more negative- pulls the voltage further away from NA EK

When voltage is so far from sodium potential, the sodium equilibrium pulls more in

Question 56

“Driving Force

Answer 56

To better understand this, we need to introduce the concept of “Driving Force”:
All objects/particles need energy to move
Energy for moving charged particles is referred to as electrical potential energy or voltage
Ions in solution move in response to both voltage and
random kinetic energy (i.e., diffusion along gradients)
Thus, the net energy for moving a charged ion through solution depends on both the membrane voltage, and that particular ion’s concentration gradient
i.e., the driving force

Can use Nernst to find voltage needed tp counter diffusion gradient
If membrane voltage doesn’t equal EK, then have enough energy to move ions
Nernst equation explains this energy dichotomy:
Gives the voltage (Eion) required to exactly counter the diffusion of an ion along its concentration gradient

Q: If RMP (a.k.a. Vm ) is equal to -74.9 mV, is there net energy for moving K+ ions across the cell membrane?
No
Q: What happens when Vm is not equal to Eion?
There is energy, or driving force, for ion flow

Question 57

Combining Ohm’s Law with the Nernst equation defines the “net” energy acting on a given ion type in solution in order to produce a current…

Answer 57

Iion is the ion current or flow
Vm is the membrane voltage
Eion is the ion’s equilibrium potential
Gion is the conductance for that ion
Driving force is defined as the difference between Vm and Eion, or Vm-Eion
Iionv (Vm -Eiom) * Gion
No voltage, no current
Voltage and current directly propotional
Conductance- changing the resistance and properties

Question 58

Now let’s revisit Hodgkin and Horowicz:

Answer 58

K+ has a higher membrane conductance than Na+ (GK > GNa), permitting larger current (IK), and hence a stronger influence on Vm
However, as EK becomes more negative and pulls Vm further from ENa, the driving force for Na+ current (Vm-ENa) increases
Hence, Na+ ions can exert more control over Vm
at negative voltages to counter the K+ effect

Question 59

Exercises on driving force

Answer 59

Exercise:
EK is -70 mV
Vm = -70 mV
How much driving force is there for moving K+ across the cell membrane?
0 mV
In which direction would K+ flow?
No net flux of ions across the membrane

Exercise:
EK is -70 mV
Vm = 0 mV
How much driving force is there for moving K+ across the cell membrane?
• +70 mV
In which direction would K+ flow?
Outward: +ve values for DF equate to outward cation flow

Exercise:
EK is -70 mV
Vm = -90 mV
How much driving force is there for moving K+ across the cell membrane?
-20 mV
In which direction would K+ flow?
Inward: -ve values for DF equate to inward cation flow

Exercise:
Half of the K+ leak channels are removed from the membrane
What happens to GK?
It drops by half
What happens to the K+ current?
It drops by half
What if all of the K+ channels were removed?
No more K+ current

Exercise:
EK is -50 mV
ENa is +50 mV

Vm = 0 mV

The conductance for K+ is 10x higher than for Na+
Which ion has the larger current? → K+
In which direction would the net flux of ions be greatest?
Outward
What would happen to Vm? → Become more negative

Question 60

Goldman-Hodgkin-Katz (GHK) equation

Answer 60

Let’s apply concepts we’ve learned to model cell at rest:
Only K+ and Na+ give rise to RMP
Vm is stable at RMP
Then: I = -Ina

IK = (Vm – EK(conductance_) * GK

For voltage to stay stable- outward flux of potassium must be equal and opposite to sodium current, these currents have to be equal and opposite

Solve for conductance

If potassium is positive and its moving out faster than sodium is moving in, will make more negative, it pulls in direction of larger current until reach resting voltage

Question 61

The Goldman-Hodgkin-Katz (GHK) equation cont

Answer 61

Assumes that at rest:
[Cl-] is in equilibrium (i.e., Vm = ECl)

GK(Vm-EK) = -GNa(Vm - ENa)
If currents are matched- voltage stays constant, if not moves in direction of larger current
GK and GE- leak channels
Remove the leak channels, GK/E become zero, depends on the leaving battery- controls voltage

The Goldman, Hodgkin, Katz equation estimates Vm at rest
If Cl- is not at equilibrium (Vm ≠ ECl), there will be a Cl- current
As we’ll see later, [Cl-] gradients across the membrane change during development
So, Cl- is a bit of a “traitor” in our tug-of-war analogy

Any ion that permates the membrane(leak channel) and has a gradient- can affect the voltage

Question 62

permeability vs. conductance:

Answer 62

permeability vs. conductance:
Ions permeate/are conducted through the membrane via ion channels
The terms permeation and conductance are used interchangeably, but there
is an important difference…

Question 63

The sodium-potassium exchange pump

Answer 63

Q: How is it that K+ and Na+ ion concentration gradients remain stable, even as the ions constantly flow across the cell membrane?
Maintaining Na+ and K+ ion gradients requires ion pumps
Gene-encoded transmembrane proteins that consume energy (mostly via
hydrolysis of ATP→ hence ATPases)
Pump ions against their concentration gradients
Energy-consuming transport processes are referred to as active transport

The sodium-potassium exchange pump maintains [Na+] and [K+] gradients
a.k.a. the Na+/K+ ATPase pump
Pumps 3 Na+ out of the cell for every 2 K+ pumped in
“P-type” ATPase → Phosophorylated (P-) state during the active transport cycle
Discovered by Nobel laureate J. C. Skou (1997)

Question 64

Ion translocation cycle:

Answer 64

Ion translocation cycle:
2 K+ dissociate to the inside and 3 Na+ bind
This causes ATP to bind the pump
The pump is phosphorylated (P-type)
Conformational change exposes Na+ ions to the outside
Affinity of Na+ binding decreases
The Na+ ions dissociate and 2 K+ ions bind
K+ binding triggers dephosphorylation
Conformational change exposes K+ to the inside
Affinity of K+ binding decreases, allowing it to dissociate and the cycle to repeat
Atp is hydrolyzed
Oxygen draw electrons to themselves, causing phosphotylation

The sodium-potassium exchange pump has a dimeric structure consisting of one alpha (α) and one beta (β) subunit
Humans have 4 α genes and 4 β genes
i.e., α1-α4, β1- β4

The sodium-potassium exchange pump is
“electrogenic”
Every transport cycle results in the net extrusion of 1 positive charge
Thus, the pump can contribute to the negative RMP of the cell

Question 65

R.C. Thomas’ experiment to prove that the
sodium-potassium pump generates voltage

Answer 65

R.C. Thomas’ experiment to prove that the
sodium-potassium pump generates voltage
Impaled a single large snail neuron with 5
electrodes!
The coupled Li+ and Na+ electrodes injected ions into the cell. If one injected, the other removed cations to compensate (no effect on Vm)
The Na+-sensitive electrode monitors internal Na+
levels
The current electrode allows you to control the voltage and record currents
The other electrode allow you to record membrane voltage
Neutralized rhe positive charge with chlorine, so no depolorizatuion happends
Injection of Na+, but not Li+, caused the membrane potential to drop
Blocking the pump with ouabain blocked the Na+ effect
External K+ was also required
Lithium didn’t show a response
Brought potassium in and had injection of K- requires potassium to be outside, because of pump

Recording membrane currents revealed an outward current upon application of internal Na+

Question 66

Ouabain

Answer 66

itional notes:
Ouabain, the Na+/K+ ATPase blocker, comes from a plant
Somali word waabaayo → arrow poison
Of the two pumps most expressed in the mammalian CNS (α1 and α3), the α3 subunit is 1,000-fold more sensitive to ouabain Inject sodium get hyperpolarization- pushing sodium out more than potassium is coming in

Question 67

Ouabain

Answer 67

itional notes:
Ouabain, the Na+/K+ ATPase blocker, comes from a plant
Somali word waabaayo → arrow poison
Of the two pumps most expressed in the mammalian CNS (α1 and α3), the α3 subunit is 1,000-fold more sensitive to ouabain Inject sodium get hyperpolarization- pushing sodium out more than potassium is coming in

Question 68

Goldman-Hodgkin-Katz (GHK) equation

Answer 68

Golden-Hodgkin-Katz (GHK) equation, is used in neuroscience to calculate the membrane potential of a cell.
The GHK equation takes into account the permeability of the membrane to multiple ions and their concentrations inside and outside the cell.
.This equation models how different ions contribute to the membrane potential based on their relative permeabilities and concentrations, which is key in understanding nerve cell activity and excitability.Would you like a deeper explanation of any specific part of the equation?
In the Goldman-Hodgkin-Katz (GHK) equation, the constants involved are:1. R (Gas constant): - Value: ( R = 8.314 - It represents the ideal gas constant, which relates energy and temperature in physical systems.
. T (Temperature): - This is the absolute temperature in Kelvin. - For human physiology, it’s usually around 310 K (which is approximately 37°C or 98.6°F).3. **
F (Faraday constant)**: - Value: ( F = 96,485 \, \text{C/mol} ) - It represents the charge of one mole of electrons or ions. This constant converts between chemical and electrical quantities, allowing ionic currents to be expressed in terms of voltage.These constants ensure that the GHK equation gives a value for the membrane potential in volts (or millivolts when multiplied by appropriate scaling factors).

Question 69

driving force for potassium

Answer 69

Yes, if the driving force*for potassium is larger, potassium ions will tend to move out of the cell. The driving force for any ion is determined by the difference between the membrane potential and the equilibrium potential for that ion (also called the Nernst potential).#
The driving force for an ion is the difference between the actual membrane potential of the cell and the ion’s equilibrium potential
If the driving force is positive, ( K^+ ) ions will move out of the cell.
- If the driving force is negative, ( K^+ ) ions will move into the cell.
. Equilibrium Potential for Potassium The Nernst equation determines ( E_K ), which is the potential at which there is no net movement of ( K^+ ) across the membrane.
Typically, for potassium, ( E_K ) is around -90 mV (depends on intracellular and extracellular concentrations of ( K^+ )).3. Resting Membrane Potential (V(_m)): - The resting membrane potential of most neurons is around -70 mV. - Since ( E_K ) is usually more negative than ( V_m ), there is a tendency for potassium to move out of the cell to try to bring the membrane potential closer to ( E_K ).### Scenario of Increased Driving Force:- If the driving force for ( K^+ ) increases (e.g., the membrane potential becomes more positive, further from ( E_K )), potassium will have a stronger tendency to move out of the cell.- For example: - If the membrane potential is -70 mV, and ( E_K ) is -90 mV, potassium has a moderate driving force to leave the cell. -
If the membrane potential becomes more positive, say -50 mV, the driving force becomes larger, and more ( K^+ ) ions will move out of the cell.### Why Potassium Moves Out:- **
Concentration Gradient: - Potassium ions are generally at a higher concentration inside the cell than outside (e.g., 150 mM inside vs. 5 mM outside).
This concentration gradient drives potassium out of the cell.
- Electrical Gradient: - The inside of the cell is negatively charged relative to the outside, so this electrical gradient tends to pull ( K^+ ) ions back into the cell. - However, if the driving force (the difference between ( V_m ) and ( E_K )) is strong enough, the concentration gradient typically dominates, and ( K^+ ) ions will move out of the cell to reduce the membrane potential.###

Question 70

Driving force for potassium during AP

Answer 70

Practical Example: During an Action Potential- During the repolarization phase of an action potential, the membrane potential becomes more positive due to sodium influx. This increases the driving force for potassium, causing it to rapidly exit the cell through voltage-gated ( K^+ ) channels, helping the cell return to its resting potential.### In Summary:If the driving force for potassium increases, more potassium ions will flow out of the cell. This flow is driven by both the concentration gradient (which favors outward movement of ( K^+ )) and the electrical gradient, depending on how the membrane potential relates to the equilibrium potential of potassium.

Question 71

Driving force for sodium

Answer 71

For sodium ions (Na⁺), the same principles apply as with potassium, but the direction of movement is different due to the specific equilibrium potential and concentration gradients of sodium.
Key Points for Sodium (Na⁺):1. **Equilibrium Potential for Sodium (E({\text{Na}}))**: - The equilibrium potential for sodium is typically +60 mV (this can vary slightly depending on concentrations, but it is generally positive). - This means that if the membrane potential reaches +60 mV, sodium ions would be in equilibrium, and there would be no net movement of Na⁺ across the membrane.2. Resting Membrane Potential (V(_m)): - The resting membrane potential of most neurons is around -70 mV, which is much lower than the equilibrium potential for sodium (+60 mV). - Therefore, at rest, there is a strong driving force for sodium to move into the cell.### Driving Force for Sodium:The driving force for sodium is calculated as:[\text{Driving force for Na⁺} = V_m - E{\text{Na}}]- If the driving force is positive, sodium ions will tend to move out of the cell.- If the driving force is negative, sodium ions will tend to move into the cell.#### Example:- If the membrane potential is at the resting value (( V_m = -70 \, \text{mV} )) and ( E_{\text{Na}} = +60 \, \text{mV} ), the driving force is negative: [ -70 - (+60) = -130 \, \text{mV} ] This strong negative driving force causes sodium ions to move into the cell.- If the membrane potential becomes more positive, for example during the depolarization phase of an action potential (e.g., ( V_m = +30 \, \text{mV} )), the driving force is still negative but smaller: [ +30 - (+60) = -30 \, \text{mV} ] Sodium ions would still move into the cell, but the driving force is reduced.### Why Sodium Ions Move Into or Out of the Cell:- If the driving force is negative, sodium ions move into the cell because: - Sodium ions are at a much higher concentration outside the cell (around 145 mM outside vs. 15 mM inside). - The inside of the cell is negatively charged compared to the outside, which attracts positively charged Na⁺ ions. - Sodium wants to move inside the cell to make the membrane potential more positive and reach its equilibrium potential of +60 mV.-

Question 72

Driving force for sodium during AP

Answer 72

If the driving force is positive, sodium ions move out of the cell: - This would happen if the membrane potential exceeds the equilibrium potential of sodium (+60 mV). For example, during an overshoot in the action potential when the membrane potential becomes more positive than +60 mM, sodium would then tend to move out of the cell. - However, in most physiological situations, the driving force for sodium remains negative, so sodium typically moves into the cell.### Practical Example: Action PotentialDuring the depolarization phase of an action potential:- The membrane potential becomes more positive (closer to 0 mV or beyond) as voltage-gated Na⁺ channels open, and sodium rushes into the cell.- The driving force for Na⁺ is negative during this phase, favoring inward sodium movement.In summary:- Negative driving force for Na⁺: Sodium moves into the cell (this is typical under normal resting conditions and during the depolarization of an action potential).- Positive driving force for Na⁺: Sodium would move out of the cell (this is rare and would only occur if the membrane potential exceeds the equilibrium potential for sodium, such as in extreme conditions).

Question 73

sodium-potassium ATPase pump

Answer 73

sodium-potassium ATPase pump** (also known as the Na⁺/K⁺-ATPase or sodium-potassium pump) is an essential membrane-bound enzyme found in most animal cells. It plays a critical role in maintaining the electrochemical gradient across the cell membrane by actively transporting sodium (Na⁺) and potassium (K⁺) ions against their concentration gradients. This pump is crucial for many physiological processes, including maintaining the resting membrane potential and enabling proper nerve and muscle function.### How the Sodium-Potassium ATPase Pump Works:- Active Transport: The pump uses energy from ATP hydrolysis to transport ions against their concentration gradients.- For each cycle of the pump: - 3 Na⁺ ions are moved out of the cell. - 2 K⁺ ions are moved into the cell.- ATP hydrolysis provides the energy required for this transport. The breakdown of ATP into ADP and an inorganic phosphate (Pi) drives the conformational changes in the pump necessary to move the ions.### Steps in the Na⁺/K⁺ Pump Cycle:1. Binding of Na⁺ Ions: - The pump binds 3 Na⁺ ions from the cytoplasm (inside the cell). 2. Phosphorylation: - ATP binds to the pump and is hydrolyzed, releasing energy. This energy causes the pump to phosphorylate itself (attach a phosphate group), changing its shape. 3. Na⁺ Transport: - The conformational change in the pump moves the 3 Na⁺ ions out of the cell and releases them into the extracellular fluid. 4. Binding of K⁺ Ions: - The pump now binds 2 K⁺ ions from the extracellular fluid. 5. Dephosphorylation: - The phosphate group is removed from the pump, causing it to return to its original shape. 6. K⁺ Transport: - As the pump resets, it moves the 2 K⁺ ions into the cell, completing the cycle.### Importance of the Sodium-Potassium ATPase Pump:1. Maintaining Resting Membrane Potential: - By moving more Na⁺ out than K⁺ in (3 Na⁺ out, 2 K⁺ in), the pump creates a net negative charge inside the cell. This is vital for maintaining the resting membrane potential of cells, especially in neurons and muscle cells. 2. Electrochemical Gradient for Nerve Signals: - The Na⁺/K⁺ gradient created by the pump is crucial for the generation and propagation of action potentials in neurons. It allows for the rapid depolarization and repolarization of the membrane necessary for nerve signaling. 3. Cellular Volume Control: - The pump helps regulate osmotic balance by controlling the ion concentration inside and outside the cell, preventing excessive water from entering or leaving the cell. 4. Secondary Active Transport: - The Na⁺ gradient established by the pump is also used for secondary active transport of other substances, such as glucose and amino acids, through symporters and antiporters.### Clinical Significance:1. Ouabain and Digitalis: - The sodium-potassium pump is a target for cardiac glycosides like ouabain and digitalis, which inhibit its activity. This can increase intracellular sodium levels, leading to an increase in intracellular calcium and stronger cardiac muscle contractions. These drugs are used in treating heart conditions like heart failure. 2. Hyperkalemia or Hypokalemia: - Abnormalities in K⁺ levels (e.g., hyperkalemia – too much K⁺ in the blood, or hypokalemia – too little K⁺) can affect the function of the sodium-potassium pump, leading to disturbances in nerve and muscle function.### Energy Consumption:The sodium-potassium ATPase pump consumes a significant portion of a cell’s energy (up to 40% in neurons), reflecting its importance in maintaining proper cellular function.In summary, the sodium-potassium pump is vital for maintaining the correct ion balance across cell membranes, enabling essential processes such as electrical signaling in neurons, muscle contraction, and the regulation of cell volume.

Question 74

Mapping the neural ”connectome”

Answer 74

Mapping the neural connectome of an organism, such as the nematode Caenorhabditis elegans (C. elegans), is a crucial step toward understanding how neural circuits give rise to behavior. This comprehensive mapping provides a framework for exploring the functional roles of neurons and the pathways through which information flows. Here’s a detailed overview of the significance of connectomics in C. elegans and what we have learned from it:The Connectome of C. elegans 1. Complete Mapping: • The connectome of C. elegans, consisting of 302 neurons and over 7,000 synaptic connections, was fully mapped in the early 1980s by researchers like John White and colleagues. This monumental achievement provided the first complete neural wiring diagram of any organism. 2. Neurons and Their Functions: • The C. elegans nervous system is composed of various neuron types, including sensory neurons, motor neurons, and interneurons. Each type plays a specific role in processing information and controlling behavior. • The simplicity of the C. elegans connectome allows for more straightforward analyses of the relationship between neuronal connections and behavioral outcomes.

Question 75

Luigi Galvani (1737–1798)

Answer 75

Luigi Galvani (1737–1798) was an Italian physician, physicist, and philosopher who is widely credited with the discovery of bioelectricity in the late 18th century. His pioneering work laid the foundation for our understanding of how electrical phenomena are involved in biological processes. Here’s an overview of Galvani’s contributions and significance in the field of bioelectricity:Background and Discoveries 1. Early Experiments: • Galvani began his experiments with the dissection of frogs in the late 1700s. He observed that when the muscles of a frog’s leg were exposed to electrical sparks, they would twitch and contract. • This led him to propose that there was a form of electricity inherent in the tissues of the frog, which he termed “animal electricity.” 2. The Frog Experiment: • In one of his famous experiments, Galvani connected the frog’s leg muscles to a metal conductor. When an electric current passed through the conductor, the frog’s leg twitched as if responding to a stimulus. • He concluded that the muscle contractions were due to a special form of electricity generated within the living tissue itself, rather than an external source. 3. Galvanism: • The phenomenon he observed became known as “galvanism,” which refers to the effects of electric currents on living tissues. This term later evolved into the field of bioelectricity. • Galvani’s work highlighted the role of electrical signals in muscle contraction and nervous system function, suggesting that electrical impulses are crucial for physiological processes.

Question 76

Optical recording

Answer 76

Had a nerve and a stimulation electrode and a recording electrode
This thing recording triggers an action potential, causing it to propogate and go to the recorder, taking snapshots at different points of time and pointing it together
Optical recording is a powerful technique used in neuroscience and other biological fields to visualize and measure cellular and tissue activity in real-time. This method employs light-based technologies to monitor the dynamics of neural activity, often at a high temporal and spatial resolution. Here’s an overview of optical recording, its methods, applications, and significance in research.Overview of Optical RecordingOptical recording techniques utilize various optical methods to capture biological signals, typically fluorescence, emitted by specific indicators or dyes. These methods allow researchers to observe processes such as action potentials, calcium dynamics, and synaptic activity in living cells or tissues.Key Techniques in Optical Recording 1. Calcium Imaging: • Calcium indicators are fluorescent molecules that change their fluorescence properties in response to calcium ion concentration. When neurons become active and calcium ions enter the cell, the resulting increase in calcium concentration can be visualized using fluorescence microscopy. • Common calcium indicators include: • Fura-2: A ratiometric dye that changes fluorescence based on calcium binding. • GCaMP: A genetically encoded calcium indicator that emits fluorescence when calcium binds to it, allowing for monitoring of neuronal activity in vivo. 2. Voltage Imaging: • Voltage-sensitive dyes can be used to detect changes in the membrane potential of neurons. These dyes exhibit changes in their fluorescence in response to changes in voltage, allowing researchers to visualize action potentials and graded potentials in real time. • Recent developments in genetically encoded voltage indicators (GEVIs) have also been made, enabling more precise measurements of membrane potential changes. 3. Optogenetics: • While primarily used for manipulating neuronal activity, optogenetic techniques can also incorporate optical recording. Neurons are genetically modified to express light-sensitive ion channels (e.g., channelrhodopsins), allowing researchers to control neuronal firing with light while simultaneously monitoring activity using calcium or voltage imaging. 4. Fluorescence Microscopy: • Advanced fluorescence microscopy techniques, such as two-photon microscopy and light sheet microscopy, enable deep tissue imaging with minimal photodamage, providing high-resolution images of neuronal networks and activity in living organisms.
Advantages of Optical Recording 1. High Spatial and Temporal Resolution: • Optical recording techniques can achieve high-resolution imaging at the cellular and subcellular levels, allowing for precise measurement of neuronal activity dynamics. 2. Non-Invasiveness: • Many optical recording techniques can be performed in living tissues without causing significant damage, enabling the study of biological processes in real-time. 3. Simultaneous Monitoring of Multiple Neurons: • Advanced imaging technologies allow the simultaneous recording of activity from large populations of neurons, providing insights into network dynamics.Challenges and Limitations 1. Photobleaching: • Fluorescent indicators can lose their signal over time due to photobleaching, which can limit the duration of imaging sessions. 2. Signal-to-Noise Ratio: • Achieving high signal-to-noise ratios can be challenging, especially in densely packed neural tissues. 3. Complexity of Analysis: • The data generated from optical recordings can be vast and complex, requiring sophisticated computational tools for analysis and interpretation.

Question 77

Hodgkin, Huxley, Cole and the voltage-clamp technique

Answer 77

The Voltage-Clamp TechniqueOverview: • The voltage-clamp technique allows researchers to control the membrane potential of a neuron while measuring ionic currents flowing across the membrane. This is crucial for understanding how ions move through channels and how those movements generate electrical signals.Procedure: 1. Setting Up the Clamp: • A microelectrode is inserted into a neuron, allowing precise measurements of the membrane potential. • The experimenter can then hold the membrane potential at a desired level, independent of the natural changes that occur during neuronal activity. 2. Measuring Ionic Currents: • By applying specific voltage steps, researchers can measure the resulting ionic currents that flow through the membrane channels. This is done by using a feedback loop that adjusts the current passing through the electrode to maintain the set membrane potential. 3. Analysis of Currents: • The data collected can show how different ions contribute to the overall membrane potential and the dynamics of action potentials.Contributions to Understanding Action Potentials 1. Hodgkin-Huxley Model: • Hodgkin and Huxley used the voltage-clamp technique to identify the role of Na⁺ and K⁺ ions in generating action potentials. They discovered: • Depolarization Phase: When the neuron is stimulated, Na⁺ channels open, causing an influx of Na⁺ ions, leading to rapid depolarization. • Repolarization Phase: Following depolarization, K⁺ channels open, allowing K⁺ ions to exit the neuron, resulting in repolarization. • They formulated a set of differential equations that describe the conductance of these ions, which is still widely used in computational neuroscience today. 2. Ionic Currents: • Hodgkin and Huxley characterized the time-dependent and voltage-dependent properties of the ionic currents, showing how they contribute to the rapid rise and fall of the action potential. • They distinguished between transient inward current (primarily Na⁺) and delayed outward current (primarily K⁺), demonstrating how these currents are responsible for the action potential’s characteristic shape. 3. Significance of the Model: • The Hodgkin-Huxley model provided a quantitative framework for understanding excitability in neurons and became a foundational element in neurophysiology. It has since been expanded and adapted for various types of neurons and other excitable cells.

Question 78

Erwin Neher, Bert Sakmann and the patch-clamp technique

Answer 78

The patch-clamp technique is a powerful electrophysiological method used to study the electrical properties of individual ion channels, cells, or small cellular patches. Developed in the late 1970s and early 1980s by Erwin Neher and Bert Sakmann, this technique has become a cornerstone in cellular and molecular physiology. Here’s an overview of the patch-clamp technique, its methodology, types, applications, and significance in neuroscience and other fields.### Overview of the Patch-Clamp TechniqueThe patch-clamp technique allows researchers to measure ionic currents that flow through individual ion channels with high temporal and spatial resolution. By isolating a small patch of membrane, it provides insights into the biophysical properties of ion channels, their conductance, gating mechanisms, and pharmacological characteristics.### Key Components and Methodology1. Setup: - Microelectrodes: Glass pipettes with a very fine tip (1-2 micrometers in diameter) are filled with an electrolyte solution and used to make a tight seal with the cell membrane. - Amplifier: A high-impedance amplifier records the ionic currents while maintaining the desired voltage across the membrane patch.2. Sealing the Membrane: - The pipette is brought into contact with the cell membrane, and negative pressure is applied to create a high-resistance seal (gigaseal) between the pipette and the membrane. This minimizes the leakage of current.3. Recording Ionic Currents: - Once a seal is established, different configurations allow for various types of measurements: - Cell-Attached Mode: The pipette remains attached to the cell, allowing for the recording of currents through channels that are still part of the cell membrane. - Whole-Cell Mode: The pipette is used to rupture the membrane patch, allowing the interior of the cell to be electrically accessed. This configuration provides a way to measure total cellular currents. - Inside-Out Mode: The pipette is withdrawn, pulling a membrane patch from the cell, allowing for the study of the intracellular environment’s effects on channel activity. - Outside-Out Mode: The pipette is withdrawn slightly after breaking the membrane, resulting in a patch of membrane that is exposed to the external solution. This allows for the study of the effects of extracellular ligands or conditions on channel activity.### Applications of the Patch-Clamp Technique1. Ion Channel Characterization: - The patch-clamp technique is primarily used to study the properties of ion channels, including their conductance, gating kinetics, and ion selectivity.2. Pharmacology: - Researchers can investigate how different drugs or compounds affect ion channel function, helping in drug development and understanding the mechanisms of action for various pharmacological agents.3. Neurophysiology: - The technique is used to study the properties of neurons and synapses, including synaptic currents, action potentials, and receptor currents. This is essential for understanding neuronal excitability and signaling.4. Cellular Physiology: - Beyond neurons, the patch-clamp technique can be applied to other cell types, such as cardiac myocytes and muscle cells, to study their electrical properties and responses.5. Disease Models: - Researchers use patch-clamp recordings to investigate ion channel dysfunctions associated with various diseases, such as epilepsy, cardiac arrhythmias, and cystic fibrosis.### Advantages of the Patch-Clamp Technique1. High Resolution: - The patch-clamp technique offers exceptional temporal (millisecond) and spatial (micrometer) resolution, allowing for the detailed analysis of ion channel dynamics.2. Versatility: - Various configurations of the technique enable the study of different aspects of ion channel function and cellular activity.3. Quantitative Measurements: - The ability to precisely control the voltage and measure the resulting ionic currents allows for detailed characterization of channel properties and behavior.### Limitations of the Patch-Clamp Technique1. Technical Expertise: - Performing patch-clamp recordings requires considerable skill and training, as obtaining a high-quality seal and maintaining stable recordings can be challenging.2. Limited Sample Size: - The technique typically focuses on single channels or small patches of membrane, which may not always represent the behavior of entire cells or tissues.3. Accessibility: - The patch-clamp technique can be difficult to apply to certain cell types, particularly those that are small, round, or difficult to stabilize, such as some types of primary neurons.### SummaryThe patch-clamp technique is a fundamental tool in electrophysiology that allows researchers to explore the intricacies of ion channel function, neuronal activity, and cellular physiology. Its high-resolution capabilities have made it indispensable for advancing our understanding of the electrical properties of cells and the roles of ion channels in health and disease. The pioneering work of Erwin Neher and Bert Sakmann in developing this technique has had a profound impact on neuroscience, pharmacology, and cell biology, providing insights that continue to shape our understanding of cellular signaling and excitability.

Question 79

There are 3 general paradigms for electrophysiological

Answer 79

Wide acess- can control voltage of cell
Thank you for the clarification! Here’s a concise overview of the three general paradigms for electrophysiology based on your description:### 1. Extracellular Recording (Single or Multiple Electrodes)Overview: - This method involves placing electrodes outside the cell to record the electrical activity of neurons or groups of neurons. It can capture action potentials and local field potentials.Key Features:- Technique: Electrodes are placed near the cell membrane but do not penetrate it.- Measurement: Records voltage changes or ion fluxes occurring along the outside surface of the cell, reflecting the summed electrical activity of multiple cells or regions.Applications:- Studying network activity and synchrony in populations of neurons.- Investigating spatial and temporal patterns of electrical signals in brain regions.—### 2. Intracellular Sharp Electrodes (Single or Double)Overview: - This paradigm utilizes sharp glass microelectrodes that penetrate the cell membrane to measure the membrane potential and ionic currents directly within a single cell.Key Features:- Technique: Sharp electrodes can be inserted into the cell cytoplasm to measure intracellular voltage or currents.- Measurement: Records voltage changes and ion fluxes across the cell membrane, providing insights into the cell’s electrical properties.Applications:- Analyzing individual neuronal responses to synaptic inputs.- Investigating cellular excitability and action potential generation at the single-cell level.—### 3. Whole-Cell Patch ElectrodesOverview: - This technique involves creating a high-resistance seal between a glass pipette and the cell membrane to gain access to the intracellular environment.Key Features:- Technique: After achieving a gigaseal, the membrane patch can be ruptured to allow full access to the cell interior.- Measurement: Records voltage and ion fluxes across the cell membrane with the ability to manipulate the intracellular environment (e.g., changing ionic concentrations).Applications:- Studying the ionic currents through specific ion channels and their gating mechanisms.- Investigating intracellular signaling pathways and synaptic responses in detail.—### SummaryThese three paradigms—extracellular recording, intracellular sharp electrodes, and whole-cell patch electrodes—represent essential techniques in electrophysiology, each providing unique insights into the electrical properties of cells and networks. These methods are crucial for understanding neuronal behavior, synaptic transmission, and the overall functioning of neural circuits.

Question 80

optical recording…
protein engineers combined:

Answer 80

Took gfp and copupled it to calmodulin, when muscle binding happens it tuggs on the gfp causing confirmational change and increasing fluorescence

Optical recording is an innovative electrophysiological technique that utilizes fluorescent proteins to visualize and measure the activity of neurons and other cells in real time. Here’s a detailed overview of optical recording, particularly focusing on the example you provided regarding the engineered protein GCaMP.### Overview of Optical RecordingDefinition: Optical recording involves the use of genetically encoded fluorescent indicators to measure changes in cellular activity, such as calcium ion (Ca²⁺) concentrations, action potentials, or other physiological signals, through fluorescence imaging.### Key Components1. Fluorescent Proteins: - Green Fluorescent Protein (GFP): A widely used fluorescent marker derived from the jellyfish Aequorea victoria, which fluoresces green when exposed to specific wavelengths of light. GFP can be genetically encoded to express in specific cell types or regions of interest.2. Calcium Sensor Proteins: - Calmodulin (CaM): A calcium-binding messenger protein that detects intracellular calcium levels and plays a crucial role in various signaling pathways. When Ca²⁺ levels rise, CaM changes conformation and activates downstream effectors.3. Engineering of Indicator Proteins: - GCaMP: This is a genetically encoded calcium indicator (GECI) that combines GFP with calmodulin and a peptide derived from the myosin light chain kinase (M13 helix). GCaMP increases its fluorescence in response to elevated intracellular calcium levels.### Mechanism of GCaMP1. Calcium Influx and Action Potentials: - When a neuron generates an action potential (AP), there is an influx of calcium ions (Ca²⁺) into the cytoplasm. This increase in intracellular calcium is crucial for various cellular processes, including neurotransmitter release.2. Activation of Calmodulin: - The rise in Ca²⁺ levels leads to the binding of calcium to calmodulin (CaM). Once CaM binds Ca²⁺, it undergoes a conformational change that allows it to interact with other proteins.3. Binding to the M13 Helix: - The activated CaM binds to the M13 alpha helix, a peptide sequence derived from myosin light chain kinase. This binding results in a structural change in the GCaMP protein.4. Increased Fluorescence: - The conformational change in GCaMP alters the proximity of the GFP moiety, enhancing its fluorescence. As a result, the intensity of the emitted fluorescence increases with rising Ca²⁺ levels.### Applications of Optical Recording1. Real-Time Imaging of Calcium Dynamics: - Optical recording techniques using GCaMP allow researchers to visualize calcium dynamics in living neurons or tissues during various physiological processes, such as synaptic transmission, sensory responses, or behavioral tasks.2. Studying Neural Activity: - GCaMP and other fluorescent indicators enable the study of neuronal network activity, providing insights into how groups of neurons communicate and process information.3. In Vivo Studies: - Optical recording can be applied in living organisms, allowing for the monitoring of calcium signaling in real-time within intact neural circuits, contributing to our understanding of brain function in physiological and pathological conditions.### Advantages of Optical Recording- Non-Invasive: Optical methods are generally less invasive than traditional electrophysiological techniques, making them suitable for in vivo studies.- High Spatial Resolution: Imaging techniques provide high spatial resolution, allowing researchers to analyze activity in specific cell populations or even subcellular regions.- Real-Time Monitoring: The ability to visualize dynamic changes in intracellular calcium levels or other signals in real time enhances our understanding of cellular processes.### SummaryOptical recording, exemplified by the GCaMP calcium indicator, represents a powerful approach in electrophysiology that combines genetics, protein engineering, and imaging technology. This technique provides valuable insights into cellular signaling, neural activity, and the dynamics of calcium influx during action potentials, significantly advancing our understanding of cellular physiology and neural circuits.

Question 81

circuit diagram of the excitable cell

Answer 81

Cell membrane is a capacitor
Resistor= ion channel
Battery= ion gradients that supply energy for biological events
Ground- electrophysoloical reporting- ground is the earth, connect it to ground to dump electrons there and avoide electric surge or can take electrons from the earth
Here’s a description of a basic circuit diagram representing an excitable cell, like a neuron, which illustrates its electrical properties and components:### Circuit Diagram Components1. Resting Membrane Potential (Em): - Represents the baseline potential across the cell membrane, typically around -70 mV in neurons.2. Ion Channels: - Voltage-Gated Sodium Channels (Na⁺): Represented as resistors that open when the membrane depolarizes, allowing Na⁺ ions to flow into the cell. - Voltage-Gated Potassium Channels (K⁺): Represented as resistors that open during depolarization and repolarization, allowing K⁺ ions to flow out of the cell.3. Leak Channels: - Represented as passive resistors for Na⁺ and K⁺, allowing for a small amount of ion flow at rest, contributing to the resting potential.4. Capacitance (C): - The membrane itself acts like a capacitor, storing charge. It is represented as a capacitor in the circuit diagram. The capacitance relates to the ability of the membrane to hold an electrical charge.5. Stimulus (I): - A current source that represents an external stimulus or synaptic input that depolarizes the membrane.### Simplified Circuit DiagramHere’s a simplified textual representation of what the circuit diagram might include:

               -----                |   |                |   |   [ C ] (Capacitance: Membrane)                |   |                -----                 |                   |  ----[Rleak]----                   |    (Leak Channels)                 |                   |              ------          ---------           |      |        |         |          [ Na+   |--------|   K+    |           |      |        |         |            ------          ---------                 |                    |                       -----                    |     |                   |     |                     -----                        |                         ---                     -----                -----                (Em)

### Explanation of the Components:- Capacitance (C): Represents the ability of the cell membrane to store charge and responds to changes in voltage. When the cell is depolarized, it affects the stored charge across the membrane. - Leak Channels (Rleak): Passive resistors for Na⁺ and K⁺ that allow ions to flow across the membrane, influencing the resting membrane potential and contributing to the cell’s excitability.- Voltage-Gated Channels: - Na⁺ Channels: Upon reaching a threshold potential (usually around -55 mV), these channels open, causing rapid depolarization due to Na⁺ influx. - K⁺ Channels: After depolarization, these channels open, allowing K⁺ to exit the cell, resulting in repolarization.### Functioning of the Circuit1. Resting State: The cell maintains a resting membrane potential due to the balance of ion concentrations inside and outside the cell, primarily regulated by Na⁺ and K⁺ leak channels.2. Depolarization: When stimulated (I), if the stimulus is strong enough to reach the threshold, Na⁺ channels open, causing an influx of Na⁺ ions. This is represented as a current increase across the capacitance, leading to rapid depolarization.3. Repolarization: Following the peak of the action potential, K⁺ channels open, allowing K⁺ ions to exit the cell, causing the membrane potential to return toward its resting state.4. Return to Resting Potential: The cell eventually returns to its resting potential, and the cycle can repeat with subsequent stimuli.### SummaryThis simplified circuit diagram illustrates the basic electrical properties of an excitable cell, such as a neuron. It highlights the roles of various ion channels, capacitance, and the effects of stimuli on the cell’s membrane potential, contributing to our understanding of how action potentials are generated and propagated in excitable tissues.

Question 82

visual processing in the retina to review how information
flows through neural circuits:

Answer 82

Visual processing in the retina is a complex yet elegantly organized system that transforms light signals into electrical signals, which are then transmitted to the brain for further processing. The retina contains multiple layers of neurons, each playing a distinct role in the visual pathway. Here’s a review of how information flows through neural circuits in the retina, focusing on key components and processes involved in visual processing.### Structure of the RetinaThe retina consists of several layers of cells, each contributing to the processing of visual information:1. Photoreceptors: - The outermost layer contains rods and cones, the two types of photoreceptor cells. - Rods are sensitive to low light levels and are responsible for scotopic (night) vision, while cones are responsible for photopic (day) vision and color perception. - Phototransduction occurs here: when light hits the photoreceptors, it triggers a biochemical cascade that converts light into electrical signals.2. Bipolar Cells: - These cells connect photoreceptors to ganglion cells. They receive input from the photoreceptors and transmit signals to the ganglion cells. - Bipolar cells can be classified as ON (depolarize in response to light) and OFF (hyperpolarize in response to light) types, allowing for different pathways based on light intensity.3. Ganglion Cells: - The innermost layer of the retina, ganglion cells receive input from bipolar cells and transmit the final output of the retina to the brain via their axons, which form the optic nerve. - Each ganglion cell has a receptive field that is influenced by the surrounding photoreceptors and bipolar cells.4. Horizontal and Amacrine Cells: - Horizontal cells provide lateral inhibition, allowing for contrast enhancement and spatial resolution by integrating signals from multiple photoreceptors. - Amacrine cells connect bipolar cells to ganglion cells and play a role in complex processing, such as motion detection and temporal aspects of visual stimuli.### Information Flow in Retinal Neural Circuits1. Phototransduction: - Light photons strike the photoreceptors, causing a conformational change in the photopigments (e.g., rhodopsin in rods). This initiates a signaling cascade that hyperpolarizes the photoreceptors, reducing their release of glutamate.2. Bipolar Cell Activation: - The decrease in glutamate release alters the activity of bipolar cells. ON bipolar cells (which express mGluR6 receptors) are activated (depolarize) in response to reduced glutamate, while OFF bipolar cells (which express AMPA receptors) are inhibited (hyperpolarize).3. Ganglion Cell Response: - The depolarized ON bipolar cells then excite ON ganglion cells, whereas OFF bipolar cells excite OFF ganglion cells. This segregation allows the retina to encode both light increments and decrements. - Ganglion cells generate action potentials based on the net excitatory and inhibitory inputs they receive, with the final output conveyed through the optic nerve.4. Lateral Inhibition: - Horizontal cells enhance contrast by inhibiting adjacent photoreceptors, which sharpens the spatial resolution of the image. This lateral inhibition is crucial for edge detection and improving visual acuity.5. Temporal Processing: - Amacrine cells modulate the signals from bipolar cells to ganglion cells, allowing for temporal processing and motion detection. They can alter the firing patterns of ganglion cells in response to changes in light over time.### Output to the Brain- The axons of the ganglion cells form the optic nerve, which carries the visual information to the lateral geniculate nucleus (LGN) of the thalamus. From there, signals are relayed to the primary visual cortex (V1) for further processing.- The visual information is processed in a parallel manner, allowing for different features (e.g., color, motion, contrast) to be analyzed simultaneously.### SummaryVisual processing in the retina is a sophisticated network of neural circuits that convert light into electrical signals. Through the interplay of photoreceptors, bipolar cells, ganglion cells, and interneurons (horizontal and amacrine cells), the retina encodes critical visual information such as light intensity, contrast, and motion. This processed information is then transmitted to the brain for further interpretation, contributing to our perception of the visual world. Understanding these neural circuits is fundamental to neuroscience and has implications for visual disorders and therapies.

Question 83

Cell Theory,

Answer 83

The Cell Theory, developed in the early 19th century, is a fundamental concept in biology that describes the properties and functions of cells. The theory was formulated primarily by scientists Matthias Schleiden and Theodor Schwann in 1838, building on earlier work by Robert Hooke and Anton van Leeuwenhoek. Here are the key components of the Cell Theory:Key Principles of Cell Theory: 1. All Living Organisms Are Composed of Cells: • This principle states that all forms of life, whether unicellular or multicellular, are made up of cells. Cells are the basic unit of structure in living organisms. 2. The Cell Is the Basic Unit of Life: • Cells are the fundamental units of life. All biological functions and processes occur within cells, and they are the smallest units capable of sustaining life. This includes processes such as metabolism, energy production, and reproduction. 3. All Cells Arise from Pre-existing Cells: • This principle emphasizes that new cells are produced from the division of existing cells, rather than arising spontaneously. This idea was later supported by the work of Rudolf Virchow in the mid-19th century, who famously stated, “Omnis cellula e cellula,” meaning “every cell originates from another cell.”

Question 84

reticular theory

Answer 84

The Reticular Theory was a historical concept in biology that proposed that the nervous system was made up of a continuous network of interconnected fibers, rather than being composed of discrete, individual cells. This theory was prominent in the late 19th century and was largely associated with the work of Camillo Golgi, an Italian physician and scientist.Key Features of Reticular Theory: 1. Continuous Network: • The theory suggested that neurons and their processes formed a reticulum or mesh-like network throughout the nervous system. According to this view, the nerve cells were interconnected, allowing for the transmission of signals through a continuous protoplasmic network. 2. Camillo Golgi’s Work: • Golgi developed a staining technique (known as the Golgi stain) that allowed for the visualization of neurons in their entirety. His observations led him to conclude that nerve cells were interconnected in this continuous fashion. • Golgi’s work earned him a share of the Nobel Prize in Physiology or Medicine in 1906, along with Santiago Ramón y Cajal, who had a contrasting view. 3. Opposition by Santiago Ramón y Cajal: • Cajal, also a Nobel laureate, proposed the Neuron Doctrine, which argued that the nervous system is made up of individual cells (neurons) that communicate via synapses. He provided detailed observations of neuronal structure, emphasizing the existence of discrete cells. • Cajal’s findings were supported by further research, leading to the widespread acceptance of the Neuron Doctrine over Reticular Theory. 4. Decline of Reticular Theory: • As more advanced techniques in microscopy and staining were developed, it became clear that neurons were indeed individual cells that communicated through specialized junctions (synapses). • The discovery of neurotransmitters and the understanding of synaptic transmission further invalidated the Reticular Theory.

Question 85

Neuron Theory, also known as the Neuron Doctrine,

Answer 85

Neuron Theory, also known as the Neuron Doctrine, is a fundamental principle in neuroscience that states that the nervous system is composed of discrete, individual cells called neurons. This theory revolutionized the understanding of the structure and function of the nervous system and is widely accepted in the field today. Here are the key components and historical context of the Neuron Theory:Key Components of Neuron Theory: 1. Individual Cells: • The Neuron Theory posits that neurons are the basic structural and functional units of the nervous system. Each neuron operates independently and is separated from other neurons by small gaps (synapses). 2. Neuronal Structure: • Neurons consist of three main parts: • Cell Body (Soma): Contains the nucleus and organelles, responsible for the cell’s metabolic activities. • Dendrites: Branched extensions that receive signals from other neurons and convey them toward the cell body. • Axon: A long, thin projection that transmits electrical impulses (action potentials) away from the cell body to other neurons or target tissues. 3. Communication through Synapses: • Neurons communicate with each other via synapses, where neurotransmitters are released from the axon terminal of one neuron and bind to receptors on the dendrites of another neuron, facilitating signal transmission. 4. Functional Specialization: • Neurons can be functionally classified into different types based on their roles, such as sensory neurons (transmit sensory information), motor neurons (control muscle movements), and interneurons (connect and process information between other neurons).

Question 86

Fluorescent dyes

Answer 86

Fluorescent dyes are powerful tools in neuroscience and cell biology for visualizing and studying live neurons and their activities. Here’s a detailed overview of how these dyes work, their applications, and their advantages over traditional staining methods like the Golgi stain:### Mechanism of Action:1. Fluorescent Properties: - Fluorescent dyes contain molecules with electrons that can absorb specific wavelengths of light (usually ultraviolet or visible light). - When these electrons absorb energy, they move to higher energy orbitals. As they return to their original, lower energy state, they emit light at a longer wavelength, a phenomenon known as fluorescence.2. Absorption and Emission: - The excitation light source (such as a laser or UV lamp) stimulates the dye, causing it to emit fluorescence. This emitted light can be detected using specialized imaging techniques, such as fluorescence microscopy.### Advantages Over Golgi Stain:1. Live Cell Labeling: - Unlike the Golgi stain, which primarily labels fixed cells and tissues, fluorescent dyes can be used to label live neurons. This allows researchers to study dynamic processes, such as neuronal activity, development, and interactions in real-time.2. Injection and Diffusion: - Fluorescent dyes can be injected directly into neurons, where they diffuse or are transported throughout the cell. This enables researchers to visualize the entire structure of the neuron, including dendrites and axons, and study their morphology and function.3. Tracking Synaptic Transmission: - Some fluorescent dyes are capable of traveling through gap junctions, which are specialized connections between neighboring neurons that allow for direct electrical and chemical communication. - By using these dyes, researchers can track synaptic transmission and study how signals propagate between connected neurons, providing insights into neural circuits and network activity.### Applications in Neuroscience:1. Visualizing Neuronal Morphology: - Fluorescent dyes can be used to visualize the structure of neurons, including dendritic trees, axonal pathways, and synaptic terminals.2. Studying Neuronal Activity: - Certain fluorescent dyes can respond to changes in ion concentrations (such as calcium or sodium ions), allowing researchers to monitor neuronal activity and signaling in real time.3. Tracing Neural Circuits: - By injecting fluorescent dyes into specific brain regions or cell types, researchers can trace the pathways of neurons, mapping out neural circuits and their connections.4. Assessing Cellular Processes: - Fluorescent dyes can also be used to study various cellular processes, such as migration, division, and apoptosis (programmed cell death), in live or fixed tissues.### Summary:Fluorescent dyes are invaluable tools in modern neuroscience, allowing for the dynamic visualization and study of live neurons. Their ability to label living cells, diffuse throughout neuronal structures, and travel through gap junctions provides unique insights into neuronal function, synaptic transmission, and the intricate networks that underlie brain activity. This contrasts with traditional methods like the Golgi stain, making fluorescent dyes essential for advancing our understanding of neural processes and circuitry.

Question 87

Protein and mRNA localization

Answer 87

Marker genes- genes that the molecule expresses, makes it that specific neuron
Ex glutamate and glu1
Ex gaba and gab1
Make synthetic mrna molecule- make complimentary mrna- will bind specifically to mrna in cell, apply probe and the complimentary cells will bound with the cells ecxpressing that gene and use dye \

Protein in situ- make antibodies, take binding region and replicate it then insert it into another organism, the immune system will make antibodies for this antidote
Conjugate antibodies with a label

The use of genetic techniques to label and identify neurons has become an essential approach in neuroscience research. These methods allow researchers to study the expression and localization of specific genes and proteins within neurons. Here’s a detailed overview of how marker genes, in situ hybridization, and immuno-labeling are utilized in neuroscience:### Marker Genes1. Definition: - Marker genes are genes that are specifically expressed in certain cell types, allowing researchers to identify and label those cells of interest. For instance, genes may be chosen that are exclusively expressed in particular types of neurons.2. Applications: - Marker genes can be used to trace specific neuronal populations, study their development, and understand their functions within the nervous system.### In Situ Hybridization1. Purpose: - In situ hybridization (ISH) is a technique used to detect specific mRNA molecules within cells or tissues. It helps to visualize the spatial expression patterns of genes.2. Process: - A labeled antisense RNA probe is designed to be complementary to the target mRNA. - This probe hybridizes to the mRNA within the cells, allowing for detection. - The labeled probes can be tagged with fluorescent markers or other labels to visualize the hybridization.3. Limitations: - While ISH effectively shows where mRNAs are localized (mostly in the soma), it does not provide information about where the corresponding proteins end up after translation, as proteins may be transported to various cellular compartments or released at synapses.### Immuno-labeling1. Purpose: - Immuno-labeling (or immunohistochemistry) is a method used to detect specific proteins within cells or tissues. This technique allows researchers to visualize protein distribution and localization.2. Process: - Antibodies are generated against specific protein epitopes (short sequences of amino acids unique to the protein). These antibodies are designed to bind specifically to the target protein. - After tissue sections are prepared, the antibodies are applied, where they bind to the target proteins. - Detection can be achieved using various methods, such as fluorescent tags, enzyme-linked detection, or colorimetric assays, enabling visualization of the protein localization.3. Advantages: - Immuno-labeling provides direct information about the presence and localization of proteins, allowing researchers to see not just where the proteins are synthesized (in the soma) but also where they are functioning within the cell (in dendrites, axons, synapses, etc.).### SummaryTogether, these techniques—marker genes, in situ hybridization, and immuno-labeling—provide powerful tools for studying the identity and function of neurons. By combining mRNA detection (which shows gene expression) with protein detection (which shows protein localization), researchers can gain a more comprehensive understanding of neuronal identity, function, and connectivity within the nervous system. This dual approach is crucial for elucidating the roles of specific neuronal populations in health and disease, as well as understanding complex neural networks.

Question 88

Fluorescent proteins

Answer 88

Fluorescent proteins are proteins that emit fluorescence when exposed to specific wavelengths of light. They are extensively used in biological research for visualizing cellular structures, tracking processes in live cells, and studying protein interactions. Here’s a detailed overview of fluorescent proteins, their characteristics, applications, and some common examples:### Key Characteristics1. Fluorescence Mechanism: - Fluorescent proteins contain chromophores, which are the light-absorbing components. When exposed to excitation light (often UV or blue light), the chromophore absorbs energy, elevating its electrons to a higher energy state. As the electrons return to their ground state, they emit light at a longer wavelength, resulting in fluorescence.2. Color Variability: - Fluorescent proteins come in various colors, enabling the simultaneous labeling of multiple targets. The color of fluorescence depends on the specific chromophore and its structure.3. Genetic Encoding: - These proteins can be genetically encoded and expressed in specific cells or tissues, allowing researchers to visualize dynamic biological processes without the need for external dyes.### Common Examples1. Green Fluorescent Protein (GFP): - Originally derived from the jellyfish Aequorea victoria, GFP emits green fluorescence when exposed to blue or UV light. It has become a widely used tool in molecular and cellular biology.2. Enhanced GFP (EGFP): - A modified version of GFP with improved brightness and stability, allowing for better visualization in live-cell imaging.3. Red Fluorescent Protein (RFP): - Derived from coral species, RFP emits red fluorescence. Variants include mCherry and mKate, which are commonly used for multicolor labeling.4. Yellow Fluorescent Protein (YFP): - A variant that emits yellow fluorescence, used in combination with GFP and RFP for multiplex imaging.5. Blue Fluorescent Protein (BFP): - Provides blue fluorescence but is less commonly used due to lower brightness compared to GFP.### Applications in Research1. Live-Cell Imaging: - Fluorescent proteins enable real-time visualization of cellular processes such as cell division, migration, and signaling pathways without disrupting the cell.2. Gene Expression Studies: - By linking fluorescent proteins to specific promoters, researchers can study the expression patterns of genes in different cell types or developmental stages.3. Protein Localization: - Fusing fluorescent proteins to proteins of interest allows researchers to track their localization within cells, providing insights into their functions and interactions.4. FRET (Fluorescence Resonance Energy Transfer): - FRET is a technique that uses two different fluorescent proteins to study interactions between proteins. When the two proteins are in close proximity, energy transfer occurs, indicating an interaction.5. Tracing Neural Circuits: - In neuroscience, fluorescent proteins are used to label specific neuronal populations, allowing researchers to study their connectivity and functions within neural circuits.### Advantages- Non-invasive: Fluorescent proteins allow for the study of live cells without the need for fixation or destruction of the sample.- Stability: Once expressed, fluorescent proteins provide a stable signal over time, facilitating long-term imaging.- Multiplexing Capability: The variety of available fluorescent proteins allows for the simultaneous study of multiple targets, enhancing experimental complexity and data richness.### SummaryFluorescent proteins are invaluable tools in modern biology, enabling scientists to visualize and study cellular processes in real time. Their ability to be genetically encoded, combined with their diverse colors and stability, makes them essential for a wide range of applications, from basic research to advanced techniques in cell biology, developmental biology, and neuroscience. Their versatility continues to drive innovations in imaging technologies and experimental designs.

Question 89

Brainbow

Answer 89

The Brainbow technique is an advanced genetic labeling method used to visualize and differentiate individual neurons in the brain. Developed by researchers, including Jeff W. Lichtman and Josh R. Sanes, at Harvard University in the mid-2000s, Brainbow allows for the labeling of thousands of neurons with distinct colors, providing unprecedented insight into the complexity of neural circuits and connectivity. Here’s a detailed overview of the Brainbow technique, its mechanisms, applications, and advantages:Key Features of Brainbow 1. Genetic Encoding: • The Brainbow technique utilizes a combination of fluorescent proteins (such as variants of GFP, RFP, and YFP) that can be expressed in individual neurons. By using multiple fluorescent proteins, each neuron can be labeled with a unique color. 2. Cre-Lox System: • Brainbow relies on the Cre-Lox recombination system, a genetic engineering tool that allows for targeted manipulation of genes in specific cell types. • In the Brainbow approach, a genetic construct containing multiple fluorescent protein coding sequences is designed such that the expression of these proteins is dependent on the presence of the Cre recombinase enzyme. 3. Random Expression: • When Cre is expressed in a population of neurons, it randomly recombines the genetic construct, resulting in the expression of different combinations of fluorescent proteins in each neuron. This randomness leads to a vast array of color combinations, enabling each neuron to be uniquely colored. 4. Color Diversity: • The range of colors available allows researchers to differentiate between many individual neurons within the same tissue section. Depending on the specific Brainbow variant used, it can label hundreds to thousands of neurons with distinct colors.Applications of Brainbow 1. Mapping Neural Circuits: • Brainbow allows scientists to visualize and trace complex neural circuits, helping to elucidate how neurons are connected and interact with one another. This is particularly useful for studying the organization of circuits in various brain regions. 2. Studying Neuronal Development: • The technique can be used to investigate how neuronal connections form during development, providing insights into the processes of synaptogenesis and axon guidance. 3. Analyzing Neuronal Plasticity: • Researchers can study changes in neuronal connectivity and structure in response to environmental changes, injury, or disease, contributing to our understanding of neuroplasticity. 4. Understanding Brain Disorders: • Brainbow can help in visualizing alterations in neuronal circuits associated with neurological and psychiatric disorders, aiding in the identification of pathological changes in the brain.

Question 90

Tracing neural circuits

Answer 90

Tracing neural circuits is a critical aspect of neuroscience that involves mapping the connections and pathways between neurons to understand how information is processed in the brain. This approach provides insights into the organization, function, and dynamics of neural networks, which are essential for various behaviors, cognition, and responses to environmental changes. Here’s an overview of the methods, applications, and significance of tracing neural circuits:Key Techniques for Tracing Neural Circuits 1. Anterograde and Retrograde Tracing: • Anterograde Tracing: Involves labeling neurons and tracking the pathways of their axons as they project to their target areas. This is often done using substances like biotinylated dextran amine (BDA) or viral tracers that are taken up by the neuron and transported along the axon. • Retrograde Tracing: Involves labeling the target neurons to see which presynaptic neurons project to them. This can be achieved using tracers like fluorogold or viruses that enter the cell bodies of neurons and travel back to the source. 2. Fluorescent Proteins: • The Brainbow technique and other fluorescent protein methods allow researchers to label neurons with distinct colors, providing a visual representation of neuronal connections and circuit dynamics. This is particularly useful for studying the organization of complex circuits.

Question 91

Serial electron microscopy (EM) reconstruction*

Answer 91

Serial electron microscopy (EM) reconstruction** and connectomics are advanced techniques used to map the intricate networks of neurons and their connections in the brain. Together, they provide detailed insights into the structure and organization of neural circuits, enhancing our understanding of brain function and connectivity. Here’s an overview of these techniques, their methodologies, applications, and significance in neuroscience:### Serial Electron Microscopy (EM) Reconstruction#### Key Features1. High Resolution: - Serial EM provides ultra-high-resolution imaging of biological tissues at the nanometer scale, enabling the visualization of cellular structures, including synapses, dendrites, and axons.2. Tissue Preparation: - Tissue samples are fixed, dehydrated, and embedded in resin to preserve the ultrastructure. Thin sections (typically 50-100 nm thick) are cut from the embedded tissue using an ultramicrotome.3. Imaging Process: - Each section is imaged using an electron microscope. The images from successive sections are taken in a systematic manner, allowing for the reconstruction of three-dimensional (3D) structures.4. Image Alignment and Reconstruction: - The acquired 2D images are aligned and combined using specialized software to create a 3D reconstruction of the tissue. This process often involves sophisticated computational algorithms to accurately stitch the images together.### Connectomics#### Definition- Connectomics is the comprehensive study of the connectome, which is the complex network of connections between neurons in the brain. It involves mapping the synaptic connections between neurons to understand how information is processed and transmitted within neural circuits.### Applications of Serial EM and Connectomics1. Mapping Neural Circuits: - These techniques are essential for creating detailed maps of neural circuits, revealing how neurons communicate and form functional networks. This is crucial for understanding brain functions such as perception, memory, and learning.2. Understanding Synaptic Plasticity: - By visualizing the structure of synapses and their connections, researchers can investigate mechanisms of synaptic plasticity, which underlies learning and memory.3. Studying Development: - Serial EM and connectomics can be used to study how neural circuits develop over time, providing insights into the formation and refinement of synaptic connections during critical periods of brain development.4. Investigating Neurological Disorders: - These techniques allow scientists to explore alterations in neural connectivity associated with neurological and psychiatric disorders, helping to identify potential targets for therapeutic intervention.5. Comparative Connectomics: - Connectomics can also be used to compare the neural circuits of different species, contributing to our understanding of evolutionary changes in brain structure and function.### Challenges and Considerations1. Data Volume: - Serial EM generates large amounts of data, making data management, storage, and analysis significant challenges. Advanced computational techniques and high-performance computing resources are often required.2. Complexity of Analysis: - The analysis of the resulting 3D reconstructions requires sophisticated software tools and skilled personnel to accurately identify and classify synaptic connections.3. Labor-Intensive: - The processes involved in serial EM, including sample preparation and imaging, are time-consuming and require meticulous attention to detail.### Significance of Serial EM and Connectomics- Understanding Brain Function: By providing detailed maps of neural circuits, these techniques help to elucidate how specific circuits contribute to various brain functions and behaviors.- Advancing Neuroscience Research: The insights gained from connectomics have the potential to revolutionize our understanding of brain architecture and organization, offering new avenues for research into brain disorders.- Interdisciplinary Approaches: The integration of connectomics with other techniques (such as electrophysiology and imaging) fosters a more comprehensive understanding of how neural networks function.### SummarySerial electron microscopy reconstruction and connectomics are powerful tools in modern neuroscience, enabling researchers to map the complex networks of neuronal connections in unprecedented detail. By elucidating the structural basis of neural circuits, these techniques contribute to our understanding of brain function, development, and the mechanisms underlying neurological disorders. As technology continues to advance, the integration of connectomics with other research methods holds great promise for uncovering the mysteries of the brain.