Health and the nervous system module 1

Glia

• Traditionally thought of a support/maintenance cells.

• Modern neuroscience is finding an increasing role for glia in regulating the development and function of the nervous system.

Neurons

• The basic unit of the nervous system

• An electrically excitable cell that receives, integrates, and propagates information.

Neural circuit

Neurons can be connected in series and parallel (just like the circuits of a computer) to process inputs and generate an output.

Soma

Cell body; contains nucleus & other organelles

Axon

Long process that propagates action potentials to send outgoing signals

Dendrites

Receive input

Nucleus

Contains DNA

Axon Initial Segment

Where action potentials form

Axon terminal

The site of synaptic transmission

Neural information flows from

Dendrites to axons (big to small)

Theory of dynamic polarization

Dendrite => Soma => Axon

Leak Channel

A channel that remains open and allows ions to freely pass through

Gated Channel

A channel that remains closed until a certain condition releases gating mechanism (e.g. ligand, voltage, mechanical, temperature, light, etc.)

Selective

Only let through one type of particle

non selective

will let though several types of particles

Chemical gradient

The difference in solute concentration across membrane

electrical gradient

The difference in charge across membrane

Electrochemical gradient

• The balance between forces that determine the direction and amount of ions to flow

• Electrochemical gradients drive movement across membranes

• Electrochemical gradients determine both the direction a solute will move across a membrane and the magnitude of the net movement

• When the chemical and electrical gradients are in agreement about where a solute should go, the force is even greater than it would be if either gradient acted alone

• When the chemical and electrical gradients think the solute should go in opposite directions, they partially or can even completely cancel each other out

Membrane potential

is the difference in charge (voltage) across the membrane

Resting membrane potential

is the membrane potential of the neuron at rest

Why are neurons electrically polarized (negatively charged)?

• Shouldn’t the electrical gradient cause the charges on the inside of the cell to be roughly the same relative to the outside of the cell?

  • There are different concentrations of ions on the inside vs. the outside of the cell. This creates chemical gradients that oppose the electrical gradient.

Nerst Equation

tells us the equilibrium potential of an ion

The Goldman-Hodgkin-Katz equation

calculates Vm

How does information flow across a neuron

• Electrical signaling

  • The movement of ions across the membrane changes the local electrical potential, creating a signal that can propagate through a neuron.

Depolarization

• A change in the distribution of charges that makes cell less negative

• Opening sodium channels depolarizes neurons

Hyperpolarization

• A change in the distribution of charges that makes cell more negative

• Opening potassium channels hyperpolarizes neurons

What do voltage-gated channels create?

action potential

Action potential

• Action potentials are brief, rapid changes in electrical potential (voltage) across a cell's membrane, primarily found in nerve and muscle cells. They serve as the fundamental mechanism for transmitting signals and information throughout the body. 

• All or nothing

synapse

• A synapse is a place where two nerve cells come in very close proximity to one another and send signals.

• Often synapses are depicted at the end of an axon through which an action potential propagates.

• A synapse is defined simply as a junction between two nerve cells across which impulses pass.

• This means that despite synapses being depicted in cartoons and many textbooks as at the end of an axon, synapses are everywhere.

gap junction

An electrical synapse can sometimes be called a gap junction

connexins

An assembly of 6 proteins called connexins come together to create a channel with a pore that ions can use to flow across an electrical synapse.

How ions travel

ions travel directly across electrical synapses

• An electrical synapse can sometimes be called a gap junction

• An assembly of 6 proteins called connexins come together to create a channel with a pore that ions can use to flow across an electrical synapse.

• Newer studies have shown that other molecules, such as ATP and some second messengers can also travel directly across gap junctions.

How are neurotransmitters used?

Neurotransmitters are used to signal across chemical synapses

chemical synapses

• In a chemical synapse the following occurs:

  • An action potential causes a wave of depolarization to travel through a neuron

  • Near to a synapse, that depolarization causes the opening of voltage gated calcium channels

  • An increase in intracellular calcium causes vesicles containing neurotransmitters to fuse to the membrane

  • Neurotransmitter is released into and travels across the synaptic cleft

  • Neurotransmitters bind to receptors on the postsynaptic cell

What is a vesicle

• Release content trough exocytosis

• They have a lipid bilayer structure just like the plasma membrane

• Vesicles are like shipping boxes - they are packages of materials that can be sent throughout the cell

• In our case, we’re focusing on vesicles containing neurotransmitters or neuropeptides

Challenges of neurotransmission

Neuronal communication has some additional features that require precise regulation of secretion:

• Neurotransmitters must be released in a specific place (the synapse)

• Neurotransmitters must be released rapidly at a precise time (following the action potential)

• Sometimes, you need a lot of neurotransmitter to be released at once 

SNARE complex

The SNARE complex is a group of proteins that bring the vesicle in close proximity to presynaptic membrane, facilitating exocytosis

Calcium

• Calcium serves as the signal that activates the SNARE complex to trigger neurotransmitter release 

Calcium has a very low basal concentration in the neuron so it’s presence is a potent signal

v-SNAREs

are associated with the vesicle

t-SNAREs

are associated with the target membrane (aka presynaptic membrane)

docked vesicle

When v-SNAREs and t-SNAREs come together to bring a vesicle close to membrane, that vesicle is considered a docked vesicle

calcium

• The action potential travels down the neuronal process until it reaches a synapse, where there is a high concentration of voltage gated calcium channels that open when the action potential arrives

• When voltage-gated calcium channels open, calcium enters and binds to nearby synaptobrevin and  synaptotagmin (a component of the SNARE complex) causing the complex to bring the docked vesicle even closer to the target membrane and trigger exocytosis/neurotransmitter release

Ionotropic receptors

ion channels

metabotropic receptors

are receptors that activate a chain of intracellular secondary messengers, they are usually called G-Protein Coupled Receptors (GPCRs)

The effect of a neurotransmitter is determined by:

the effect of the receptor it acts on

GPCRs

• G-protein receptors are simply receptors in the membrane that are coupled to g-proteins 

• The type of g-protein they are coupled to and the cellular environment determine the effect of receptor activation

  • Some are inhibitory (ex. G_ɑi)

  • Some are stimulatory (ex. G_ɑs)

GABA

• The action of GABA is usually inhibitory

  • This is the most common ‘inhibitory’ neurotransmitter

• There are ionotropic GABA_A receptors that flux chloride

  • In fully developed animals opening a chloride channel is inhibitory because chloride ions flow into the cell

  • In development, the concentration of ions is different such that there is more chloride ions inside the cell and so opening a chloride channel would cause them to flow out of the cell and be excitatory

• There are metabotropic GABA_B receptors are coupled to inhibitory g-proteins

Glutamate

• The action of glutamate is excitatory

  • This is the most common ‘excitatory’ neurotransmitter

• There are ionotropic glutamate receptors that flux Na⁺, K⁺, and possibly Ca²⁺

• There are also metabotropic glutamate receptors (mGluRs)

Acetylcholine

• The action of acetylcholine is excitatory

• Nicotinic acetylcholine receptors (nAChRs) are ionotropic

  • These channels open to allow Na⁺ and Ca²⁺  to flow into the cell, and K⁺ to flow out of the cell

  • Nicotine activates them

• Muscarinic acetylcholine receptors (mAChRs) are metabotropic

  • Muscarine (in poison mushrooms) activates them

Neuromoduaters

Neuromodulators tune the effects of neurotransmitters

• Neuromodulators can enhance or diminish the effectiveness of other neurotransmitters or signaling

• They do not act directly to open or close an ion channel

• They generally work through GPCRs, which initiate a signaling cascade that can cause a variety of changes in a cell (both short and long term)

• The two most well known neuromodulators are serotonin and dopamine

Meta Analyses

a statistical technique that combines the findings of multiple individual studies to draw an overall conclusion about a specific research question

• A meta-analysis can be conducted as part of a systematic review or on it’s own using just a few studies

  • A meta-analysis can be conducted using as few as two studies

  • Usually the analysis is conducted on randomized controlled trials

• A meta-analysis pools data from multiple studies and runs high-powered statistical tests on that data to answer a research question

Systematic Reviews

a type of literature review that uses a rigorous and transparent process to identify, assess, and synthesize all available research evidence related to a specific research question

• A systematic review is an objective, reproducible method to find answers to a certain research question, by collecting all available studies related to that question and reviewing and analyzing their results.

• During the systematic review process, the quality of studies is evaluated, and a statistical meta-analysis of the study results is conducted on the basis of their quality.

Meta-Analyses vs Systematic Reviews

A systematic review is a comprehensive and structured search, appraisal, and synthesis of all available evidence on a specific research question.

A meta-analysis is a statistical method used to combine and analyze data from multiple studies, often within the context of a systematic review, to produce a single, more precise estimate of an effect

Scientific method process

observation/question; research topic area; hypothesis; test with experiment; analyze data; report conclusions

Computed Tomography (CT or CAT Scan)

• Uses x-rays which are sent through a patient’s body and quickly rotated around a tube

• This generates a “slice” called a tomographic image

• The slices can then be digitally assembled to form a stack, which can be used to generate a 3D image of the portion of the body that was scanned.

Positron Emission Tomography

• Is used to assess the metabolic or biochemical function of tissues

• Uses a radioactive drug called a tracer

Functional Magnetic Resonance Imaging (fMRI)

• fMRI uses changes in blood flow to different parts of the brain as a proxy for neural activity

  • High neural activity induces dilation of local blood vessels… inducing blood flow in that region!

Diffusion Tensor Imaging

• A special type of MRI used to look specifically at white matter

  • It uses the Brownian motion of water molecules to determine how far and in what direction water is moving

  • Water molecules move differently depending on what type of tissue they are in

  • White matter is made up mostly of myelinated axons - this constrains the direction of water flow

• DTI gives us a fractional anisotropy (FA) value that tells us about the directionality of the diffusion of water in the tissue 

Electron Microscopy workflow

Sample preparation, cryo-EM grids setup, Cryo-EM imaging, Data collection, data pre-processing, map reconstruction, model building, structural analysis

transgenics

We can create genetically modified organisms that have perturbations in specific endogenous (natural) genes or have a gene of interest added

transgene

a gene which is artificially introduced into the genome of another organism

Electrophysiology experiments

Electrophysiology experiments measure the electrical properties of neurons

• You can measure:

  • An individual neuron’s activity

  • Multiple neurons at once

    • extracellular electrodes

  • Large-scale electrical activity in the brain

    • Electroencephalogram (EEG) - non invasive, can be done in humans

Calcium imaging

• calcium is a proxy for neural activity

• GCaMP, a fluorescent Ca²⁺ sensor

Immediate early genes (IEGs)

 genes that are strongly upregulated in neurons that have been recently (strongly) activated

• By upregulated we mean that there is an increase in their transcription

• Because translation follows transcription, we can also then reasonably expect an increase in the protein product the gene encodes

antibody stain

• An antibody stain for c-fos can tell you which regions of the brain were active

  • Antibody staining links a fluorescent molecule with the protein of interest through a series of 1 or two antibodies

Lesions

Lesions are areas of tissue that have been damaged due to injury or disease.

Random mutagenesis

 feed animal dangerous drugs, blast em with radiation, look for behavioral phenotypes, figure out which gene it was (old fashioned way)

Knockout mutant

delete a gene

Knock-in mutant

add in a new gene

Loss of function mutation

alter the content of a gene to make it/the protein product less functional

gain of function mutation

alter the content of a gene to make it/the protein product have a function it normally doesn’t (e.g. always active)

CRISPR/Cas9

Tool for gene editing

• Can target any known gene (using a “Guide RNA” of your design)

• Can either knockout a gene, knock in a gene, or edit single base pairs of gene

Clinical trial phase 1

focus on safety and the proper dosage

Clinical trial phase 2

Focus on effectiveness and side effects

Clinical trial phase 3

Compares the new treatment to existing treatment

Clinical trial phase 4

Treatment is approved and available. Long-term effects are observed

uncontrolled trial

An uncontrolled trial compares an endpoint after treatment to the baseline before the treatment

• Known to produce a seemingly greater effect of the treatment than controlled studies

• Doesn’t count for self-resolution (or unrelated decline)

• Usually used to determine the pharmacokinetic properties of a new drug:

  • Liberation

  • Absorption

  • Distribution

  • Metabolism

  • Excretion

Placebo

patients are given an inert substance

no treatment

follow the course of the disease in patients not given any treatment

active treatment

patients are given a known, effective drug

unblinded study

the patient and the researchers both know what treatment the patient is receiving

Blinded study or single-blinded study

the patient does not know what treatment they are receiving but the researchers does

Double-blinded study

• the patient and the main researcher both do not know what treatment the patient is receiving 

  • A supervisor, colleague, or computer will know so that the results can be “unblinded” at the end of the study and conclusions can be made

  • This is the most scientifically rigorous option!

Randomized control trial

best type of clinical trial

• Have a control - a placebo or an active drug control

• Randomly assign patients to a treatment or control group

  • There are different ways to randomize - but this is beyond the scope of this course

• Often use some form of blinding - but check the specific study

null hypothesis

is an assertion that whatever effect we are studying does not exist, or that two groups are the same.

alternative hypothesis

proposes that there is some difference or effect.

t-test

• A t-test is used to compare the means of two groups

• T-tests are performs on quantitative (numerical data)

t-test assumptions

• Your data is roughly normally distributed 

• Your two groups have roughly the same variance (spread of your data) 

unpaired t-test

independent

paired t-test

related

one-tailed t-test

• Use a one-tailed t-test when you care about the direction of the difference

  • Example: our class height is taller than the average height of students in the Harvard Summer School.

two-tailed t-test

• Use a two-tailed t-test when you just want to know if there’s a difference between the groups (in either direction)

  • Example: our class height is different than the average height of students in the Harvard Summer School.

ANOVA

• Like a t-test, the ANOVA compares the means between groups

• An ANOVA can compare the means across more than two groups

• ANOVA also assumes the data are normally distributed and have similar variances

Chi-square

• Chi-Squared Tests (χ²) are like t-tests but they compare categorical values

  • Heads vs tails

  • Gender and type of shoe

• They make no assumption about distribution of the data

p-value

a test statistic typically determines the probability of the observed result (or a more extreme result) occurring by random chance, if no association exists.

type 1 errors

• occurs when the null hypothesis was rejected but it is actually true

  • we said there is a difference, but there is not

  • false positive

• P-values tell us the probability of making a type 1 error

• Note: while it’s generally thought that having a higher sample size is better, the false positive rate can increase with increasing sample size

type 2 errors

• A type 2 error occurs when the null hypothesis is accepted but it is actually NOT true

  • we said there was no difference but there is 

false negative

Power analyses

• The probability of not making a Type 2 error is called the power of the statistical test

  • In science we talk a lot about p-values and much less about power. Both are important!

Power is affected by

• Sample size

• Significance level

• The “true” difference between your groups

• Variability in the population

• The type of test you run (especially when it comes to tailed tests)