neuro exam two

specific animal models…

…are suited for specific questions

squid and snails

basic biology of neurons, synaptic transmission and plasticity

cats and primates

visual system

rats and mice

neuropharmacological and behavioral studies (most common)\`

worm

(C. elegans), aging and development

fruit fly

(D. melanogaster), synapse formation and genetics related to synapse formation

zebrafish

(D. rerio), development and drug screening

health research extension act(1985)

provided strict guidelines for the care of animals used in biomedical and behavioral research; the goals of this legislation include humane animal maintenance and experimentation that limits both the use of animals and animal distress

ACUC

animal care and use committee, has the ability to veto any studies that do not meet the predetermined criteria

ACUC reviews each scientific protocol with 3 considerations in mind

1. the research should be relevant to human or animal health, the advancement of knowledge, or the good of society
2. alternative methods such as computer simulations that do not require animal subjects must be considered
3. procedures should avoid or minimize discomfort, distress, and pain

behavioral assays

understand the inner workings of the brain through behavior

behavioral observation

simple observation of untrained behavior → tremors, salivation, defecation, catalepsy, response to tail pinch, changes in eating or drinking

catalepsy

seizure with loss of consciousness accompanied by the rigidity of the body

motor activity

changes in motor activity produced by a drug

open field test

place and animal in a large box and record its activity for 5-10 minutes (measure distance traveled or whether it spent more time in the center versus the periphery)

analgesia

reduction of perceived pain without loss of consciousness

pain assessment

measures an animal’s avoidance of a noxious stimulus

tail flick test

record withdrawal reflex, how long it takes the animal to perceive the stimulus

hot plate test

place the animal on an enclosed hot plate and measure the latency to lick its paw; record how long it takes for an animal to perceive the stimulus

Von Frey test

apply filament to animal paw and record withdrawal reflex, record how long it takes for the animal to perceive the stimulus

mazes

measure memory, learning, or motivations in an animal (cognition); contain a start location and an end location that contains a small piece of food or another reward

t-maze

an animal is given the opportunity to explore the maze and locate the reward, in subsequent trials, learning in evaluated (latency to goal and errors made are some variable are measured)

anxiety assessments

measures anxiety-like behavior through induction of fear or unconditioned reaction to avoid bright lights or heights

elevated plus/zero mazes

measure the time spent in enclosed arms versus in open arms

light-dark box

measure the amount of time spent in the light box versus the dark box

depression assessments

measures depressive-like behavior; there is no one task that mimics all symptoms of depression. Instead, different tasks measure specific aspects of the disorder

forced swim test

animal is placed in a cylinder of water, measure the time spent swimming

sucrose preference test

animal is given the choice between sucrose water versus regular water, measure the amount of each consumed (depressed = no preference)

tail suspension test

suspend mouse by tail and record time spent mobil

learned helplessness

expose animals to aversive events over several periods of time, ability to make an appropriate response that could alter an aversive event when placed in a new situation is measured

maternal separation

separate the mother from the pups

chronic mild unpredictable stress

expose animals to unpredictable micro-stressors for 12-30 days, following the stressors, animals show several behaviors similar to depressed individuals

resident-intruder paradigm

introduce an unfamiliar male (intruder) into the home cage of another male (resident) for 10 minutes; observe the behavior of the resident

assessments of fear

measures the freezing response (mice freeze when they scared)

fear conditioning

classical conditioning → classically condition tone to footshock, measure freezing response to tone

fear-potentiated startle

classically condition neural stimulus to footshock, measure startle response

transgenic

one or more DNA sequences from another species have been introduced by artificial means (random)

* new gene added, native gene is still present
* can’t control where new DNA integrated into the genome
* can use to express in specific cells, determine effects on cell or circuit function

knockout

genetically engineered to lack one or more specific genes (targted)

knock-in

a foreign or mutated gene replaced the normal gene (targeted)

why use transgenic mice?

* introduces genes for inducing or inhibiting neural activity
* ablate cells by expressing toxin or toxin receptors
* put in genes measuring neural activity
* mark specific organelles
* trace axons and dendrites with membrane-bound fluorescent reporters

How to make transgenic mice:

* genes of interest are injected into fertilized mouse eggs
* eggs are implanted in the uterus of a surrogate mother
* offspring are assessed for evidence of mutations
* chimeric mice are mated with wild-type mice
* offspring produce some heterozygous mice that will be mated with other heterozygous mice to produce mice homozygous for the transgene

how to make KO/KI mice

* create targeting vector
* place in embryonic stem cells, select for proper incorporation
* place stem cells into blastocyst
* insert blastocyst into a female mouse
* offspring are assessed for evidence of mutation
* mosaic mice are mutated with wild-type mice
* offspring will contain both knock-out/knock-in and wild-type mice

why make KO/KI mice?

* can inactivate a specific gene or inactivate a gene in a specific group of cells
* inactivate at a specific time in development (if earlier knockout is lethal)
* can replace a gene with the modified version of the gene (knock-in) - determine the effect of mutations in disease genes

CRISPR-Cas 9

a method based on a natural system used by bacteria to protect themselves from infection by viruses and plasmids

* use guide RNA to identify a specific genomic sequence to be modified
* introduces Cas-9 (enzyme) - which cuts out the specific section of DNA
* a new section of DNA is inserted

Optogenetics (in vivo)

* technique for controlling neural firing via light
* light activated ion-channels are inserted into the cell membrane
* light excite (channelrodospins) or inhibits (halorhodopsins) specific neurons in living organisms
* used to study addiction or learning

DREADD

chemogenetics - ligand binds to G protein-coupled receptors leading to more prolonged mechanism of action compared to optogenetics; ligand administered either activates or suppresses the inserted receptor; geneticalyy engineered receptor is sterotaxically targeted to specific brain cells (via viral vectors)

immunohistochemistry (in vitro)

* use a preservative to fix the brain
* cut tissue slices and incubate with antibody in solution
* antibody attaches to antigen whenever it is present
* visualize where the tagged antibody is

antibody

protein produced by white blood cells that bind to specific proteins in the brain

Western Blot

* homogenize (grind up) tissue sample
* use gel electrophoresis to separate proteins based on size and place onto a blotting membrane
* incubate membrane in antibody of interest
* add second antibody to provide a chemical reaction to create a detectable product
* quantify protein

ELISA

* antigen/sample (BLOOD SAMPLE) is added to plate
* blocking buffer is added to block remaining protein-binding sites
* next a suitable primary antibody is added
* a suitable secondary antibody — HRPO conjugate is then added which recognizes and binds to the primary antibody
* TMB substrate is added and is covered by HRPO to detectable form

PCR

* measure small amounts of RNA or DNA
* used to compare expression of genes in different neurons, different stages of development, mutant animals or disease tissue
* tiny amounts of tissue needed
* can be done in situ (in the original place)
* make multiple copies
* qualitative — allows reading the result as presence or absence

Quantitative PCR

quantifies the amount of DNA amplified in each cycle

DNA Sequencing

general lab technique for determining the exact sequence of nucleotides or bases in a DNA molecule

RNA Sequencing

a sequence technique which uses next-generation sequencing to reveal the presence and quantity of RNA in a biological sample at a given moment — analyzing and continuously changing cellular transcriptome

synapse

specialized junction where one part of a neuron contacts and communicates with another neuron or cell type

pre-synaptic neuron

before synapse, releases neurotransmitters

post-synaptic

target cell, where neurotransmitter binds, after the synapse

gap junctions (electrical synapse)

narrow gap that is spanned by clusters of connexins; allow ions (and small molecules) to pass directly from the cytoplasm of one cell to the cytoplasm of another cell

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bidirectional and allow for fast communication

help control behaviors that require fine timing (like motor control)

connexins

6 subunits of connexins form a connexon

connexon

2 connexons combine to form a gap junction

Otto Loewi

established the concept of chemical transmission between nerves when he identified AcH released from the vagus nerve

synaptic cleft

contains extracellular matrix proteins that help organize the synapse and enzymes which can break down neurotransmitters

active zones

site of neurotransmitter release (located on pre-synaptic neuron)

docked vesicles

stores neurotransmitters (pre-synaptic neuron)

secretory granules/large dense core vesicles

store proteins (pre-synaptic neuron)

post-synaptic densities

contain receptors and associated proteins

axodendritic

synapse between axon and dendrite

axosomatic

synapse between axons and cell bodies

dendrodendritic

synapse between dendrites and dendrites

gray’s type I

asymmetrical thickness (usually excitatory)

grays type II

symmetrical (usually inhibitory)

astrocytes

present at synapses and participate in regulation of signaling

mixed synapse

contains gap junctions and chemical components

neuromuscular junction

* occurs between motor neurons and muscles
* the largest synapse in the body
* transmission is fast and reliable
* contains similar structures to CNS synapses
* lots of active zones

amino acids

GABA and Glutamate and Glycine

monoamines

dopamine

serotonin

epinephrine

norepinephrine

AcH

muscle movement, arousal (alertness/attention), reward, learning and memory

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NT at neuromuscular junction, also used by many circuits in autonomic nervous system

* made by all motor neurons in spinal cord and brain
* important in attention and memory
* modulatory role - influence the effects of other chemical messengers

GABA

main, inhibitory NT

* present in many areas of the brain such as cortex and midbrain
* most present in local circuit interneurons, but some can be on projection neurons

Glutamate

main, excitatory NT

* most excitatory neurons in brain are glutamatergic
* involved in many circuits including learning and memory (long term potentiation) and motor function
* excitotoxicity by glutamate can occur during a brain injury

Dopamine

executive functions (attention, inhibition, working memory, behavioral flexibility), motor control, motivation, arousal, reward

serotonin

regulation of mood, appetite, sleep, memory/learning, emotional behavior, aggression, feeding behavior

epinephrine

fight or flight, present in few neurons, regulate cardiac and respiratory function

norepinephrine

alertness, arousal, readiness for action, feeding behavior, sleep, mood, learning/memory, reduce background noise and increase response to strong simulation

dual neurotransmission

release two or more different NT together

peptide NT

synthesized in the soma and transported in secretory granules via fast axonal transport

monoamines NT

synthesized at axon terminals, loaded into vesicles by specific vesicular transporters

Dale’s Principle

a given neuron contains and released only one neurotransmitter and exerts the same functional effects at all of its termination sites

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* DEBUNKED = some neurons makes different types of NT

exocytosis

how vesicles release their contents; occurs within .2 millisecond of calcium influx

transient docking

vesicles are docked at the active zone

V-SNARES

located on the vesicle

T-SNARES

located on the membrane of the pre-synaptic terminus

endocytosis

vesicles membranes are recycled

clathrin-mediated endocytosis

widely accepted, slowest

kiss-and-run

fastest mode of endocytosis

perisynaptically

neurotransmitters are released from locations near the synapse

extrasynaptically

neurotransmitters are released from non-synaptic sites

gliotransmitters

released by astrocytes in response to receiving signals from neurons, this signals back to neurons and influences signaling at one sometimes more synapses

receptors

specialized proteins that NT bind to

ionotropic

* c\`omposed of 4-5 subunits
* not as selective as voltage-gated channels
* the pore is usually closed until the ligand binds; after the ligand binds, it changes conformation and channels open within milliseconds
* can produce EPSPs or IPSPs depending on the receptor

is membrane potential is less than 0 (reverse potentials)

depolarization