NEUR-201 Exam 1

Population

entire group researcher wants to understand

Sample

subset of which measurements are collected

Descriptive Statistics

summarizes observable sample data

EX: mean firing rate of 50 recorded neurons was 22 Hz

Inferential statistics

draws conclusions about larger population

EX: treatment appears to increase firing rates in comparable neurons

Random Sampling

members have equal chance of being selected

EX: randomly selected individual from a population registry

Representative Sampling

deliberately matching important population characteristics

EX: recruiting participants in proportions matching population

Convenience Sampling

selecting easily accessible subjects (can skew data)
EX: studying volunteers from one class

Independent variables

what researcher manipulates

(EX: drug treatment)

Dependent variable

measured outcome

(EX: number of apoptotic cells)

Categorical variables

variables that can be put into categories

(EX: drug/control, cell type, etc)

Continuous variables

numerical quantities

(EX: membrane voltage, reaction time, neuronal firing rate, etc)

Experimental study

manipulates variable to test its effect

(EX: administering different drug dosages and measuring neuronal activity )

Correlational study

measures existing variables to determine whether they are associated

(no manipulation involved)

(EX: recording naturally occurring protein expression & neuronal activity to examine their association)

Somatic intervention

manipulates brain/NS to measure behavior

Behavioral intervention

manipulates behavior/experience to measure brain activity

Frontal lobe

involved in planning, decision-making, movement, impulse control, speech production

(impaired frontal lobe = impaired planning & behavioral regulation)

Parietal lobe

involved in somatosensory processing & spatial awareness

(impaired parietal lobe = impaired spatial processing)

Temporal lobe

involved in hearing, language comprehension, memory, recognition, etc

(impaired temporal lobe = difficulty in memory/or language comprehension

Occipital lobe

visual processing

(impaired occipital lobe = visual processing difficulties)

Hippocampus

aids in formation of new memories & learning

(impaired hippocampus = memory impairment)

Amygdala

aids in emotional processing (especially threat)

(impaired amygdala = altered responses to emotionally significant stimuli)

Hypothalamus

area of temperature, thirst, hormone, sleep regulation

(impaired hypothalamus = disrupted homeostasis)

Thalamus

area of sensory & motor relay/regulation (except smell)

(impaired thalamus = altered sensory processing)

Basal ganglia

aids in movement control & reward-related processing

(impaired basal ganglia = movement regulation difficulties)

Neuron

receives & transmits electrical and chemical information

Astrocyte

supports neurons, supplies nutrients, and regulates NTs

Microglia

aids in immune surveillance, inflammatory responses, and synaptic pruning

Oligodendrocytes

produces myelin around CNS axons

Schwann Cells

produces myelin around PNS axons

OPCs

progenitor cell that aid toward producing oligodendrocytes

Histology

studying microscopic structure of tissues

(staining techniques allows researchers to determine which cells are present & what cells are doing)

IBA1 identifies?

Microglia (also some related myeloid cells)

IBA1 localizes where?

Cytoplasm/membrane (common microglial marker)

DAPI identifies?

DNA-containing nuclei

DAPI localizes where?

cell nucleus (nuclear location) (DNA stain not primary antibody cell marker)

NeuN identifies?

most mature/post-mitotic neurons

NeuN localizes where?

localizes mainly cell nucleus of mature neurons

GFAP identifies?

Astrocytes

GFAP localizes where?

Cytoplasmic filaments of astrocytes in CNS

TMEM119 identifies?

specific cell marker of resident brain microglia

(specific marker for microglia)

TMEM119 localizes where?

mainly in membrane-associated structures (plasma membrane, ER, etc)

(helps distinguish residential microglia from infiltrating macrophages)

Olig2 identifies?

Oligodendrocyte lineage cells

Olig2+ localizes where?

nuclear (cell nucleus)

PDGFRα (NG2) identifies what?

oligodendrocyte progenitor cells

PDGFRα localizes where?

membrane

CC1 (APC) identifies what?

mature oligodendrocytes

CC1 (APC) localizes where?

in cell body (useful for mature oligodendrocyte identification)

MBP identifies what?

myelin and mature oligodendrocyte-associated structures

MBP localizes where?

in myelin (stains sheath; doesn’t directly count cell bodies)

what does Ki67 tell us?

active cell cycle is occurring (cell is proliferating/cycling)

what does BrdU/EdU tell us?

cell is undergoing DNA synthesis

What does CC3 tell us?

cells is undergoing apoptosis

Double positive mean whats?

combination of interpretations

(EX: IBA+ and CC3+ means that the cell is an apoptotic microglia)

Antibodies

y-shaped proteins that target molecules called antigens

Primary antibody

targets & binds to target antigen directly

Secondary antibody

binds to primary antibody and carries fluorescent label

primary host and secondary target must?

Match between each other

(EX: rabbit anti-IBA1/goat anti-rabbit)

3 Things to look out for in two-marker experiments

1. primary antibodies cannot be same species (makes it difficult to distinguish targets in ordinary indirect staining)

2. using the same species antibodies on its tissue (can cause issues)

3. using two fluorophores that have overlapping emission spectra

Direct immunofluorescence

• fluorophore is attacked to primary antibody

• fewer staining steps

  • often has lower signal amplification

  • less flexibility in secondary-antibody selection

Indirect immunofluorescence

• fluorophore is attached to secondary antibody

• more staining steps

  • can amplify fluroscent signal

  • many fluorophores can be used with one primary

Immunohistochemistry

antibody staining performed on tissue (cellular organization is preserved)

Immunocytochemistry (ICC)

antibody staining of isolated cells (EX: neurons grown in culture)

FFPE (Formalin-Fixed Paraffin Embedded-Tissue(

prep: fixation and paraffin embedding

sections: 3-5 µm

Tissue morphology: excellent

antigen retrieval: often required

lipid preservation: poorer

storage: stable paraffin blocks

Frozen Tissue

prep: freezing/fixation with cyroprotection

sections: 5- 50 µm

tissue morphology: good (can have freezing artifacts however)

antigen retrieval: unnecessary

lipid preservation: better than FFPE

storage: usually cold storage

Histology staining preparation

1. collect & prepare tissue

   1. dissect tissue (fix/freeze as appropriate)

2. pretreat specimen

   1. remove paraffin and retrieve antigens (if FFPE protocol requires it)

3. block nonspecific binding

   1. reduce unwanted antibody

4. apply primary antibodies

   1. allows target-specific antibodies to recognize antigens

5. apply secondary antibodies

   1. bind fluoroscent/other detection labels to primary antibodies

6. counterstain and image

   1. use DAPI (if needed), mount specimen, & visualize labeled cells

Microscopy

allows researchers to examine biological samples at scale that cannot be seen/resolved by naked eye

Magnification Formula

M_total = M_objective x M_eyepiece

(63x objective & 20x eyepiece = 1260x resolution)

Resolution

how closely spaced two points can be while still appearing separate

Simplified resolution equation

Poor resolution means?

two points appear merged

Better resolution means?

two points appear separate

Brightfield microscopy

uses transmitter light to observe cells and tissue structures

(useful for naturally colored or stained specimen)

Widefield fluoroscence

illuminates entire field simultaneously

(fast & effective for thin samples/out-of-focus light creates blur in thick samples)

Laser-scanning confocal microscope

scans sequentially to produce detailed optical sections

(excellent for fixed, thicker, multicolor tissues, but relatively slow)

Spinning-disk confocal microscope

uses many pinholes to image quickly with optical sectioning

(good for fast live-cell imaging in samples within its practical depth range)

When to use Widefield?

thin culture & fast fluoroscent imaging

When to use laser-scanning confocal microscope?

thick fixed brain tissue & fine detail

When to use spinning-disk confocal microscope?

Fast imaging requiring optical sections

When to use Brightfield?

standard visible tissue staining

Voltage

electrical potential difference

Current

flow of electrical charge

Resistance

opposition to current

Conductance

how easily current flows

Ohm’s Law

V = I R

g = 1/ R

( Voltage (V) = pressure; current (I) = flow)

(Resistance (R) = restriction; conductance (g) = ease of flow)