MCB 170 Midterm 2

electrophysiology

• study of electrical activity in neurons

• use electrodes

  • by nearby neurons or inside them

  • extracellular/intracellular

voltage clamp

• Cole, Hodgkin, Huxley - studied w/ squids because you can see their axon

• cells held at certain membrane potential and measure current through voltage-gated ion channels

  • study how action potential is impacted by ion flow/currents and if currents depend on voltage/membrane potential

• on segment of axon

  • reference electrode outside

  • recording electrode inside

  • current-electrode sets membrane potential

Steps of voltage clamp

1. depolarize axon to set membrane potential

2. voltage-gated sodium channels open —> Na+ entering cell

3. equal but opposite charge (aka - ) added to keep at 0mV

4. voltage-gated Na channels inactivate as sodium ones open —> K+ entering cell

5. equal but opposite charge (aka + ) added to keep at 0 mV

electroencephalogram (EEG)

• measure electrodes on surface of cell to pick on activity of multiple neurons

• less invasice

• study sleep

patch clamping

measure electrical current by ion channels by clamping onto the membrane/channels

local field potential (LFP)

• measure electrodes directly in brain & tissue to pick of activity of multiple neurons

• more invasive

• can pick up dendritic activity

staining & microscopy

• preserve and cut tissue

• use stain to visualize cells

• formalin (FFPE): used to cut tissue

golgi stain

• staining tech. using Ag

• detailed, but only look at a small subset of neurons

nissl stain

• staining technique using cresyl violet

• binds to nucleic aids and ribsomal DNA to examine the cell body

immunohistochemistry

• primary antibody directed onto antigen on cell of interest

• secondary binds to primary

  • tagged w/ fluorescent dye for visualization

  • strengthens signal

electronmicroscopy (EM)

• view thin sections of tissue - ultra-structure

  • view w/ fine detail & resolution

• view molecular structure (ex. myelinating axons)

connectomics

determine structure of NS by mapping neural connections

neuroatomical track-tracing techniques

• antereograde: cell body —> axon

• retrograde: axon —> cell body

• fluorescent + immunohistochemistry + EM

• see what neurons lead to which one

• harder to see circuits

viruses and tracing techniques

• infect neurons and spread to other neurons

• question of toxicity of viruses (need to find safe ones)

• use antero and retro directionality

• more specific than conventional tracing (can cause false positives)

• viral vectors + immuno & fluorescent proteins to help visualize

model organisms

• compare effects w/o worrying about ethics

  • ex. mice

model organisms and studying neurons/glia

• use fluorescent proteins (light shined will have light shine back)

  • genetic engineering in cells of interest

GCaMP

• engineered Ca²⁺ indicator

• fluorescent light glows when Ca²⁺ binds, showing neuronal activity

in vivo

testing within live organsim

in vitro

• in glass/dish cell culture allows for study cellular environment outside of the organism

• can manipulate one cell type and see how it responds to drugs, chem. cues, etc.

• can harvest DNA, RNA. proteins

  • see change in gene expression/proteins

  • see genes involved in growth & injury

• glia also isolated (microglia/astrocytes)

embryonic origin of NS

formed by ectodermal layer germ layer

formation of CNS from ectodermal layer

• folding of neural tube —> vesicles forming regions of the brain

• neural plate neuralizes first

  know order

formation of PNS from ectodermal layer

• neural crest folds to from the neural plate

• neural crest cells are derived from neural fold

  • migrate to periphery to create other cells (peripheral neurons & Schwann cells)

radial glial cells

• parents cells (progenitors) for other cell types (neurons & glia)

  • neurons 1st, then glia

neuronal migration

• raidal glial cells provide scaffold of newborns to migrate

• neural precursors (cells that become neurons) migrate along radial glial cells

• start at ventricular zone and migrate up

  • process, trailing, migrating, leading formation

symmetrical cell division

one parent radial glial cell = two daughter radial glial cells

asymmetrical cell division

one parent radial glial cell = one neural precursor daughter cell (which will become

neuron or glia) + one daughter radial glial cell

techniques to study radial glial cells

• fluorescent protein (GFP)

• viruses

• video microscopy to study w/ timelapsing

order layers of neurons in the cerebral cortex form

neurons migrate from deepest layer —> top (inside —> out)

way neurons know where to go

• axons sense neurotrophic factors: guidance cues and growth factors

  • bind to specific receptors on growth cone: growing tip of axon/dendrite

    • no receptor = grows towards nowhere

  • cause cytoskeleton to remodel to grow towards/away (attracted/repulsed)

  • goal: to go to muscle, organ, skin, other neurons

• neurite: growing extension of neuron that becomes dendrite/axon

• use Ca²⁺ indicator dye to show how calcium is important for signaling movement of growth cone and axon

motor neurons traveling long distance to find target

• cross over midline from one side of cell body to another at the pyramidal decussation in corticospinal track

  • require series of repulsive/attractive gradient cues to guide it

  • target secretes factors promoting growth of axons towards them

• take foreverrrrrr tho ;-;

polarity by guidance cues

• semaphorin 3A is an example of guidance cue protein

• determines polarity

• basil end (low S-3A; repulsed); apical end (high S-3A; attracted)

how do synapses form

when the axon reaches the target!

formation of motor neuron synpases @ neuromuscular junctions

• motor neuron axon secretes factors (Agrin?) to receive ACh

  • sets up post-synaptic side

• action potential —> synapse —> ACh release

• muscle fibers open so Na⁺ enters muscle fiber

  • EPSP so muscle contracts

pruning

• form of neuroplasicity occurring when too many synapses made in development

• made redundantly for functionality (in case something goes wrong)

synapse elimination at neuromusclar junction (PNS)

• before: muscle fibers receive input from multiple motor neurons

• after:

  • AChR blockage in small area (toxin) causes synapse to be eliminated, so neuron retracks

  • one neuron per fiber (one neuron can connect to multiple fibers though)

  • determined by activity: stronger input causes specific neuron to be kept

    • loss of all input = death

synapse elimination in CNS

• neuron-neuron connection

• fliopodium - portion of neuron - contracts axon

  • vesicle recruits on pre-synaptic side

  • postsynaptic NT receptors form clusters

• microglia do pruning by engulfing the synapse

trophic support

• provide nourishment to target tissue by secretion by other tissues

• lowered strength connections means lowered trophic support so the cell dies

• “support” —> tells cell to keep going <3

• limited supply means ones more likely to survive get it

what do memory & learning impact?

#plasicity

Aplysia study

• studied because of fewer nerves & simple circuits

• study gill withdrawal reflect

1. stimulating siphon —> sensory neuron releases glutamate —> stimulated motor neuron —> withdrawal

• habituation: constant weak signals result in no response (no withdrawal and lowered glutamate release)

2. shocking tail —>sensory neuron serotonin level increase —> glutamate released from 1st circuit —> stimulated motor neuron —> withdrawal

• strengthen withdrawal response

• sensitization of tail if touched

olidendrocytes and response to injury (CNS)

• demyelination of myelin changes action potential propagation

  • can lead to Multiple Sclerosis (MS)

• olidendrocyte precursor cells (OPCs): stimulated to promote remyelination following injury

  • make into olidendrocytes

  • MS - inflammatory environment makes inhibits OPC from becoming olidendrocytes

astrocytes and response to injury (CNS)

• become reactive following injury

• GFAP gene expression increases

• astroglyosis: proliferate/divide and create scars

• inc. immune cell infiltration & BBB maintenance/repair

• coral peripheral immune cells/amplify inflammatory response

microglia and response to injury (CNS)

• typical function: reg. neuronal activity, synaptic plasticity,, BBB maintenance

• after injury:

  • large-scale cytokine prod. (tell cells to move/act + coord. immune response)

  • recruit peripheral immune cells

  • pathogen destruction

  • debris clearence

  • tissue repair

  • microglia changes shape

neurovascular unit and response to injury (CNS)

• after traumatic brain injury (can lead to neurodegen. disorders if constant)

  • reactive astroglosis

  • neuronal death

  • olidendrocyte dysfunction

  • microglia activates

  • pro-inflammatory cytokines

• damage can be caused by peripheral vascular problems (can lead to dementia)

• hypotension, high BP as cell walls lose plasicity

• BBB breakdown w/ natural aging

PNS nerve regeneration

• by Schwann cells, immune fibroblasts (mesenchymal) ,macrophages

• Schwann cells migrate along vasculature, taking axon to target as it grows along track made by it

• repair Schwann cells help axon regrow to debris

• everything past injury is GONE

• Bands of Bünger: forms new bridge for axon to grow w/ factors to help w/ regrowth

CNS nerve regeneration

• inhibitory to axon regeneration

  • CNS molecules inhibiting its growth

    • ex. astrocytes secreting extracellular matrices telling it not to grow

• no macrophages to clean debris