Connectome I Research Techniques

Front 1

Psychology according to Simon

Back 1

subjective experience of neural activity

Front 2

A 95% CI (confidence interval) is calculated as

Back 2

xi ±1.96Xsd

Front 3

Pharmacology acc to Simon

Back 3

Interface between neurochemistry and function

Front 4

Neuroanatomy

Back 4

the physical organisation of neural circuits

Front 5

Gene expression

Back 5

the synthesis of chemicals, neurotransmitters and ion channels

Front 6

Circuits dictate

Back 6

function

Front 7

If we think of Neurons as the unit of computation action potentials are

Back 7

the unit of information

Front 8

Properties emergent from the circuit should be evident in

Back 8

populations of neurons that compose that circuit

Front 9

Objective of functional study

Back 9

to verify that circuit components (i.e. neurons) behave in a way consistent with the predicted behaviour of the circuit as a whole

Front 10

To understand the ‘how’ of neuronal circuitry you need to know

Back 10

which individual neurons are involved – single neuron resolution is essential

Front 11

Neuronal activity ( definition)

Back 11

the temporal profile of action potential (spike) discharge

Front 12

Features of neuronal activity

Back 12

binary event- all or nothing, size unimportant- it’s all about the timing, streams of data

Front 13

Concepts of neuronal activity

Back 13

ongoing discharge, evoked discharge, firing rate, volley

Front 14

Brain imaging technologies / neuronal circuitry

Back 14

current technologies: spatial resolution 2-3mm/ 8-27microm, temporal solution 10-30 ms / action potential 4 ms, future technologies: theoretical limitations (magnet power) $$$, experimental limitations

Front 15

EEG measurement of neuronal activity

Back 15

can’t record deep in the brain, spatial resolution even worse than fMRI, at least it measures electrical activity

Front 16

Active neurons generate

Back 16

electrical fields / electrophysiology

Front 17

Electrocorticography ECoG

Back 17

measures local field potentials, wobbles and beta rhythms interesting but do not offer insight into the behavioural mechanisms of cells

Front 18

Extracellular recording

Back 18

electrode measuring 30 microns in diameter, measuring activity of a single neuron, binary info on action potentials

Front 19

Intracellular recording

Back 19

electrode measuring 50 nanometres measuring inside the cell, measures action potentials but also post synaptic potentials

Front 20

Electrophysiology

Back 20

branch of research measuring the electric field generated by excitable cells to measure their activity directly

Front 21

Considerations in Electrophysiology

Back 21

Electrode size determines the number of neurons recorded, invasive – ethical considerations, painstaking – one neuron at at time, BUT allows to directly monitor neuronal activity

Front 22

Two approaches to electrophysiology

Back 22

in vitro, in vivo

Front 23

In vitro

Back 23

samples of living brain tissue from experimental animals ( or human brain biopsy), cut into slices 0.3mm thick, kept alive in artificial cerebrospinal fluid, connectivity intact

Front 24

In vivo

Back 24

mostly anaesthetised experimental animals ( although implantable recording devices become available for animals or human subjects), expose brain and penetrate with electrode mounted on motorised drive

Front 25

The main physiological driver of breathing

Back 25

CO2, respiratory rate is highly sensitive to carbon dioxide (much more sensitive than to oxygen)

Front 26

CO2 concentration in expired air

Back 26

3.5 -5%

Front 27

Introduction of >3.5% CO2 into breathing canal in rats leads to

Back 27

almost immediate increase (20 sec delay) of breathing intensity and rate

Front 28

Same effects as increase of CO2 rate is evoked by

Back 28

acidification of the brain stem

Front 29

Acidity in body fluid is important because

Back 29

it changes the shape of proteins – denaturation

Front 30

Natural acidity level / physiological range

Back 30

7.42 – 7.46, needs to be regulated very carefully

Front 31

Relation of acidity in brainstem to neuronal firing

Back 31

the lower the pH, the faster the firing and the higher the amplitude see image

Front 32

Hypothesis emerging from brainstem –acidity observations

Back 32

neurons somewhere in the brainstem sense acid/CO2 and drive respiratory responses to acidosis, but where?

Front 33

Patients missing Phox2b gene suffer from

Back 33

congenital central hypoventilation syndrome, Ondine’s Curse – insensitivity to CO2

Front 34

Immunohistochemistry

Back 34

research approach that makes proteins visible

Front 35

Immunohistochemical detection of Phox2b showed

Back 35

dense cluster of nuclei in the caudal ventral brain stem as well as wider spread in lateral, ventral rostral area see image

Front 36

Improved Hypothesis on CO2/ acidity effects in brainstem

Back 36

ventral brain stem Phox2b-expressing neurons in the ventral brainstem are CO2 sensitive and underlie respiratory responses to acidosis

Front 37

Retro-trapezoid nucleus RTN is located on the

Back 37

ventral medullary surface

Front 38

Are RTN neurons sensitive to CO2 in vivo:

Back 38

record RTN Neuron in anaesthised rat -> change CO2 -> does neuronal activity increase see image

Front 39

Phrenic nerve controls

Back 39

diaphragm contraction, breathing

Front 40

While measurement of RTN neurons showed a clear correlation between CO2 and firing rate this is not direct evidence because

Back 40

the cell could be stimulated by another cell that is detecting (maybe far away in another brain region

Front 41

Advantage of measurement of RTN neurons sensitive to CO2 in vitro

Back 41

we can add synaptic blockers that eliminate the possibility of neurons being stimulated from somewhere else

Front 42

How do we confirm that CO2 sensitive RTN neurons contain Phox2b?

Back 42

electrically charged phosphorescent dye is injected via recording electrode, positive charged dye pulse through electrode

Front 43

Biotinamide was used as a functional marker indicating

Back 43

the firing of the cell, when overlayed with Phox2b expression, yellow in red indicate green Phox2b marker underneath

Front 44

Neuronal function aspects

Back 44

sensitivity to stimuli, relationships of activity to network outputs, pharmacological sensitivity

Front 45

Neuronal structure aspects

Back 45

morphology, neurochemical content, genetic profile

Front 46

Neuronal activity influences

Back 46

protein transcriptions: scaffolding, synaptic plasticity, calcium dependent messenger pathways

Front 47

Immediate early Genes

Back 47

normally quiescent class of genes that are activity gated, therefore biochemical markers of neuronal activity, Genes (and their protein products) that are rapidly (within 20min) and transiently upregulated by periods of activation

Front 48

With immediate early genes protein expression can be

Back 48

an index of cell activity

Front 49

Two major IEGs in neuroscience

Back 49

c-Fos and Arc

Front 50

Theory of Fos Expression

Back 50

When synaptic driveGlutamatergic input -> neuronal activity -> biochemical cascade of secondary messengers -> drives transcription of Fos – protein

Front 51

Fos expression in behaving animals reflects a summation or integreation of neuronal activity-dependent calcium influx over seconds to minutes and only ___________ ______________ activity over this time frame will increase calcium levels enough to induce Fos

Back 51

strong, prolonged

Front 52

Example of Fos expression in rats exposed to Cats and controls

Back 52

see image

Front 53

Advantages of Fos expression activity marking

Back 53

technically simple, great for binary questions, cheap, high throughput ( whole lot of cell tissue can be processed in one go)

Front 54

Disadvantages of Fos expression activity marking

Back 54

all or nothing ( cells are on or off), timecourse is difficult, stimulus needs to be long lasting, never quite know which aspect of stimulus you’re measuring, assumes that fos-expression corresponds to neuronal activity, some cells express fos more readily than others (very little in spinal chord)