Pain Quiz 3

Question 1

Central pain control - How normal pain/touch is sent: CNS?

Gate control theory:
- Convergent input in dorsal horn:
- Inhibitory interneurons local spinal cord interneurons:
- Activation of Non-nociceptive fibers(Abeta):
- Just small fiber pain input coming in from the periphery the inhibitory interneuron is

What happens in the spinal cord during chronic pain?

Answer 1

c inhibits to send pian and A beta excites to block the inhibitory interneuron

Central pain control - How normal pain/touch is sent: CNS controls the perception of pain is due to dedicated circuits modulating perception info from peripheral to spinal cord

Gate control theory:
- Convergent input in dorsal horn:
Nociceptive(C fibers) and Non-nociceptive(Abeta fibers) input and synapse same inhibitory neuron

• Inhibitory interneurons local spinal cord interneurons:
  When these are firing, pain signal is not being sent
• Activation of Non-nociceptive fibers(Abeta) can interfere with signals with pain fibers so inhibiting pain in the dorsal horn:
  Sending information from periphery it activities the inhibitory interneuron which keeping the gate closed so pain into not sent even if our small fiber neurons are sending input the large fiber activation is able to override that and keep the gate closed. if the touch info is able to enhance the inhibitory interneuron activity it can close the gate and stop pain from being sent but when stimuli is sig or intense enough to override this inhibition the gate can open and pain can be sent
• Just small fiber pain input coming in from the periphery the inhibitory interneuron is inhibited this reduces and blocks the inhibition of out projection neuron so gate is open and pain is sent

What happens in the spinal cord during chronic pain? Sensitization!

Question 2

Question: In a healthy and functional system, the “gate” acts to reduce painful sensations sent to the brain.

C-fiber input fill in blank (inhibits or excites) the inhibitory interneuron, which in turn fill in blank (increases or decreases) the inhibition of the projection neuron. This fill in the blank (stops or allows) pain info to be sent. A-beta input fill in blank (inhibits or excites) the inhibitory interneuron, which in turn fill in blank (increases or decreases) the inhibition of the projection neuron. This fill in the blank (stops or allows) pain info to be sent.

What do you think will happen to the stimulus-response function in sensitization compared to normal?

True or False: peripheral sensitization can resolve after injury OR can lead to chronic pain.

Answer 2

C-fiber input fill in blank (inhibits) the inhibitory interneuron, which in turn fill in blank (decreases) the inhibition of the projection neuron. This fill in the blank (allows) pain info to be sent. A-beta input fill in blank (excites) the inhibitory interneuron, which in turn fill in blank (increases) the inhibition of the projection neuron. This fill in the blank (stops) pain info to be sent.

What do you think will happen to the stimulus-response function in sensitization compared to normal? Shift to the left

True: peripheral sensitization can resolve after injury OR can lead to chronic pain.

Question 3

What is sensitization?

What can happen in the periphery?
Sensitization at the level of the Nociceptors:

What about in the spinal cord?

Answer 3

What is sensitization? Sensitization is a leftward shift in a stimulus response function: decrease threshold, increase in magnitude of response to a previously effective stimulus. Decreased threshold so increase in response magnitude

What can happen in the periphery?
Sensitization at the level of the Nociceptors:
- Increased excitability decrease threshold
- Enhanced responsiveness increase magnitude to response
- Sometimes increased baseline activity AP sent without input
Sensitizing events and agents:
- Excessive noxious chemical, thermal, or mechanical stimulation
- Inflammatory mediators and algesic agents

What about in the spinal cord?
- Central Sensitization
- Neurophysiological changes that lead to increased responsiveness to normal or subthreshold input
- In dorsal horn these changes include strengthening of synapses and disinhibition
- Neuroinflammation in the dorsal horn promotes central sensitization - hyperalgesia and allodynia

Question 4

Central Sensitization includes all of the following:

Answer 4

Windup: Facilitation of C-fiber evoked responses in dorsal horn neurons following repeated stimulation - C fibers send constant AP can elicit over excavation of dorsal horn so they are winding up or turning up dorsal horn - long lasting

LTP(enhance connection)/Synaptic potentiation: Intense noxious stimulation leads to potentiation of glutamatergic synapses (spinal LTP).
NMDA/AMPA mediated at some synapses.
Increase of AMPA receptors at synapses - takes less glutamate to excite so now have an enhanced response.

Latent Sensitization:
Acute inflammatory injury leads to mechanical hypersensitivity that abates 10 days later
Delivery of naltrexone, a mu-opioid receptor antagonist to block, reinstates hypersensitivity, indicating long-term activity of endogenous opioids
Acute injury like a burn

Disinhibition: Reduced excitatory drive to inhibitory interneurons
Increase in intracellular Cl as a result of down regulation of potassium chloride co-transporter leads to reduced inhibitory transmission and in some cases conversion of inhibitory transmission to excitatory

Question 5

During central sensitization, fill in blank (dorsal horn or somatosensory cortex) neurons exhibit fill in blank (increased or decreased) thresholds and fill in blank (increased or decreased) magnitude of response to effective stimuli. Inhibitory interneuron activity is fill in blank (increased or decreased), leading to fill in blank (increased or decreased) activation of second order neurons.

What Does Central Sensitization Look Like?

Answer 5

During central sensitization, fill in blank (dorsal horn) neurons exhibit fill in blank (decreased) thresholds and fill in blank (increased) magnitude of response to effective stimuli. Inhibitory interneuron activity is fill in blank (decreased), leading to fill in blank (increased) activation of second order neurons.

What Does Central Sensitization Look Like? Electrophysiology and Behavior

Question 6

Functional changes in dorsal horn circuitry classification during Central Sensitization
4 things

Answer 6

LT: low threshold (CMH, AMH etc) - increase hyper response

WDR: wide dynamic range - all types of receptors - increase enhanced

NS: nociceptive specific - high threshold for temp and mechanical - increase so the threshold has lowered

Responses evoked by brushing, pressing, pinching and squeezing the skin are illustrated

Question 7

Electrophysiological and Behavioral Correlates of Central Sensitization

Following injection of the inflammatory mediator Carrageenan into the hindpaw?

Following initiation of arthritis, the injured subject elicits?

Explain the differences between input into LT (low threshold), WDR (wide dynamic range) and NS (nociceptive specific/HT) dorsal horn neurons. What is the activity pattern of these neurons normally? What is the activity pattern of these neurons following sensitization?

Answer 7

Following injection of the inflammatory mediator Carrageenan into the hindpaw, second order dorsal horn neurons exhibit reduced threshold and enhanced excitability. - lower threshold and enhanced responses

Following initiation of arthritis, the injured subject elicits reduced mechanical withdrawal thresholds that are attenuated following injection with an analgesic.

LT respond to non-noxious with low activity,WDR respond to both noxious and non-noxious with increasing activity with intensity, and NS respond only to noxious.

Question 8

Increase of Pro-nociceptive molecular markers in dorsal horn following injury?

This was measured in multiple ways:

Answer 8

Following inflammatory injury, dorsal horn neurons increase expression of Cox-2(target of ibuprofen or aspirin), a part of the prostaglandin pathway

This was measured in multiple ways:
Western blot for protein
QPCR mRNA
IHC protein

Question 9

Substance P (neuropeptide) and Pain

released where following what? causes? receptors?

Spinal neurons that possess the Substance P receptor can be eliminated: 4 things

Answer 9

SP is released into the spinal cord following activation of nociceptors - dorsal horn neuron second order express receptor NK1

Spinal application of SP causes hyperalgesia

Spinal application of SP causes sensitization of dorsal horn neurons - wind up

SP receptors:
The NK1 receptor is a G protein-coupled receptor - metabotropic are slower and longer lasting
G protein-coupled receptors undergo internalization upon agonist binding

Spinal neurons that possess the Substance P receptor can be eliminated:
- SP receptors undergo internalization following agonist binding
- Conjugate a toxin (saponin) to SP and use receptor internalization as the portal of entry into specific cells
- Saporin inhibits protein synthesis —> cell death
- Loss of NK1-containing neurons and effect on pain and central sensitization

Question 10

Central sensitization: Immune involvement?

The following things can lead to central sensitization following injury??

Answer 10

Microglia – macrophages adapted to the CNS
Homeostatic and reparative functions
Mediate host defense

The following things can lead to central sensitization following injury:

Constant c-fiber situation, reduced inhibitory drive, long term potentiation of dorshal horn neurons, immune activation, increased nociceptive NT expression

Not!!! More interneuron activation or anti-inflammatory cascade

Question 11

Following burn injury or something painful, the first initiation is to grab your arm because of the gate theory. What is gate theory? Why does this work? After the burn injury how might peripheral sensitization develop? How might this develop into central sensitization?

Answer 11

Gate theory bc pain info is inhibitory to the inhibitory neurons causing an excitatory response for the C-fiber and A-beta - Abeta override C-fiber creating inhibitory response so pain can not be sent.

C-fiber synapse on inhibitory interneuron and synapse on the projection neuron in dorsal horn L 1 and 2 this goes up to the brain. When this is active this means pain info is being sent. A-beta is non nociceptive mechanical going through the dorsal column medial lemniscal pathway to the brain and sends collateral. C-fiber inhibits the interneuron whereas A-beta excites the interneuron. Hyperpolarizing projection neurons further away from threshold so will take greater excitatory input to overcome it so it does not cure pain.

Inhibitory input blocking projection neuron forms needing pain and it is A-beta canceling out C-fiber activation.
Example is like itching which blocks AP from sending info to the brain.

Peripheral sensitization cavaliers are releasing histamine from mast cells and CGRP which vasodilation that increase blood flow to area in order to increase circulating immune cells which causing inflammation, warm, swelling, and redness - hallmark of peripheral goes beyond the actual injury site so red around the burn site itself. In the Peripheral tissue but once get to the spinal cord and see long term change now in central sensitization.

Peripheral sensitization to develop central sensitization are the four hallmarks of central sensitization - windup, spinal LTP, latent sensitization, and disinhibition.

Question 12

Define Central Sensitization:?

Answer 12

Define Central Sensitization: (pain lasting longer than the healing of injury in the spinal cord and brainstem):

Neurophysiological changes that lead to increased responsiveness to normal or subthreshold input In dorsal horn these changes include strengthening of synapses and disinhibition Neuroinflammation in the dorsal horn promotes central sensitization.
Hallmarks of central and all four do not have to happen to be central and more than one can occur at the same time.
Peripheral somatosensory cortex changes in fibromyalgia alters your perception of pain and not seeing all of the jarmaks in the perception part of our pain triad.

Question 13

Wind Up: ?

Spinal LTP: ?

Latent Sensitization: ?

Disinhibition: ?

Answer 13

Wind Up: Enhanced postsynaptic response mediated by C-fibers - peptidergic and non peptidergic enhance the response
Includes facilitation through NMDAR/AMPAR, peptide receptors(substance P and CGRP and NK1 is what substance p binds to), and ion channels(K,Na)
Mediated specifically by repeated C-fiber stimulation
Can lead to central sensitization

Spinal LTP: mediated via glutamate using glutamatergic NMDA/AMPA that bind glutamate
NMDA allow Na and Ca though but need NMDA for magnesium block to open for long term potential because of the Ca
AMPA bind glutamate and allow Na through for depo
Enhanced response in postsynaptic because more receptors for glutamate to bind to me
Don’t difference between C fibers or A-delta glutamate release so either high threshold mechanical repsortes or C fibers CMH (not specific about source of glutamate)
Facilitation of signaling specifically through NMDAR/AMPAR - glutamate ligand gated
Can lead to central sensitization

Latent Sensitization:
Occurs after injury has healed/pain is gone
Can lead to central sensitization

Disinhibition:
Reduction of inhibitory interneuron activation
Can lead to central sensitization

Question 14

Problem #1 Experimenters were trying to investigate the role of stress in inducing latent sensitization. They used the CFA model of inflammatory pain to induce mechanical allodynia that lasted for 15 days and then abated by three weeks post-injury. Injection of CFA and is an acute temporary pain that stops eventually and is useful for latent sensation because the further along the longer the pain.

If you were designing an experiment to examine this, what are several ways you might try to do so?

To experimentally model stress, researchers injected corticotropin-releasing factor (CRF) following recovery of injury. What does CRF do? What is the HPA-axis?

What does the CFA model do or look like?

What do the above graphs show about the role of CRF in eliciting latent sensitization?

Answer 14

If you were designing an experiment to examine this, what are several ways you might try to do so? Predatory encounter or smells like using the urine to stress the mouse out, Inject cortisol - naltrexone, Sound and temp stress, Changing circadian rhythms for light dark signals, Social, mental, environmental, or chemical ways to induce stress.

CRF Peptide hormone in stress response - involved in endocrine response and relates to the HPA axis. Negative feed loop between hypothalamic and pituitary adrenal axis - CRF released by adrenal gland - part of our stress response autonomic nervous system apart of the feeding, flight, fight, and reproductive behavior - can be visceral organs but also release by sympathetic onto peripheral nociceptor targets

What does the CFA model do or look like? Inflammatory pain to induce mechanical allodynia

What do the above graphs show about the role of CRF in eliciting latent sensitization? Saline before CRF and after contralateral and ipsilateral are the same. Reduced withdrawal threshold so enhanced response so increase magnitude so less needed to respond in ipsilateral and contralateral. Injecting CFA into hindpaw causing reduction in withdrawal threshold in ipsilateral and not contralateral.

Investigate stress to induce latent sensation which is acute inflammation injury that you heal from it and no longer in pain and then some event happens like when have an old injury can flare up that causes mechanical allodynia and they gave them naloxone to try to restart the pain and what is the mechanism. They tried to figure out how stress was able to restart pain after an injury. If we were trying an experimental model to induce stress so what are mice stressed out by predators smells, sound/temp/light or dark, and inject cortisol. CRF peptide hormone that induce corticotropin to release the CRF which induces the release of cortisol into the bloodstream so tried to cause a stressful event and is part of the HPA axis that has to do with the hypothalamic pituitary adrenal axis which the normal adrenal gland has communication with the sympathetic response to reduce into the bloodstream but hypothalamus can induce release of cortisol directly into the bloodstream in an intensive stress situation.
Before given CRF or saline, is the inflammatory molecule to induce an inflammatory pain model inject into feet traisnet pain that lasts for a few weeks after the inject only in ipsilateral not the contralateral, groups that got CRF or saline had almost identical threshold and same recovery. Injecting either CRF or saline into verterics over a period of 150 mins tested von fry that saline in ventricles did not see any significant changes in ipsilateral or contralateral paw.
CRF group saw with the stress response that stress can cause medicinal allodynia so reducing the release of cortisol with CRF itself cause medical alloying in the contralateral paw and the ipsilateral paw has even greater drop so both paws see drop in the withdrawal threshold but more significant in ipsilateral compared to contralateral. Possible that CRF is reducing descending inhibitory input - neurons in brainstem that express opioids onto dorsal horn neurons to act as a pain relief bc exogenous drugs only work bc in your body you already have the rector making for it. So maybe CRF is inhibiting the inhibitory brain stem neurons, injecting lidocaine to block the input on the brainstem which tells us about latent sensitization that it is still inducing the latent but not as extreme or intense. Ipsilateral paw is causing latent but as severe as original injury but also inducing contralateral drop, that is what we don’t see with the naloxone model and don’t see drop with contralateral paw. The disinhibition of brainstem neurons might be part of what is happening with CRF.
Potential mechanisms of CRF induced latent is - injecting CRF post injury can cause a dip in threshold on both sides and the mechanism is disinhibition so something to do with the molecules that get released during the stressful period - CRF induce latent that there disinhibition of brainstem inhibitory neurons - reduction of activity if the inhibitory neurons in the dorsal horn and brainstem so inhibiting and inhibitory neurons. Just bc brainstem lidocaine kinda shows the same thing doesn’t prove anything would need to see more signaling. Fluids in high concentration like to defuse and injecting lidocaine is not a sufficient way because you need to have CRF to show that it is actually a part of this response. Latent is breaking leg then in a period of pain and then reach remission then back to normal pain threshold then something happens and have reintroduction to that pain and you recovering bc you release endogenous opioids and not bc the nociceptors are not sending pain receptors but the endogenous is sending to stop nociceptors os if you block endogenous it brings back that pain. Stress response blocks endogenous opioids receptors which could induce the latent.

Question 15

How do immune cells excite peripheral nociceptors and spinal cord neurons

Cytokine:?

Chemokine?

Answer 15

Cytokine: small any secreted protein released by immune cells that affects the behavior of nearby cells bearing receptors - autocrine action or paracrine action(nearby cells) or distance cells (endocrine action)
Interleukins are cytokines (IL-2 – interleukin 2) - pro/anti inflammatory

Chemokine(subtype of cytokine): small, secreted cytokines typically responsible for chemotaxis - attract and guide migration of other cells
Any cell can produce cytokines, not just immune cells
Chemokines recruit immune cells from blood into tissue

Question 16

Peripheral mediators: Innate immune cells - non specific immune response includes chemical, physical, and cellular defenses against pathogens - immediately respond and prevent pathogens

6 things

Answer 16

Hours to respond

Lack specificity

No memory - no antibodies just generic response bc can not remember

Mast cells: postponed in tissue near blood vessels, release mediators of vasodilation/inflammation, associated with allergy responses

Macrophage: phagocytose pathogens, dead cells/debries, present antigens for adaptive immune cells for activation, produce chemokines and cytokine

Dendritic Cell: Processes and present antigen to T cells to enable activation, produce chemokines and cytokines

Question 17

Peripheral mediators: Adaptive immune cells - also called acquired or specific

8 things

Answer 17

Days to respond - around 96 hours to respond so slow

High specificity for a specific pathogen

Form memory - long lasting effect bc B and T cells

Only found in vertebrate

Can make errors and attack itself so autoimmune diseases

B cell: antigen (foreign protein), produce antibody, present antigen to T cells

CB8+T cell “Cytotoxic” T cell: antigen (foreign peptide), recognize peptide presented on MHC class 1 (all cells express), kill infected cells

CD4+ T cell “Helper” T cell: Antigen(foreign peptide), recognize peptide presented on MHC class 2 (only antigen presenting cells express), produce cytokines to help activate B cells and CD8+T cells

Question 18

Question: Which peripheral immune cell produces antibodies to pathogens? fill in blank (B cells; CD4 T cells; Macrophages; Dendritic cells) Are they part of the innate or adaptive immune system? fill in blank (innate or adaptive)

5 Hallmarks of inflammation?

Involvement of the immune system in peripheral sensitization:?

Question: Postulate the events that occur after a cut becomes infected.
- What innate immune cells are immediately active following the infection?
- What cells produce antibodies to the infection?
- What might nociceptors release to induce swelling in the area?
- How might nociceptors become activated and send pain signals?

Answer 18

Question: Which peripheral immune cell produces antibodies to pathogens? fill in blank (B cells Are they part of the innate or adaptive immune system? fill in blank (adaptive)

Hallmarks of inflammation: hear, redness, swelling, pain, loss of function - cause by cascade of immune cells

Involvement of the immune system in peripheral sensitization: tissue injury and infection causing inflammation via leakage of plasma but also infiltration of immune cells such as T cells, mast cells, macrophages, release inflammatory mediators such as Brady Canaan, prostaglandins, ATP, and proinflammatory then Ca cAMP second messengers and then release kinases to increase neuronal excitability to send higher frequency of AP in response to stimuli. Dual activation and interplay of nociceptors in the immune system ensures rapid localized initiation of innate defenses, recruitment and priming of immune cells, and nosoffensive or protective behaviors to prevent additional damage and allow healing.

Question: Postulate the events that occur after a cut becomes infected.
- What innate immune cells are immediately active following the infection? Mast cells, Macrophages, and Dendritic Cells
- What cells produce antibodies to the infection? B Cells
- What might nociceptors release to induce swelling in the area? Proinflammatory cytokines
- How might nociceptors become activated and send pain signals? interplay of nociceptors in the immune system ensures rapid localized initiation of innate defenses, recruitment and priming of immune cells, and nosoffensive or protective behaviors to prevent additional damage and allow healing

Question 19

What about the Central Nervous System??? In peripheral have multiple immune cells but in CNS only microglia

derived?

Microglia enter the brain when?

In the mature brain, they are found ?

Each cell seems to occupy ?

After a pathological event?

Answer 19

Bone marrow derived cells - monocyte lineage same precursor macrophages

Microglia enter the brain during development

In the mature brain, they are found almost evenly dispersed throughout the CNS

Each cell seems to occupy a defined territory

After a pathological event, these cells undergo a transformation from queisecent to an activated or inflammatory phenotype which take ameboid shape
- Transformed cells acquire amoeboid shape
- Have the capacity to migrate, proliferate(multiply), and phagocytose

Question 20

Microglia

Constitutes how much of total CNS glia?

Resident immune cells cells?

Network of surveillance cells
respond to?

Pro- and anti? factors??

Microglial activation in the spinal cord following peripheral nerve injury ?

What are things that you predict microglia might release that can activate nearby neurons and increase hyperexcitability?

Answer 20

Constitutes 20% of total CNS glia population, on average 10,000 microglia per mm3 (in mouse)

Resident immune cells cells – first line of defense in CNS
Antigen presenting cells, phagocytose debris, communicate with nearby neurons

Network of surveillance cells
respond to slightest alteration in their environment

Pro- and anti-inflammatory - microglia look fat and large
- Release a number of pro-inflammatory factors when reactive, e.g. cytokines, chemokines prostaglandins, growth factors, NO (nitric oxide)

Microglial activation in the spinal cord following peripheral nerve injury - white matter, ventral horn, but mostly dorsal horn - sciatic nerve L3-L5 not above or below not entire spinal cord for microglia

What are things that you predict microglia might release that can activate nearby neurons and increase hyperexcitability? microglia can excite each other by proinflammatory cytokines - prostaglandins, ATP
Paracrine fashion

Question 21

First order neurons release neurotransmitters that _______________ second order neurons and microglia. Microglia then releases ______________ to activate neurons pre and post synaptically. This causes neurons to show _____________ and can lead to ______________.

Answer 21

excite

cytokines

hyperexcitability

pain

Question 22

P2X4 receptors induced in spinal microglia gate mechanical allodynia after nerve injury - which side and produce?

Microglia can switch from undirected surveillance to targeted movement of processes: ?

After injury microglia display signs of ?

Inhibition of microglia using “general” glia?

Factors that serve as neur-glia messengers and contribute to experimental pain states?

Answer 22

Nerve injury → P2X4R increase on ipsilateral side specifically in spinal microglia

Knock-down of spinal P2X4R activity suppresses tactile allodynia

Spinal administration of microglia, in which P2X4R is induced and stimulated, produce tactile allodynia in naïve rats

Microglia can switch from undirected surveillance to targeted movement of processes:
Microglia are rapidly activated after exposure to ATP

After injury microglia display signs of an enhanced reactivity in a number of different experimental models of pain

Inhibition of microglia using “general” glia inhibitors such as minocycline or specifically targeting ATP receptors attenuates(reduce) pain-like behavior in models

Cytokines and chemokines such as TNF and IL-1 beta, but also lipids, growth factors and ATP, have been identified as factors that serve as neur-glia messengers and contribute to experimental pain states

Question 23

Macrophages: 4

B Cells: 2

CD4 T Cells: 4

Mast Cells: 2

Dendritic Cells: 2

CD8 T Cells: 2

What is the difference between the initiate and adaptive immune system:?

What is difference between the peripheral and CNS immune system:?

Answer 23

Macrophages: release inflammatory, phagocyte debris/pathogens, produce chemokines and cytokines, presents antigens to T-cells

B Cells: produce antibodies, present antigen to T-cells

CD4 T Cells: recognize antigen peptide on MHC(either class), produce cytokines to help activate B cells and CD8+T cells, produce chemokines/cytokines (chemokines release inorder to recruit more cells so attract and cytokines can act on Toll receptors on the cells in questions and on immune cells), present antigens to T-cells

Mast Cells: release mediators of vasodilation/inflammation, associated with allergy responses for histamine (both dangerous allergies and normal)

Dendritic Cells: present antigens to T-cells, produce chemokines and cytokines

CD8 T Cells: kill infected cells and recognize antigen peptide on MHC 1 and 2

What is the difference between the initiate and adaptive immune system: adaptive slower is specific with memory to antigen being slow and initiative is fast, no memory no specific short lasting. B cells make the antibodies in the adaptive.

What is difference between the peripheral and CNS immune system: In the peripheral have multiple immune cells like macrophages, mast cells, dendritic cells, and those in adaptive such as B cells, CD4 and CD the T cells but in CNS only microglia but they can’t make antibodies but are antigen presenting cells so adaptive can interact with microglia - only innate immune cell in CNS bc they share the same lineage as macrophages and travel into the early nervous system by macrophages before creation of the BBB has to do with timing of development.

Question 24

What is a complement system? Is it a part of the innate or adaptive immune system?

C3aR1 expression and figure out which cell type expresses the receptor. They are stained for neurons (NeuN), microglia (Iba1), and astrocytes (GFAP).
Based on the figures below, which cell type expresses C3aR1?

Answer 24

The complement system is a part of the innate immune system as it is a group of proteins found in the blood and tissues that, when activated, work together to detect and respond to pathogens, damaged cells, or immune complexes. There are several pathways (classical, lectin, and alternative) through which the complement system can be activated, and each leads to a cascade of events where proteins interact in sequence, amplifying the response. It’s a part of the immune system that functions by activating a cascade of protein interactions when it detects pathogens, damaged cells, or immune complexes, leading to their destruction through various pathways like the classical, lectin, and alternative pathways. - complement existing circulating immune cells and complement their functions - primarily inflammation

microglia (Iba1)

Question 25

After determining the microglia cell type that expresses C3aR1, they wanted to examine TLQP-21 specific activation of dorsal horn cells. They did calcium imaging in spinal cord slices, and they used different markers to confirm which cell type TLQP-21 is activating.

What is Fura2-AM used to label?

How does Fura2-AM imaging show cellular activation?

What is SR-101 used to label?

What does the graph below show regarding Fura2-AM and SR-101 overlap?

Answer 25

What is Fura2-AM used to label? calcium ion-binding fluorochrome used in living cells to measure cytosolic calcium mobilization

How does Fura2-AM imaging show cellular activation? measure changes in calcium concentration which is a key indicator of cellular activation; when a cell is activated its intracellular calcium levels often rise leading to a detectable increase in fluorescence - increase in bound Ca for a period of time and a decrease in the unbound Ca and then graph the relative ratio of bound vs unbound Ca which will show actual raw peak point bc they diff fluorescent profiles

What is SR-101 used to label? red fluorescent dye that labels astrocytes primarily and some oligodendrocytes(located closer to axons) and we were looking at cell bodies on dorsal horn gray matter so that would be astrocytes - SR-101 just a marker not a functional dye

What does the graph below show regarding Fura2-AM and SR-101 overlap? If no Fura-2AM is filling SR-101 positive cells, can you rule out signaling in astrocytes occurring? Why or why not? There is no overlap between the two which means when looking at Ca imaging in these slices then you can not rule out signaling for astrocytes bc cant stay if they are active they just are not taking up the dye - SR-101 have not taken up the Fura2-AM dye. Astrocytes are not the cells likley to take up TLQP-21 bc they don’t have the rector; it is more likely microglia.

Question 26

Now that they determined that none of the positive Fura2-AM signals would be in astrocytes, they tried to figure out specifically if they could look at microglial signaling. As there is not an effective dye to look at microglia while calcium imaging, they wanted to use a genetic model, with a mouse that expresses GFP in their microglia. Iba1 is microglia and GFP is an unknown cell type of target cell and fluorescent marker protein.

Based on the images below, do you think they found an effective model to label microglia? Why or why not?

Now that researchers found a genetic model that had the ability to visualize microglia at the same time as calcium imaging, look at the image below.

Is there overlap in GFP and Fura-2?

Does every Fura-2 positive cell overlap with Iba1?

On the right shows a GFP positive/Fura2 positive cell in (n) and a GFP negative/Fura2 positive cell in (o). What is your interpretation of these results?

Answer 26

Based on the images below, do you think they found an effective model to label microglia? Why or why not? Yes bc the overlap is high with 120 cellular profiles per section of Iba1 and GFP overlap. There are some Iba1 positive microglia on each slice that don’t express GFP, when doing Ca imaging looking at Fura2 signaling Ca fluxes are not happening in the astrocytes so only looking at microglia and neurons and the microglia are label with GFP and can be confident that a positive GFP is cell is microglia but there is a positively that a microglia that doesn’t express GFP but takes up Fura so off change it could still be a microglia and not neuron

Is there overlap in GFP and Fura-2? Yes

Does every Fura-2 positive cell overlap with Iba1? What do you think this means? Not every cell overlap which means that each one does not to be microglia and we know are not astrocytes so could be neurons

On the right shows a GFP positive/Fura2 positive cell in (n) and a GFP negative/Fura2 positive cell in (o). What is your interpretation of these results? Positive cell microglia respond to GFP TLQP-21 and little glutamate and the neurons do not respond to GFP but they do respond to the glutamate a lot. N are microglia and o are neurons.