Investigations into ASIC2b Expression and Implications for

Investigations into ASIC2b Expression and Implications for Neuronal Death

A Senior Honors Thesis

Presented in Partial Fulfillment of the Requirements for graduation with research distinction in Biology in the undergraduate colleges

of The Ohio State University

by Kirsten Loomis

The Ohio State University

June, 2009

Project Advisor: Dr. Candice C. Askwith, Department of Neuroscience

Investigations into ASIC2b Expression and Implications for Neuronal Death Kirsten G. Loomis

Introduction Acquired brain damage is a leading cause of death and long-term disability

throughout the world. It can be caused by a number of pathological events including

traumatic injury, hypoxia, infection, inflammation, seizure, and stroke. Approximately 5.3

million people in the United States live with disabling brain damage, representing 2% of

the total population [1]. By understanding the mechanisms which lead to cell death

following cerebral insults and injuries, we will be better able to develop treatments to

prevent brain damage.

Extracellular Loop

Transmembrane Domain

Figure 1. ASIC protein in a cell membrane. Three ASIC subunits come together to form the functional ion channel. This may be composed of homomultimers (one subunit) or heteromultimers (different subunits).

Extracellular Loop


Extracellular Loop


Figure 1. ASIC protein in a cell membrane. Three ASIC subunits come together to form the functional ion channel. This may be composed of homomultimers (one subunit) or heteromultimers (different subunits).

Brain damage is caused by death of neuronal cells (neurons) which generate

electrical impulses and form the circuitry of the brain as well as death of glia, the

supportive, structural cells of the brain. Surprisingly, the majority of neuronal death

which causes brain damage occurs after the initial injury in a process called “secondary”

damage [2]. This secondary damage occurs due to the pathophysiological changes that

occur minutes, hours, and days after the initial injury [3, 4]. This long time frame allows

ample opportunity to intervene and pharmacologically prevent secondary neuronal

damage. However, no effective

pharmacological treatments exist to

prevent secondary neuronal damage

and previous potential treatments

have failed in clinical trials [5]. This

suggests that our current

understanding of secondary damage

is incomplete.

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Recently, a novel mechanism of neuronal death causing secondary injury has

been proposed. It is established that the brain becomes acidic during the time of

secondary injury following cerebral injuries [6, 7]. Acidosis develops gradually and the

severity of injury often correlates with the severity of acidosis. Brain tissue acidosis has

been shown to play a large role in neuronal death in several models of acquired brain

injury [8, 9]. Recent data shows that prolonged acidosis can cause neurons to die due

to specific activation of a family of proteins called the acid-sensing ion channels [8-10].

C-terminusN-terminusASIC2a

Transmembranedomain 1


ASIC2bN-terminus C-terminus



ASIC4Transmembrane

domain 1Transmembrane

domain 2

N-terminus C-terminus

Splice site

Splice site

Figure 2. Differences in the ASIC subunits that modify ASIC1a expression characteristics.







ASIC4Transmembrane


domain 2


Splice site

Splice site








ASIC4Transmembrane


domain 2


Splice site

Splice site







ASIC4Transmembrane


domain 2





ASIC2bASIC2bN-terminus C-terminus



ASIC4Transmembrane


domain 2


Splice site

Splice site


Acid sensing ion channels (ASICs) are found in neurons and normally function in

learning and memory, as well as fear-related behaviors [11, 12]. These proteins form

channels in the cell membrane and allow charged ions to pass into the cell, producing

electrical current. ASICs are activated in response to extracellular protons; a high proton

concentration during acidosis causes channels to rapidly activate. Prolonged acidosis

induces inappropriate activation of ASICs, a flood of calcium enters the cell upsetting

the normal cellular physiology, and neurons die [9, 10]. In mouse models of stroke,

animals lacking the specific ASIC

subunit ASIC1a show substantially

less brain damage [9].

Administration of an ASIC1a

blocker following stroke in mice

also decreases brain damage by

60% [13]. Recent data indicate that

ASIC1a activation may also play a

role in neuronal death during

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multiple sclerosis [8]. Thus, ASIC1a is likely involved in many forms of acquired brain

damage. Yet, there are other ASIC subunits expressed throughout the brain which play

an unknown role in acidosis-induced

neuronal death.

3.

?

3.

?

ASIC channels are composed

of 3 distinct acid-sensing subunits

[14] (Figure 1). ASICs can be

homomeric (composed of 3 identical

subunits) or heteromultimeric

(composed of different subunits) [15,

16]. There are six different ASIC

subunits: ASIC1a, ASIC1b, ASIC2a,

ASIC2b, ASIC3 and ASIC4. ASIC1a,

2a, 2b and 4 are expressed in the

brain (Figure 2). ASIC1a homomultimers and ASIC1a/2a heteromultimers produce

current. To date, only ASIC1a homomultimers are known to contribute to acidosis-

induced death. ASIC2b, a splice variant of ASIC2, does not form proton gated

homomeric channels, but heteromultimerizes with other subunits with unique

characteristics. Our data indicate that ASIC1a and ASIC2b form heteromultimeric ion

channels, but the function of ASIC1a/ASIC2b heteromultimeric channels remains

unknown.

My project has focused on understanding the role of ASIC2b in central neurons.

Our data suggests that ASIC2b may be neuroprotective (Figure 3). My goal has been

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(1) to identify how ASIC2b relates to neuronal death and (2) to asses ASIC2b

expression in the brain using real-time polymerase chain reaction (qPCR). Relative

ASIC2b expression levels in response to specific conditions have not yet been

assessed. We hypothesize that changes in the expression levels of ASIC2b under

different conditions will affect susceptibility to acidosis-induced death.

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1. Experiment of ASIC2b in Neuroprotection

ASIC1a contributes to neuronal death following prolonged acidosis [9]. ASIC2a

can form functional homomultimers, however, ASIC2a homomultimeric channels require

pH < 5.0 solution for activation [15]. ASIC2a can form heteromultimers with ASIC1a

channels with reduced sensitivity to protons than ASIC1a homomultimers, and plays an

unknown role in neuronal damage. The role of ASIC2b is even less understood. We

have analyzed the electrophysiological characteristics of ASIC1a -/- hippocampal

neurons in culture (Figure 4). We discovered that acid-evoked current was absent in

the ASIC1a -/- hippocampal

neurons as compared to

wild type neurons. Acidosis

assays were also conducted

on ASIC1a -/- hippocampal

neurons. In wild type

neurons, acid evokes

approximately 30-35%

death compared to a no-

acid control. In ASIC1a -/-

hippocampal neurons

sustained approximately

equal death to the no-acid

control indicating acidosis-induced death is dependent on presence of ASIC1a and,

thus, proton gated current (figure 4). We conducted a study in which ASIC2 -/-

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hippocampal neurons were

transfected with ASIC2b

(Figure 5). In empty vector

transfected neurons, current

was evoked similar to that of

wild-type hippocampal

neurons. In ASIC2b

transfected ASIC2 -/- hippocampal neurons current was absent. We also found a

decrease in cell death in ASIC2b transfected wild type (ASIC2 +/+) hippocampal

neurons following acidosis (Figure 6). Only 5-10% acid-induced neuronal death was

observed in the ASIC2b transfected neurons as compared to 35-45% acid-induced

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death in empty vector transfected cells. This implies that ASIC2b is neuroprotective in

acidosis-induced neuronal death, and could lead to less neuronal death following stroke.

2. Relative expression of ASIC2b in the brain

We assessed RNA levels as a first step in determining relative contribution of

ASICs in the brain as specific antibodies to individual ASIC subunits are not yet

available. It is known that ASIC1a can form functional homomultimers that contribute to

acidosis-induced neuronal death. ASIC2a, 2b and 4 also form heteromultimers with

ASIC1a (Figure 7), and all homomultimeric and heteromultimeric channels are

expressed in neurons. If ASIC2b is forming heteromultimers with ASIC1a and these are

neuroprotective, the abundance of ASIC2b relative to the abundance of other subunits

may affect acidosis induced neuronal death. We chose to investigate ASIC expression

in specific brain regions of animals of different sex, age, and exposure to psychological

stress. Relative findings will

need to be further examined

to determine ASIC2b’s

contribution to ASIC function

and neuroprotection.

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Brain Region: Different areas of the brain are differently susceptible to ischemic

injury, as well as differently susceptible to acidosis. We chose to look at the

hippocampus, the cortex, and the striatum. It is known that the hippocampus is more

susceptible to selective neuronal loss and injury [17] even after global ischemic events

in the brain. In studies of gerbils and rats, selective neuronal damage in the

hippocampus can

be observed after

occlusion of blood

flow to the brain

after 3 minutes

and 10 minutes

respectively. Rats

sustained

selective neuronal

damage to the

cortex and striatum after 20-30 minutes of occlusion, indicating these tissues are less

susceptible to selective neuronal loss and injury than the hippocampus [18]. The

striatum has also been analyzed to have high presence of ASIC4 protein [19], [20].

Using real-time PCR (qPCR) we analyzed ASIC mRNA expression of the hippocampus,

cortex, and striatum for ASIC expression (Figure 8).

Real-time PCR is used to quantify starting material in a sample, and uses

fluorescent DNA binding dyes to measure a threshold of detection above background

fluorescence at a certain cycle in the PCR. This is referred to as the cycle threshold, or

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Ct of a sample. Data are reported as the change in cycle threshold (∆Ct) calculated by

ASIC Ct minus the Ct of the housekeeping gene glyceraldehyde 3-phosphate

dehydrogenase (GAPDH): a lower ∆Ct value thus means greater expression as the

cycle threshold was reached earlier, and a difference in ∆Ct value of 1.0 indicates a

doubling or 100% increase in product. We find higher ASIC4 (∆Ct of 5.2) expression in

the striatum (Figure 8) relative to hippocampus or cortex. This indicates that our

methods are able to detect differences in ASIC mRNA expression. We found that

ASIC2b was the most abundant mRNA message of the ASIC subunits overall across all

brain regions with ∆Ct values of 5.6 in the cortex, 6.3 in the hippocampus and 6.6 in the

striatum. ASIC2b also did not fluctuate as dynamically in expression as the other

subunits across brain regions. ASIC1a was expressed most greatly in the striatum (∆Ct

= 5.5), followed by the cortex (∆Ct = 6.1) and then the hippocampus (∆Ct = 7.9). In the

striatum ASIC2a and ASIC2b have ∆Ct values of 7.6 and 6.6, respectively; thus in the

striatum ASIC4 (∆Ct = 5.2) and ASIC1a seem to have the highest presence. ASIC2a

was most greatly expressed in the cortex (∆Ct = 6.7), but is less abundant than ASIC1a

or ASIC2b in that tissue. Overall, for the cortex and the hippocampus,

ASIC2b>1a>2a>4 and for the striatum, ASIC4>1a>2b>2a. These results suggest that

(1) ASIC4 subunits likely contribute mainly in the striatum, (2) in the cortex and

hippocampus, there is less ASIC1a expression relative to other ASIC subunits,

suggesting less ASIC1a homomultimers are forming and more ASIC1a/2b and

ASIC1a/2a heteromultimers are being formed.

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Effects of Age: It is known that following stroke or ischemic injury, older

individuals are at greater risk for stroke and sustain greater neuronal damage than

younger individuals. ASIC expression may also change during development. Thus,

studies investigating the levels of ASIC2b expression were conducted across differently

aged animals (Figure 9). Three mice, ages 4 days, 6 weeks and 12 months were tested

for relative abundance of ASIC1a, 2a, 2b, and ASIC 4 subunit expression. It was found

that the 4 day animal had greater expression of all ASIC subunits except for ASIC4 in

the cortex. In the cortex, ASIC2b levels decline with age (∆Ct = 4.3 in day 4 pup, ∆Ct =

5.3 in the 6 week animal, ∆Ct = 5.7 in the 12 month animal). Yet, in the hippocampus,

ASIC2b levels were found to be lowest (∆Ct 5.5) in the 6 week old animal. ASIC2a

showed a similar pattern in the hippocampus to ASIC2b, except less abundant. Also,

the typical pattern observed in relative abundance of ASIC subunits is 2b>1a>2a>4 for

11


all ages and tissues but the hippocampus of the 12 month sample (2b>2a>1a>4).

Although ASIC1a levels do not decrease in hippocampus of the 12 month sample

compared to the 6 week sample, since ASIC2a is in greater abundance, it may compete

with the other most available subunits to form heteromultimers, thereby shifting

heteromultimer composition in that tissue. The most striking contrasts are the vastly

more abundant ASIC1a and ASIC2b expression levels in the day 4 animal across

tissues compared to other ages.

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Effects of Sex Difference: It is known that males and females differ in

incidence of stroke and in stroke outcome [21]. Women, while less likely than men to

have strokes until the age of 85, constitute more than half of the stroke related deaths

[21]. Estrogen is also known to be neuroprotective in brain damage after stroke in

animals but the translation of this to patients has not yet occurred [22]. We thus

analyzed the cortex, hippocampus, and striatum tissues of male and female animals to

investigate differences in ASIC subunit expression (Figure 10). Two wild type mice of

the same age (seven months), one male and one female were used in this trial. We

found that in the hippocampus,

females have greater expression

of all ASIC subunits, especially

ASIC2b and ASIC1a. There is

a greater difference in ASIC1a

expression in the hippocampus

(a difference of 1, or 100%

increase in the female) of

ASIC1a, followed by ASIC2b (a

difference of 0.8, or 80%

increase in the female) and

ASIC2a (a difference of 0.6, or

60% increase in the female).

Though increased ASIC2b

expression may mean more

13


neuroprotection, there is a greater increase in ASIC1a expression which suggests an

overall excess of neurotoxic ASIC1a. In the cortex, however, the only significant

difference is in ASIC2b levels, which are slightly decreased in females (∆Ct = 5.6 in

male, 5.9 in female).

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Effects of Acute Stress: Psychological stress has been studied to contribute to

a poorer prognosis after stroke [23]. We hypothesized this mechanism may be

mediated partially through ASICs. RNA from six age-matched wild-type mice that were

used for an acute stress procedure preceding RNA extraction was isolated. Three of

the mice received the acute stress treatment while the other three animals were used as

controls. The method of acute stress was restraint. The RNA was harvested and

analyzed for change in ASIC expression (Figure 11). No significant changes in the

relative abundance of ASICs were observed between stressed and non-stressed

forebrain samples.

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Effects of Chronic Stress: Chronic stress was also investigated for the same

reasons mentioned above. Chronic stress, however, would stress the animals daily by

restraint for two weeks. In this experiment, right before euthanasia, all animals were

stressed by restraint in order to verify the stress effect by measuring corticosterone

levels. Since no significant differences in ASIC expression were found between the

acutely stressed and non-stressed animals, the ASIC expression of the acutely stressed

animals may be similar to

a non-stressed animal.

Thus data presented is a

comparison of chronically

stressed animals to

acutely stressed animals

(Figure 12). We found no

significant difference in

ASIC expression with

chronic stress.

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Discussion

Although ASIC2b has been described for over twelve years, the role of this ASIC

subunit remained elusive [24]. On its own, ASIC2b doesn’t form proton gated currents

and when co-expressed with other ASIC subunits in the central nervous system, no

observable effects were reported. Here we find that expression of ASIC2b in cultured

neurons dampens proton gated currents and prevents acidosis-induced neuronal death.

Although the mechanism of action is unknown, there are two likely hypotheses: (1)

ASIC2b form heteromultimers with ASIC1a which are proton-insensitive or (2) ASIC2b

forms heteromultimers with ASIC1a that do not get to neuronal cell surface. Further

studies are necessary to distinguish the mechanism of neuroprotection.

In looking at ASIC expression, several limitations using our method must be

acknowledged. First, mRNA expression is an indirect measurement of protein levels.

mRNA analysis through qPCR has been established as a useful and valid method [25].

However, since it measures the mRNA expression message versus protein itself, we

must further quantify these studies with protein immunohistochemistry to specifically

look at protein levels. Second, we are assuming there is no selection for

heteromultimer or homomultimer formation. Third, measuring expression levels using

this tool would not distinguish (1) a change in neuron:glia ratio, as ASICs are most

abundant in neurons, and (2) whether a change in expression represents a large

change in some neurons or a small change in most neurons. We plan to perform these

experiments in more animals, and also plan to validate our findings with

electrophysiological methods and cell death assays.

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Because the role of ASIC2b in central neurons is not established, we chose to

look at relative ASIC2b expression within several important brain regions. We found

that (1) ASIC2b expression is the most abundant in the cortex and hippocampus and (2)

that ASIC2b expression is the most consistent across different brain regions. We also

looked at the other ASIC subunits found in central neurons to take into account the

competition for the homomultimeric and heteromultimeric formation that we hypothesize

happens in vivo. Within the cortex and hippocampus, we found a greater difference

between ASIC1a and ASIC2b expression in the hippocampus, which may indicate a

greater heteromultimeric formation between ASIC1a and ASIC2b in the hippocampus

as ASIC1a may not be forming as many homomultimers. The overall abundance of the

ASIC2b message leads us to believe that ASIC2b may play a prominent role in

mediating acid-sensing in neurons.

The trends observed in the age studies were, overall, that (1) ASIC expression

decreases with age and (2) the general expression trend of ASIC2b>1a>2a>4 was

reversed in the hippocampus of the oldest animal to ASIC2b>2a>1a>4. ASIC2b

declines with age in the cortex, but declines early on in the hippocampus and then

potentially even increases slightly in the 12 month old animal. If ASIC2b does in fact

increase in the later years, this could be due to the potentially neuroprotective function

of ASIC2b. ASIC2a also follows the trend of ASIC2b.

Significant differences in ASIC expression were observed between males and

females. ASIC1a, ASIC2b, and ASIC2a are more greatly expressed in females than in

males. The elevated ASIC1a levels may explain the poorer prognosis in females

following stroke as they would have greater ASIC1a activation and thus greater

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neuronal death. However, elevated ASIC2b levels conversely would suggest

neuroprotection following stroke. The sex difference in ASIC expression may also

influence stress response. Recently, the literature has suggested that ASIC1a plays a

role in depression [26]. It is known that in mouse models of depression, one can model

a depression-like state by repeatedly stressing an animal. ASIC1 -/- animals behaved

in a way that was anti-depressant. We discovered in our stress tests that the females

had much greater corticosterone levels after chronic stress than males (not shown).

The greater response in females to stress may be mediated through the fact that they

have elevated basal ASIC1a levels, in accordance with the literature. However, ASIC2b

is also elevated in females, which under our hypothesis would imply a dampening of

ASIC1a activity.

In observing stress conditions alone, we did not find acute stress or chronic

stress to influence ASIC subunit expression levels. This tells us that ASIC subunit

expression does not change with stress.

We plan to further investigate other conditions known to contribute to altered

prognosis after stroke, such as pre-conditioning for stroke. Pre-conditioning, or

experiencing a smaller, non-injurious stroke is known to be neuroprotective for stroke

prognosis if a larger stroke event occurs within a certain time frame of the pre-

conditioning [27].

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Methods

Mass culture of hippocampal neurons:

Primary hippocampal neuron cultures were prepared using previously published

methods [28]. Briefly, hippocampi were dissected from postnatal day (P) 0–P1 pups,

freed from extraneous tissue, and cut into pieces. ASIC1 knockout mice develop and

breed normally, and there are no overt abnormalities in brain morphology [28].

Dissected tissue was transferred into Leibovitz's L-15 medium containing 0.25 mg/ml

bovine serum albumin and 0.38 mg/ml papain, and incubated for 15 min at 37°C with

95% O2-5% CO2 gently blown over the surface of the medium. After incubation, the

dissected tissue was washed three times with mouse M5-5 medium (Earle's minimal

essential medium with 5% fetal bovine serum, 5% horse serum, 0.4 mM L-glutamine,

16.7 mM glucose, 5,000 U/l penicillin, 50 mg/l streptomycin, 2.5 mg/l insulin, 16 nM

selenite, and 1.4 mg/l transferrin) and triturated. Dissociated cells were then centrifuged

for 4.5 minutes at 730 rpm and M5-5 media was aspirated. Cells were resuspended in

supplemented Neurobasal-A media (1% B27 supplement containing anti-oxidants, 1%

B27supplement minus anti-oxidants, 0.5 mM L-glutamine, 0.5 mg/ml gentomycin, and

2.5 mg/l insulin, 16 nM selenite, and 1.4 mg/l transferrin). Cells were plated in 24-well

plates containing 10-mm poly-D lysine coated glass coverslips at a density of 5 x 104

cells per well. After 48–72 hours, 10 µM cytosine β-D-arabinofuranoside was added to

inhibit glial proliferation. Neurons were maintained at 37oC with 95% ambient air - 5%

CO2 for 14-21 days before experiments were performed.

Plasmid transfection of hippocampal neurons:

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Hippocampal neurons were transfected at the time of preparation using a

nucleofector kit (Amaxia) per manufacturer’s protocol. Neurons were harvested as

above, except dissociated cells were resuspended in 100 µL of nucleofector containing

3 µg of total plasmid DNA (at a 3 parts ASIC2b or vector to 2 parts GFP ratio) after

centrifugation. Following resuspension, neurons were immediately transfected via

electroporation and plated in Neurobasal-A on 10-mm poly-D lysine coated glass

coverslips as described above.

Whole-cell patch clamp on primary neurons:

To record ASIC-dependent current, neurons were perfused with extracellular

solution at varying pH values. The extracellular solution contained 140 mM NaCl, 5.4

mM KCl, 10 mM HEPES-buffer, 2 mM CaCl2, 1 mM MgCl2, 5.55 mM glucose, 10 mM

MES-buffer, 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 50 µM D-2-amino-5-

phosphonovaleric acid (AP5), 30 µM bicuculline, and 500 nM tetrodotoxin. The pH was

adjusted with 1 N NaOH. Patch electrodes were pulled with a P-97 micopipette puller

(Sutter Instrument, Novato, CA) and fire-polished with a microforge (Narishige, East

Meadow, NY). Micropipettes with 2–4 M were used for experiments. The intracellular

pipette solution contained 121 mM KCl, 10 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 10 mM

HEPES, 2 mM Mg-ATP, and 300 µM Na3GTP (pH 7.25). The membrane potential was

held constant at –70 mV. Data were collected at 5 kHz using an Axopatch 200B

amplifier, Digidata 1322A, and Clampex 9 (Molecular Devices, Sunnyvale, CA).

Neurons were continuously superfused with the extracellular solution from gravity-fed

perfusion pipes at a flow rate of about 1 ml/min. Perfusion pipes were placed 250 to 300

21


µm away from cells, and flow was directed toward the recorded cells to ensure

fast solution exchange. Stable H+-gated current was evoked by the exogenous

application of pH 6.0 or 6.5 from a holding pH of 7.4. Typically 3-4 applications of acid

solution was required before H+-gated peak current amplitude stabilized.

Acid-induced neuronal death assays:

At 14-17 days in culture, neurons were randomly divided into designated

experimental groups (see figure 4 & 5). Neurobasal media was removed before

washing cells 2 times with ECF solution (140 mM NaCl, 5.4 mM KCl, 25 mM Hepes

Buffer, 20 mM glucose, 1.3 mM CaCl2, 1.0 mM MgCl2) at pH 7.4 (pH adjusted with 1N

NaOH). Cells were then washed pH 7.4 EFC inhibitor solution containing 10 µM

Dizocilpine (MK-801), 20 µM CNQX, 5 µM nimodipine and 500 nM tetrodotoxin. Specific

acidosis interventions were performed as described in Figure 4 & 5 using ECF with

inhibitors. Within each culture, at least 2 different coverslips were used for a given

intervention. Following acidosis interventions, cells were washed 2 times with pH 7.4

ECF without inhibitors, and fresh Neurobasal culture media was returned. Neurons

were allowed to recover for 24 hours at 37o C with 95% ambient air - 5% CO2. After the

recovery period, cells were incubated in ECF containing 5 µM fluorescein-diacetate

(FDA) and 2 µM propidium iodide (PI) for 30 minutes at room temperature. For

experiments using transfected neurons, cells were treated with propidium iodide only.

Cells were then washed 4 times with Ca2+-free Phosphate Buffered Solution (PBS) and

fixed for 20 minutes in 4% paraformaldehyde. After fixing, cells were washed 2

additional times with PBS, and coverslips were then mounted on microscope slides.

22


Pictures of 5 random fields of view demonstrating PI/FDA staining were taken for each

coverslip (500-700 cells/coverslip) by an individual blinded to the experimental

conditions. For experiments using transfected neurons, pictures were taken of the entire

coverslip to quantify the maximium number of transfected cells. Images were obtained

using a Ziess Axioscope microscope at the 20x objective with 490 nm and 575 nm

fluorescent filters (to observe FDA/ GFP and PI fluorescence respectively), an Axiocam

digital camera, and Zeiss AxioVision 4.6 software. Images were pseudo colorized to

distinguish labeling (Green for FDA or GFP staining; Red for PI staining). Live (FDA-

stained cell bodies) and dead (PI-stained nuclei) cells were counted in each field by a

blinded individual. For experiments using transfected neurons, only neurons displaying

GFP expression were counted. Neuronal death was determined by averaging the

percentage of dead cells on each coverslip, thus one neuronal culture yielded one n for

a given acidosis intervention.

Isolate Tissue

RNA extraction, DNAse treatment

cDNA synthesis, RNAse treatment

Real-Time PCR

Output indicates relative amount of starting material

Figure 13. Steps necessary to perform real time PCR.

Isolate Tissue



Real-Time PCR


Isolate Tissue



Real-Time PCR


Figure 13. Steps necessary to perform real time PCR.

RNA extraction and Real-Time PCR (qPCR)

Mice were euthanized and tissues from

distinct areas of mouse brain were dissected.

RNA was extracted using the RNEasy Miniprep

kit (Qiagen) (Figure 12) or tissue was placed in

RNAlater (Qiagen) for later RNA isolation. The

extracted RNA was treated with DNAse using

the Ambion DNA-free Kit to destroy genomic

23


DNA contamination. cDNA was made from purified RNA using reverse transcription

with random hexamers, and was treated with RNAse to destroy the initial RNA using the

SYBR GreenER Two-Step qRT-PCR Kit Universal. The cDNA was quantified by qPCR,

also using the SYBR GreenER Two-Step qRT-P CR Kit Universal, on an Applied

Biosystems Step-One Plus thermal cycler. Data was normalized to glyceraldehyde 3-

phosphate dehydrogenase (GAPDH) expression and was analyzed to determine

relative ratios of ASIC subunit expression.

Acute stress behavioral assay

Mice were stressed by restraint (2h) preceding RNA extraction. Three control mice

were used and three animals were stressed, all male. After the two hour period, mice

were euthanized and RNA was extracted immediately. Samples were from forebrain

tissues.

Chronic stress behavioral assay

Animals were stressed by restraint for 2h daily over the course of two weeks while

control animals were unstressed. The day prior to euthanasia, an eye bleed was

performed on all animals to obtain blood samples for corticosterone level analysis. The

day of euthanasia, all animals including control animals were stressed by restraint for 2h,

an eye bleed performed, and the animals were euthanized. Tissues were placed in

RNAlater following manufacturer’s instructions and RNA was later extracted.

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