3.

Results and Findings

3.1. Introduction

This section presents results that answer the following research questions raised in chapter1: i) to

identify the pool for spontaneous neurotransmission activity; ii) to identify the pool for evoked

neurotransmission activity; and iii) to identify the similarity and differences of the spontaneous

and evoked neurotransmission activities. For a brief reminder, the section will: identify the pool

for spontaneous neurotransmission activity; identify the pool for evoked neurotransmission

activity; and identify the similarity and differences of the spontaneous and evoked

neurotransmission activities.

3.2. pHoenix

3.2.1. Conditions observed

Two conditions were observed in the pHoenix experiment. However, there was an observation of

thirty seconds order of pre-illumination order to obtain a succinct baseline and ensure that the

SVs were at the starting point. The first condition thus was to stimulate the cells and then

activate the pHoenix using light at the end of the ammonimpulse. The second paradigm involved

first imposing light then stimulation before ammonium at the end.

pHluorin-Arch-3-mKate2-ßHK construct modifies phoenix to target vesicles. Light-activated

proton pumps were functionally expressed in synaptic vesicle membranes using the fluorescent

proteins mKate2, located in the cytosol, and pHluorin, located in the lumen, to show protein

expression and localisation, together with luminal acidification. When we realized that Arch3's C
terminus was positioned somewhat on cytosolic side, we inserted the transmembrane portion of

the gastric H+/K+ ATPase -subunit to conserve the hydrophobic topology (Rost et al., 2015).

3.2.2. Paradigms of the experiment

Two paradigms were applied during the performance of this experiment. The first paradigm is an

exemplar that cells are first illuminated (to activate pHoenix) and then stimulated followed by

ammonium pulse. The second paradigm was an architype that cells are directly stimulated using

stimulation of certain strength. This is followed by illumination step (561nm laser light) to

activate pHoenix and at the end the ammonium pulse is given in order to visualize the total

number of SVs.

Bacterial Archaeordopsin-3 was placed between Helix three and Helix four of the

synaphopHluorin. pHluorin placed on the luminal side of the SVs was used to visualize pHoenix

expressed cells and lysosomal acidification. The vesicular SV generates synaptopHluorin an C-

terminus of Archaeordopsin extended towards the cytosolic side. betaHK was used to maintain

transmembrane morphology and control pHluorin uptake across the cell membrane. Age1 sites

were used to construct Ca2+ ATPase to the syp1-pHy1.

Polystyrene tubes were used with 10cm plates of 1.88cc-dosed hypoccampal cells. A solution of

23.61 of Transit 293 reagent was incubated for 10minutes at room temperature. cDNA followed

the incubation process. . a mixture of 5 g of lentiviral construct, 2.5 g of pCMV R.8.2 Helper 2

(Pack), and 5 g of pM D2.G Helper 1 (ENV) added to the solution and incubated. The DNA was

titrated to the solution and then incubated with 5% CO2, for 72 hours at room temperature (Rost

et al., 2015).
the synaptic terminals were saturated by Glutamate transporter 1 (VGULT). Untargeted

Archaeordopsin3-pHluorin was not saturated. The image recordings were done at intervals of 1s.

the resulting pulses evoked 250 times smaller somatics of outward electro-currents in pHoenix-

expressing cells as compared to control cells. This indicated that pHoenix is mostly retained in

the cell culture. Optical quantification of pHluorin signals in pHluorin molecules signified the

presence of only a smaller percentage of pHoenix proteins in plasma membrane. It also indicated

the presence of vesicular PH14 and effective synaptic transmission (Rost et al., 2015).

3.2.3. The mobilization of RRP- depletion by incubation (spontaneous activity) or

stimulation

The main idea in the pHoenix RRP mobilization is in relationship to its ability to manipulate

multiple cellular activities when cells are exposed to light that when exposed. Phoenix-expressed

cells illuminated on 561nm laser light expand the porous holes of the synaptic cell membrane,

increasing the sensation of the pH by the pHluorin and V-GLUTI-6 transporter activities thus it

is easier to count the number of SVs (fig. 3.1A). After bafilomycin1 (Baf) has been applied to an

isolated cultured hippocampal neuron, endocytosis of SV requires the Baf inhibitor (Fernandez-

Alfonso & Ryan, 2004), and a cell-permeant inhibitor of V-type ATPase. A Baf-poisoned

vesicle, for example, is unable to reload after releasing its "quantum" of glutamate, hence it

ceases to function. A vesicle is tallied only once if this method is used.

Synaptic vesicles are recycled after the release of specific glutamate transporter. At the

neuromuscular junction, discoloration dye identified RRP and RP. In exocytosis, the SVs are

completely depleted. CD63-pHluorin provides a medium to estimate the size of pools and the
mobilization rate at the neuromuscular junction. To estimate the size of RRP and RP, the

recruitment of SVs from the RP required 561nm laser light illumination. Since sypHy1 is

activated by immunofluorescence to trigger rapid uptake of pHluorin, synaptic currents evoked

at 10HZ were studied at room temperature. Upon nerve stimulation in synaptophysin, pHluorin

signals decline rapidly on first stimulus, slowly during subsequent 50 s, and then attained

steadiness. This indicated that pHluorin triggers rapid fusion of proteins across the

transmembrane gradient until a saturation level is attained.

Based on a series of tests, this method was found to meet all three important requirements:

Figure 3-1: Each button in RRP has around 20 SVs. Each button in RRP has around 20 SVs. We hypothesize that a 20-

minute incubation of the cells in the presence of TTX (to inhibit recurrent activity) is sufficient to deplete the

majority of SVs resident in RRP. One vesicular synapse is released every 60-90 seconds during spontaneous

movement. B) Cross-depletion of experimental paradigms: 1) Cells are lighted first, then stimulated, and finally

exposed to an ammonium pulse. 2) following pre-illumination and pre-incubation, cells are stimulated directly with
a specific strength of stimulation. Following that, an illumination step (361nm laser light) is used to activate

pHoenix, and finally, an ammonium pulse is used to visualize the entire number of SVs; the amplitudes, denoted as

A1 and A2, are compared. If SVs are pooled together, we anticipate A1 and A2 to be distinct. C) It is expected that

when three different stimulation strengths are used, there will be differences in the amplitudes achieved. We

predicted that 50AP/10Hz (ref.) stimulation can demonstrate the greatest difference between A1 and A2, since this

stimulation power is sufficient to deplete SVs in RRP.

To assess whether insufficient filling of glutamatergic vesicle release affects the release

probability, pHoenix-expressed neurons were compared to control neurons (pHoenix). On the

activation of Phoenix under 561nm laser light illumination, SypHy-infected cells showed no

effect. Also on illumination under 488 nm laser light, the untreated neurons had no effect but

neuro-activity was evident in phoenix-expressed neurons.

Both the control and pHoenix-expressed cells under 488nm laser light induction showed similar

changes in paired pulse ratio (PPR). Illumination did not have a differential effect on control

neurons. The light-activated proton pumping significantly increase the vesicular transmitter

accumulation shown by postsynaptic responses. The limited uptake of pHluorin shows that the

synaptic vesicles are nearly filled at physiological conditions.

Vesicular release probability increase with vesicular filling. However, excessive accumulation

of glutamate is associated with progressive desensitization of AMPA receptors. The observations

in glutamate control experiment suggests that glutamate is lost through a stimulus-dependent

process other than leakage. Cross-depletion of synaptic vesicle pools is determined by the

endocytosis versus exocytosis at sustained AP at synaptic nerve terminals.

3.2.4. pHoenix Activation and Stimulation at 600Aps/10Hz

This experiment involved pHoenix activation and stimulation at 600APS/20Hz using a pHoenix

trigger. In this experiment, we examined the cellular responses to pHoenix activation using

561nm laser light, followed by stimulation with 600APs at a frequency of 20Hz.   Following pre-

incubation and subsequent pre-illumination, cells are stimulated with 600APs at a frequency of

20 Hz immediately after being pre-illumination and pre-incubated. An illumination phase

(361nm laser light) is then used to activate pHoenix, followed by an ammonium pulse, which

allows you to see the total number of SVs. Experiments were carried out on a large number of

averaged locations (n=11), and signals were normalized to the ammonium signal to obtain

unbiased and optimal results. To compare to the Control, the normalized amplitudes from A)

(shown as Light/Stim) and B) (shown as Stim/Light) are added together in bar graphs (SEM).

The T-test was used to determine whether or not the differences were statistically significant.

Thus, the finding p=0.0005 shows that the differences are not statistically significant.

Considering that SVs are grouped together, we should presume that light amplitudes represented

as Stim/Light and Light/Stim are separate. As a result, the amplitudes obtained from fig.3.2B

decreased significantly as compared to those obtained from fig 3.2A.

Figure 3.2: pHoenix activation and stimulation at 600APS/20Hz. A) Cellular responses to

pHoenix activation using 561nm laser light followed by stimulation with 600APs at a frequency

of 20Hz. B) 2) After pre-illumination and pre-incubation, cells are stimulated immediately with

600APs at a frequency of 20 Hz. This is followed by an illumination phase (361nm laser light) to

activate pHoenix, and finally an ammonium pulse to view the total number of SVs. Experiments

are conducted on numerous averaged regions (n=11), and signals are normalized to the

ammonium signal. C) The normalized amplitudes from A) (shown as Light/Stim) and B) (shown

as Stim/Light) are summed in bar graphs (SEM) in comparison to the Control. The T-test is used:

p=0.0005 indicates that the differences are not statistically significant. If SVs are pooled

together, we should assume that the amplitudes shown as Stim/Light and Light/Stim are distinct.

Indeed, the amplitudes obtained from B) are slightly less than the ones produced from A).
pHluorin-based assay was used to evaluate presynaptic activity. Since pHluorin is a pH-sensitive

variant of GFP with a pK of 7.1, this fluorescent tag worked well as an SV exo- and endocytosis

reporter according to Sankaranarayanan et al. (2000). Additionally, pHluorin's excitation reached

it is maximum emission at between 500nm and 600nm. The pHluorin moiety was then fused to

the luminal part of Sys to examine SV recycling.

Nonetheless, the notion that when SVs merge with the plasma membrane during stimulation, the

fluorescence signal increases 20-fold, and freshly exocytosed pHluorin exposed to extracellular

neutral pH is dequenched was affirmed (fig 3.2A). After compensatory endocytosis and

subsequent reacidification of SVs, the pHluorin signal was re-quenched, and this resulted in a

drop in fluorescence signal. By superfusing the pHluorin-tagged SV proteins with an ammonium

chloride solution, the surface-stranded percentage was measured.

An acidic pH inside SVs quenched pHluorin while the introduction of promoters in the solution

trigerred uptake of neurons causing an external pH to dequench pHluarin. This neutralized the

pHluirin molecules that had escaped into the extracellular space.

3.2.5. pHoenix Activation and Stimulation at 100Aps/10Hz

According to figure 3.3, pHoenix illumination (561 nm laser light) and stimulation (100APs at

10 Hz) were performed at different time intervals, namely 25 seconds (figure3.3A illumination

and 3.3B stimulation) and 50 seconds (figure3.3A illumination and 3.3B stimulation) (fig. 3.3B

illumination and 3.3A stimulation). Later on, at 60 seconds, an ammonium pulse was added. The

primary objective of the alternated lighting and stimulation was to observe the normalized

amplitude (fig. 3.3C), that is, what would happen if the pHoenix was stimulated first and then

illuminated, as opposed to what would happen if it was illuminated first and then stimulated. The
following conclusions were drawn from the experiment: i) there was no significant effect of

pHoenix illumination alone, but there was a substantial effect of consecutive illumination and

stimulation regardless of which started first. ii) As shown in fig 3.3A, pHoenix illumination prior

to stimulation contributed a lower normalized normalized amplitude (0.25) than the normalized

normalized ampllitude (0.28). iii) However, as shown in Figure 3.3B, pHoenix stimulation

followed by illumination resulted in a larger normalized amplitude (0.30). Despite this,

ammonium impulse had no discernible effect on fluorescence.

Figure 3.3: pHoenix Activation and Stimulation at 100Aps/10Hz. A) Cellular responses

following activation of the pHoenix with 561nm laser light and stimulation with 100APs at

10Hz. B) 2) Following pre-illumination and pre-incubation, cells are stimulated immediately

with 100APs at a frequency of 10 Hz. Following that, an illumination step (361nm laser light) is

used to activate pHoenix, and finally, an ammonium pulse is used to visualize the total number

of SVs; experiments are conducted on numerous averaged regions (n=10), and signals are
normalized to the ammonium signal. C) Normalized amplitudes from A) (shown as Light/Stim)

and B) (shown as Stim/Light) are summed in bar graphs (SEM) in comparison to the Control.

The T-test is used: p=0.0005 indicates that there is no statistically significant difference. If SVs

all belong to the same pool, we should assume that the amplitudes represented above as

Stim/Light and Light/Stim are distinct. Indeed, the amplitudes derived with B) are approximately

250 times smaller.

Vesicular acidification was blocked by incubating hypocammpal cells in Bafilomycin. pHluorin

signals in glutermatic neurons were monitored to follow the vesicular acidification.. Pre-

incubation with 1 μM Bafilomycin1 for 20 minutes depleted the gradient of the light-driven

proton. Under 561nm laser light and 100AP 10HZ, there were strong pHluorin signals exhibited

at the synaptic terminals.

3.2.6. pHoenix Activation and Stimulation at 50Aps/10Hz

At 50Aps/10Hz, pHoenix Activation and Stimulation are carried out. Figure 3.4A shows the

cellular responses that occurred after the pHoenix was activated with 561nm laser light and

stimulated with 50APs at 10Hz, as shown in the experiment. Cells are stimulated directly in

figure 3.4B after pre-illumination and pre-incubation, using 50APs at a frequency of 10 Hz,

following the pre-illumination and pre-incubation steps. A subsequent illumination phase

(361nm laser light) is used to activate pHoenix, and a final ammonium pulse is used to visualize

the total number of SVs; studies are conducted on several averaged locations (n=10), and signals

are normalized to the ammonium signal. Figure 3.4C depicts standard error of the mean (SEM)

of normalized amplitudes from A) (shown as Light/Stim vs B) (shown as Stim/Light) and B)

(shown as Stim/Light). The T-test is employed, and the result is p=0.0005, which indicates that

there is no statistically significant difference between the groups. If all of the SVs are members
of the same pool, we should assume that the amplitudes indicated above as Stim/Light and

Light/Stim are unique from one another. In contrast to the second experimental paradigm, the

amplitudes measured in figure 3.4B are significantly smaller than the amplitudes measured in

figure 3.4A. The averaged normalized amplitudes of light/stim in A and stim/light in C

demonstrate that, as expected, the stimulation strength indicates the greatest difference between

two amplitudes.

Figure 3.4: pHoenix Activation and Stimulation at 50Aps/10Hz. A) Cellular responses following

activation of the pHoenix with 561nm laser light and stimulation with 50APs at 10Hz. B) 2)

Following pre-illumination and pre-incubation, cells are stimulated directly utilizing 50APs at a
frequency of 10 Hz. Following that, an illumination step (361nm laser light) is used to activate

pHoenix, and finally, an ammonium pulse is used to visualize the total number of SVs;

experiments are conducted on numerous averaged regions (n=10), and signals are normalized to

the ammonium signal. C) Bar graphs (SEM) of normalized amplitudes from A) (shown as

Light/Stim vs B) (shown as Stim/Light). The T-test is used: p=0.0005 indicates that there is no

statistically significant difference. If SVs all belong to the same pool, we should assume that the

amplitudes represented above as Stim/Light and Light/Stim are distinct. Amplitudes measured in

B) are much smaller than those measured in the second experimental paradigm (A). As expected,

this stimulation strength reveals the greatest difference between two amplitudes (refer to the

averaged normalized amplitudes of light/stim in A and stim/light in C).

Bursts of 50APs at 10HZ triggered an increase of pHluorin immunofluorescence resulting from

exocytosis of synaptic vesicles in pHluorin-expressed neurons. In phoenix-expressed neurons,

561 nm laser light triggered fluorescent signal. The resulting exocytosis increase pHluorin

signals. This could have resulted from membrane fusion of phoenix-expressed SVs. The

observations showed the pHoenix construct was efficiently integrated to the synaptic vesicles.

The process of pHoenix expressions did not interfere with the release of neurons.

3.2.7. Pre-stimulation (50Aps/20Hz) and Stimulation (50APS/20Hz) of the pHoenix

The pHoenix was subjected to pre-stimulation (50Aps/20Hz) and stimulation (50Aps/10Hz). In

this experiment, the pHoenix was activated with 561nm laser light and stimulated with 50APs at

10Hz, resulting in the following cellular responses: A) As depicted in fig. 3.5, cells are

stimulated with 50APs at 10 Hz immediately following pre-illumination and pre-stimulation

(50APs at 20 Hz). A subsequent illumination phase (561nm laser light) is used to activate

pHoenix, and a final ammonium pulse is utilized to visualize the total number of SVs; studies are

conducted on several averaged locations (n=12), and signals are normalized to the ammonium

signal. With bar graphs, the normalized from A) and 3.5B (shown as Light/Stim) are contrasted

with the normalized amplitudes from A) (shown as Stim/Light) (SEM). The T-test is employed,

and the result is p=0.0005, which indicates that there is no statistically significant difference

between the groups. All SVs belonging to the same group should have similar amplitudes, which

are represented in the table below as Stim/Light and Light/Stim dif. A significant difference

exists between the amplitudes observed in figure 3.5B and those measured in the second

experimental paradigm (figure 3.5A). As expected, the difference between two amplitudes (I'm

talking to the averaged normalized amplitudes of light/stim in figure 3.5A and stim/light in

figure 3.5C) is greatest when the stimulation strength is increased.

In figure 3.5, the pHoenix was stimulated and illuminated in the same manner as in figure 3.3,

i.e., illumination followed by stimulation (fig. 3.5A) and stimulation followed by illumination

(fig. 3.5B). However, the stimulation face was reduced to 50 APs/10Hz, as opposed to 100

APs/10Hz in figure 3.3. At the 60th second, ammonification remained consistent as well.

pHoenix lighting followed by stimulation (fig. 3.5A) resulted in a somewhat lower average

flourescent amplitude than the control in the experiment in 3.5C. On the other hand, stimulation

followed by illumination of pHoenix (fig. 3.5B) resulted in a much smaller normalized amplitude

than the control and light/stimulation conditions (fig 3.5C). The results above indicate that

stimulation phase played a substantial effect in pHoenix, as the amplitudes seen in the

100APs/10 Hz phase were somewhat greater than (fig. 3.3B) or closer to (3.3A) the control.
Figure 3.5: Pre-stimulation (50Aps/20Hz) and Stimulation (50APS/20Hz) of the pHoenix. A)

Cellular responses following activation of the pHoenix with 561nm laser light and stimulation

with 50APs at 10Hz. B) 2) After pre-illumination and pre-stimulation (50APs at 20 Hz), cells are

stimulated directly with 50APs at 10 Hz. Following that, an illumination step (361nm laser light)

is used to activate pHoenix, and finally, an ammonium pulse is used to visualize the total number

of SVs; experiments are conducted on numerous averaged regions (n=12), and signals are

normalized to the ammonium signal. C) The normalized amplitudes from A) (shown as

Light/Stim) and B) (shown as Stim/Light) are compared using bar graphs (SEM). The T-test is

used: p=0.0005 indicates that there is no statistically significant difference. If all SVs are

members of the same pool, we should assume that the amplitudes represented below as

Stim/Light and Light/Stim dif. Amplitudes measured in B) are much smaller than those

measured in the second experimental paradigm (A). As expected, this stimulation strength
reveals the greatest difference between two amplitudes (this is in references to the averaged

normalized amplitudes of light/stim in A and stim/light in C)

3.2.8. Control experiment findings

A wide range of observations were made when control experiments were compared to one

another (fig.3.6). The responses were obtained for control experiments using the following

stimuli: fig. 3.6A (50 APs at 10Hz stimulation), fig. 3.6B (100 APs at a frequency of 10 Hz

stimulation), and fig. 3.6C (100 APs at a frequency of 10 Hz stimulation) (600 APs at a

frequency of 20 Hz). The fluorescence signals from multiple regions are normalized and

averaged in the traces (n=10 for each condition) to produce the final results. The pre-incubation

for 20 minutes (in the presence of 1M TTX (blocks sodium channels- to prevent recurring

activity) and 5nm Bafilomycin 1a (V-ATPase inhibitor)) and pre-stimulation are not included in

the controls because they are not considered necessary. The cells were simply stimulated with 50

APs at 10 Hz, 100 APs at 10 Hz, and 600 APs at 20 Hz, as well as a combination of these

stimuli.
Figure 3.6: Comparisons from control experiments. Responses obtained for control studies

using: A) 50APs at 10Hz stimulation. B) 100 APs at a frequency of 10 Hz, and C) 600 APs at a

frequency of 20 Hz. The fluorescence signals from multiple areas are normalized and averaged

in the traces (n=10 for each condition). Pre-incubation for 20 minutes (in the presence of 1M

TTX (blocks sodium channels- to preclude recurrent activity) and 5nm Bafilomycin 1a (V-

ATPase inhibitor)) or pre-stimulation are not included in the controls. The cells were simply

stimulated with 50 APs at 10 Hz, 100 APs at 10 Hz, and 600 APs at 20 Hz, followed by an

ammonium pulse.

3.3 iGluSnFR

iGluSnFr is bright and relays stabilized imaging under illumination. In axonal-assays, IGlusnFr

is significant in detecting Ca2+, estimating and reporting excitatory synaptic release. However,
the coupling between Ca2+ presynaptic release and the transmitter collapses when vesicle pools

are depleted by sustained stimulation. This experiment was conducted to test;

3.3.1 iGluSnfr is strong enough to detect singe AP-evoked stimulus.

To visualize iGluSnFr, the construct was engineered in vitro from E. Coli Gltl and circularly

permuted GFP. The PCR product was cloned to pCDNA3-syn vector to generate pCDNA3-syn

iGluSnFr A184Vin hippocampal culture, iGluSnFr detected single field-stimulus evoked

glutamate release. iGluSnFR-expressing cells showed excitatory currents evoked by light

stimulus. The major goal was to assess the influence of different numbers of APs and

spontaneous activity on the height of the pHoenix fluorescence amplitude, as shown in figure

3.7. pHoenix stimulation with 5AP was performed between the 10th and 25th seconds, 10*1AP

was produced between the 25th and 125th seconds, and spontaneous activity was introduced

between the 125th and 300th seconds.

The experiment revealed that stimulation with 5AP resulted in only one elongated (1.8)

normalized fluorescence, but stimulation with 10*1AP resulted in 10 comparably short

fluorescence with amplitudes ranging from 1.05 to 1.2. Spontaneous activity, on the other hand,

contributed amplitudes that were significantly smaller, up to 1.05. The results show that pHoenix

activity is far more dependent on a single intense stimulation than on distributed milder

stimulations or spontaneous activity.

Figure 3-7: Comparison of the effect of using single strong APs, distributed weak APs, and spontaneous activity in

stimulating pHoenix. A: Pictorial presentation of the stimulation of the iGluSnFR at stronger AP versus weaker APs

and spontaneous activity: Graphical presentation of the AP and spontaneous activities on the pHoenix

The major goal was to assess the influence of different numbers of APs and spontaneous activity

on the height of the pHoenix fluorescence amplitude, as shown in figure 3.8. As a result,

pHoenix stimulation with 2AP was compared to single AP stimulation (fig 3.8A), and the results

were comparable to those in figure 3.7, i.e., stimulation took longer (1.3, fig 3.8A) when a

powerful AP (2AP) was utilized and shorter (1.1, fig. 3.8B) when single AP was used to

stimulate pHoenix. In figure 3.8B, the effect of a single weaker AP has been magnified. The

findings in Figure 3.8 confirm that pHoenix activity is considerably more dependent on a single

powerful stimulation than on distributed milder stimulations, as shown in Figure 3.7.

Figure 3-8: Comparison of the effect of using single strong APs and distributed weak Aps. A: the effect of using

2AP stimulation versus single APs for pHoenix stimulation. B: A magnification of the amplitudes that single APs

contribute when used to stimulate pHoenix.

Before the stimulation and illumination activities, a 2.5mm Ca2+ buffer solution containing AP5

and CNQX, TTX (incubation only), Baf, and 1mM ascorbic acid were administered to the

pHoenix to see evoked activity. Before and after the chemicals were applied, the number of

successful evoked activity events as well as the amplitudes (F/Fo) of the activities were recorded.

The number of successful occurrences was found to be 6.7 prior to the application of the

reagents, and 3.5 following the introduction of the chemicals (3.9A). This indicates that the

chemicals, both ascorbic and non-ascorbic, had a considerable impact on the evoked activities.

The chemicals generated a considerable decline in the amplitude of the evoked activity, with the

amplitude dropping from 40*10-3 to 38*10-3 F/Fo (fig. 3.9B). As a result, the chemicals had a

big impact on the amplitude of the elicited activity.

Figure 3-9: Evoked activities before and after addition of ascorbic and non-ascorbic. A: the number of successful

evoked activity events observed before and after the application of the 2.5mM ascorbic and non-ascorbic chemicals.

B: the amplitude (F/F0) of the evoked activity after the application of the ascorbic and non-ascorbic chemicals.

Nonetheless, the number of successful evoked activities remained the same (3.10A) in the

control experiment while the amplitude reduced (3.10B) from 70*10^-3 to 60*10^-3 F/Fo (fig.

3.10). This means that the addition of the ascorbic and non-ascorbic chemicals had no significant

effect on the amplitude of the evoked activity but a significant effect on the number of successful

evoked activities.
Figure 3-10: Evoked activities in the control experiment. A: the number of evoked activities did not change since

there was no addition of the reagents. B: the change in the amplitude of the evoked activity even no addition of

reagents.

Experiment 3.11 (fig. 3.11) was designed to show how AP depletion affects the amplitude and

occurrence of spontaneous activity. The two spontaneous actions were seen before the APs were

depleted (300APs/4Hz), while the stimulation curve trend steadily shifted to the negative (fig

3.11A). However, when the 300APs were depleted at 4Hz, the graph's trend began to

progressively rise. Despite this, due to the depletion, no spontaneous activity was observed (fig

3.11B). This meant that AP depletion had a big impact on the occurrence of spontaneous activity.
Figure 3-11: The effect of APs depletion on occurrence of spontaneous activity. A: Before the APs depletion, the

two spontaneous activities are recorded even though the trend of the graph falls rapidly. B: As the APs get depleted,

the normalized fluorescence becomes longer but no spontaneous activity is registered .

The measurements of the number of successful events and the amplitude of the evoked activity

before and after the administration of 1mM ascorbic and non-ascorbic reagents are carried out in

figure 3.12 a and 3.13, which are identical to those carried out in figure 3.9. The number of

successful induced activity events as well as the amplitudes (F/Fo) of the activities were recorded

before and after the chemicals were applied, and the results were similar. The number of

successful occurrences was found to be 6.7 before the reagents were applied, and 3.5 after the

chemicals were introduced (3.12A). This suggests that both ascorbic and non-ascorbic

substances had a significant impact on the elicited activities. The chemicals caused a significant
decrease in the amplitude of the evoked activity, which fell from 40*10-3 to 38*10-3 F/Fo (fig.

3.12B). As a result, the substances significantly influenced the amplitude of the induced activity.

Nonetheless, in the control experiment, the number of successful evoked activities remained

constant (3.13A), although the amplitude decreased (3.13B) from 70*10-3 to 60*10-3 F/Fo (fig.

3.13). This suggests that the ascorbic and non-ascorbic chemicals had no influence on the evoked

activity's amplitude but did have a substantial effect on the number of successful evoked

activities.
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