City University of New York (CUNY)CUNY Academic Works
Dissertations and Theses City College of New York
2019
Effect of hypoxia on spontaneous neural activity inthe cortex of neonate mouse pupsKrithikka Ravi Ms
How does access to this work benefit you? Let us know!Follow this and additional works at: https://academicworks.cuny.edu/cc_etds_theses
Effect of hypoxia on spontaneous neural activity in the cortex of neonate mouse pups
Thesis
Submitted in partial fulfillment of the requirement for the degree
Master of Science (Biomedical Engineering)
at
The City College of the City University of New York
By
Krithikka Ravi
May 2019
Approved by:
Professor Adrian Rodriguez-Contreras, Thesis Advisor
(Department of Biology, Center for Discovery and Innovation)
Professor Mitchell Schaffler, Chairman
Department of Biomedical Engineering
Effect of hypoxia on spontaneous neural activity in the cortex of neonate mouse pups
Krithikka Ravi
Department of Biomedical Engineering
Dr. Adrian Rodriguez-Contreras
(Department of Biology, Center for Discovery and Innovation)
ABSTRACT
Hypoxia caused by inadequate oxygenation has profound effects on the normal functioning of the
brain in mammals. Acute or chronic hypoxic insults occur in the brain depending on the duration
of hypoxic exposure. Hypoxia is known to occur in the human womb and exerts adverse effects
on the developing fetus. Most of the ongoing research on hypoxia is performed on rodent brain
slice taken from various brain regions using intracellular recording. Extensive work has been
carried out to understand the effects of chronic hypoxia on the developing nervous system,
specifically during intrauterine development. However, effects of acute hypoxia occurring
perinatally, on neuronal activity remain less studied. Spontaneous neural activity occurring during
the first weeks of development is important for priming the nervous system to function efficiently
when encountering sensory-evoked inputs. This calls for the need to understand the effects of acute
hypoxia on spontaneously arising neural activity in-vivo in awake and unanesthetized animals at
an age corresponding to the perinatal period in human fetus (36 – 40 weeks). This study utilized
wide-field epifluorescence imaging to indirectly record neural activity in the form of fluorescence
signals arising from a large volume of brain (from lambda suture to bregma) under normal air and
hypoxic air in SNAP-25-2A-GCaMP-6s transgenic mice at postnatal day 7. Results of this study
demonstrated a statistically significant reduction in frequency and increase in amplitude of
neuronal activity in the entire cortex under hypoxic air when compared to normal air. Bilateral
synchrony in neuronal activity was observed during normal and hypoxic conditions.
TABLE OF CONTENTS
1. Introduction………………………………………………………………………………..1
Hypoxia and its classification……………………………………………………………...1
Hypoxia as a risk factor for newborn infants……………………………………………....2
Developmental spontaneous activity in sensory systems………………………………….4
Significance of the current study…………………………………………………………..5
2. Hypothesis………………………………………………………………………………....6
3. Materials and Methods…………………………………………………………………….7
Animals…………………………………………………………………………………....7
Genotyping………………………………………………………………………………...7
Anchoring cranial windows………………………………………………………………..8
Transcranial wide field epifluorescence imaging………………………………………….9
Immunohistochemistry…………………………………………………………………..10
Image Processing………………………………………………………………………...11
Paired Sample t-test……………………………………………………………………....12
Wilcoxon Signed rank test………………………………………………………………..12
4. Results……………………………………………………………………………………13
Genotyping identified GcaMP-6s positive animals……………………………………....13
Wide-field epifluorescence imaging recorded fluorescence
peaks in GCaMP-6s positive animals and not in GCaMP-6s
negative animals………………………………………………………………………….13
Presence of bilateral synchrony in neuronal activity……………………………………...16
Hypoxia causes a reduction in frequency and increase in
amplitude of spontaneously neural activity……………………………………………....17
Immunohistochemistry………………………………………………………………….. 18
5. Discussion………………………………………………………………………………..20
6. Conclusion……………………………………………………………………………….22
7. References………………………………………………………………………………..23
LIST OF TABLES
Table 1: Effects of hypoxia on neuronal activity…………………………………………………..3
Table 2: List of reagents and oligonucleotide sequences used in PCR reaction…………………....8
LIST OF FIGURES
Figure 1: Wide-field epifluorescence imaging of spontaneous
activity in the cortex………………………………..………………………………….10
Figure 2: Results of PCR reaction………………………………………………………………...13
Figure 3: Time series of changes in GCaMP-6s fluorescence signal……………………………. 14
Figure 4: Overlap of GCaMP-6s fluorescence from left and right
cortex from SNAP-25-2A-GCaMP-6s positive animal...……………………………...15
Figure 5: Overlap of GCaMP-6s fluorescence from left and right
cortex from SNAP-25-2A-GCaMP-6s positive animal...……………………………...16
Figure 6: Bilateral cortical synchrony of intensity peaks…………………………………………16
Figure 7: Frequency distribution of fluorescence peaks………………………………………….17
Figure 8: Amplitude distribution of amplitude peaks…………………………………………….18
Figure 9: Immunohistochemistry………………………………………………………………...19
ACKNOWLEDGEMENT
It is with a deep sense of gratitude that I place on record my sincere thanks to all those who have
encouraged and supported me during the course of my research work.
This thesis work has been completed under the guidance of Dr Adrian Rodriguez–Contreras,
Associate Professor, who had tirelessly kept on motivating and encouraging me to put in my best
effort. I express my heartfelt gratitude to him for the valuable guidance, advice and encouragement
that I received from him throughout the course of the research study.
I also express my sincere gratitude to Dr. Bingmei Fu and Dr. Steven Nicoll for their valuable
inputs.
My special thanks are due to my friends Zeinab Esmaeilpour, Hash Sherif, Mahima Sharma, Erina
Hara, Lukas Hirsch, Aakriti Mittal, Pooja Kumar, Janak A Jain, Durga Shankar and Abhishek
Sanghani for their unconditional support and encouragement throughout the course of my research
study.
I would also like to thank Dr. Lucas Parra, whose class inspired me and gave me the confidence
on the usage of programming language for this project.
Finally, I would like to thank my family for their constant support and motivation.
ABBREVIATIONS
AMPA - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
NMDA- N-methyl-D-aspartate
kPa - Kilo pascal
PCR - Polymerase Chain Reaction
HIF - Hypoxia Inducible Factor
EDTA - Ethylenediaminetetraacetic acid
TBE - Tris-Borate-EDTA
BBB - Blood Brain Barrier
1
INTRODUCTION
Adequate oxygen concentration in atmospheric air is quintessential for normal functioning of the
human body. Normal atmospheric gas composition of oxygen is 21% of dry air and inspired
oxygen pressure is considered to be 19.6 kPa at sea level (atmospheric pressure is partial pressure
of constituent gases along with partial pressure of water vapor, 6.3kPa at 37℃). Normoxia is a
condition of normal oxygen composition of 21% along with other gases in the atmosphere.
Increase in altitude causes a fall in atmospheric pressure and partial pressure of oxygen. This fall
in partial pressure of oxygen leads to reduction in volume of inspired oxygen caused by reduced
driving pressure for gas exchange in the lungs (Peacock, 1998).
Hypoxia and its classification
Inadequate oxygenation leads to depletion in the required supply of oxygen concentration to the
tissues causing a condition known as hypoxia. Hypoxia can occur throughout the human body
affecting all cell types. Therefore, it is crucial for the cells and tissues to be able to detect reduction
in the partial pressure of oxygen and respond appropriately for effective survival (Shimoda, 2010).
Hypoxia is classified into two types on basis of duration of hypoxic insult, acute hypoxia and
chronic hypoxia (Bayer, 2011). Duration of acute hypoxia ranges from a few minutes to few hours
whereas chronic hypoxia extends from days to months (Hutter, 2010).
The brain is the largest oxidative organ and consumes a disproportionately large percentage of
oxygen in comparison to its total body mass. Adult human brain is approximated to take up only
2% of the total body weight, its energy consumption is 10 times more than the entire body’s energy
consumption (Erecińska, 2001). This is due to its constituent cells, neurons, that are extremely
sensitive to changes in the partial pressure of oxygen. Because of a neuron’s high sensitivity to
reduction in the partial pressure of oxygen, any reduction in oxygen saturation (or hypoxia) leads
to increased production of reactive oxygen species (ROS) in the brain (Maiti, 2006).
Condition of reduced partial pressure of oxygen manifests in the human womb during fetal
development (Hutter, 2010). Hypoxia can occur to the fetus during the conception of embryo,
gestational developmental period and delivery. Hypoxia exerts a supportive effect during
embryogenesis (until the first 10 weeks of pregnancy) by protecting the developing embryo against
oxygen-mediated damage, antioxidant enzyme catalase, peroxidase and mitochondrial superoxide
dismutase arising within placental tissue (Watson, 1998). This protective role of hypoxia changes
after the 13th week of gestation when oxygen saturation gradually increases attains a level of 60%
during the second trimester. Any decrease in partial pressure of oxygenation after the second
trimester has an adverse effect of the fetus causing a condition known as intrauterine hypoxia
(Hutter, 2010).
Chronic hypoxia in the uterus occurs from several days up to months. On the contrary, acute
hypoxia occurs for a few minutes to hours in fetal life. This is caused during maternal labor,
umbilical cord compression, placental abruption and abnormal uterine contractions, posing a major
challenge to fetal life (De Haan, 2006).
2
Hypoxia as a risk factor for newborn infants
Acute hypoxia occurring during birth, also termed as asphyxia affects 1-6 infants in every 1000
live full-term births. Brain injury caused by asphyxia perinatally, stands as one of the common
causes of morbidity and death of preterm and term fetuses, contributing to 23% of neonate’s death
across the globe (Lawn, 2005). In addition, perinatal hypoxia is also associated with increasing
the risk factor of the fetus to develop neurological disorders in the future. For example, it was
concluded from a pilot study and review of literature case study that patients affected with
schizophrenia had a history of obstetric complication in comparison with other psychiatric and
normal patients (Lewis, 1987). A perinatal asphyxia rat model study on Wistar rat pups showed
exaggerated age-related long-term memory impairment in rats that experienced perinatal
asphyxia during birth (Berg, 2000). A 2018 study (Lima, 2018) using perinatal asphyxia rat model
revealed abnormal permeabilization of the blood-brain barrier, increased mitochondrial
respiration rate, cyanosis and hypertonia in pups that were exposed to acute hypoxia (15 minutes)
around the time of their birth. The study concluded that perinatal asphyxia, causes mitochondrial
damage and alteration of brain development at a subcellular level without exhibiting any visible
changes in the morphological features of neurons.
General fetal response to hypoxia at the cellular level includes changes in the expression patterns
of hypoxia- inducible factor (HIF), a transcription factor that regulates several genes belonging
to the glycolytic pathway (Zimna, 2015). Such changes occurring at a genetic level in addition to
providing protective effects, persist throughout postnatal life and increase susceptibility to
develop neurodegenerative disorders (Nalivaeva, 2018). Changes occur in expression levels of
certain specific genes in the cortex during fetal and early postnatal development and these
changes play a direct role in postnatal and adult cognitive functioning. One such genes that plays
a significant role in early brain development is RE1- silencing transcription factor (REST) which
is also shown to play a dominant role in shaping the changes occurring during adult neurogenesis
(Lunyak, 2016). Changes in the expression levels of REST factor have shown to correspond to
mild cognitive disorders, Huntington disease, down syndrome and Alzheimer’s disease occurring
postnatally (Nho, 2015). REST factor helps in protecting integrity during normal embryogenesis
and during hypoxia REST accumulates in nucleus of hypoxia exposed cells, functioning as major
repressor of 20% of hypoxia- repressed genes, acting counter-regulatory to HIF-dependent gene
expression (Nalivaeva, 2018).
Notable change induced by hypoxia in the brain is rapid failure of synaptic transmission and
considerable membrane depolarization acting as a signal for further neuronal hyperexcitability,
irreversible neuronal dysfunction and neuronal death (Balestrino, 1995). Two hypotheses attempt
at explaining the reason for suppressed neuronal activity. The first hypothesis talks about the
central role of glutamate and excessive opening of ionotropic glutamate receptor channels in
creating a hypoxic brain damage (Choi, 1990). The second hypothesis postulates that an excessive
accumulation of calcium ions in the cytosol, caused by loss of calcium homeostasis, over
stimulates calcium dependent proteases, phospholipases and endonucleases (Lipton, 1994).
3
Most of the original work focusing on understanding the effects of hypoxia on neuronal activity
have utilized brain slices and intracellular recording technique. The reason stated by most of the
brain slice studies is that different brain regions exhibit different responses to hypoxic insult and
in order to understand how hypoxia is directly related to irreversible neuronal damage in a
particular brain region, recording using brain slices is the most straightforward approach (Nieber,
1999). Effects of hypoxia in brain slices taken from different regions of the brain are described in
Table 1.
Brain region Age and strain
of the animal
used in the
study
Technique
used in the
study
Observation
made
(Hypoxic
condition)
Normoxic
condition References
Hippocampus Sprague-
Dawley rats
(Postnatal day
14- postnatal
day 23)
Intracellular
recording of
resting
membrane
potential from
CA1 region in
hippocampal
slices (300 µm)
under hypoxic
condition (95%
N2- 5% O2) for
15-20 minutes
at 30℃ - 31℃
Initial response to
hypoxia was
membrane
hyperpolarization
along with
increase in resting
conductance
Reoxygenation of
the slices led to
post-hypoxic
hyperpolarization
followed by
normalization of
resting potential
(Hyllienmark,
1999)
Brain stem Sprague-
Dawley rats
(Postnatal day
5- postnatal
day 15)
Intracellular
recording from
transverse
brainstem slices
(400 µm) under
hypoxic
condition (95%
N2-5% CO2)
for less than 1
minute
Hypoxia induced
spreading like
depression found
in the brainstem
slices
Recovery from
hypoxia observed
during
reoxygenation
(Funke, 2009)
Retinal and
superficial
superior
colliculus
neuronal culture
Male and
female Wistar
rats (Postnatal
day 1)
Whole cell-
patch clamp
technique in
presynaptic and
postsynaptic
cells under
hypoxic
solution for a
duration of 20
minutes
Hypoxia induced
suppression of
retinocollicular
neurotransmission
in pre-synaptic
and postsynaptic
coupled neurons
Rapid recovery of
neurotransmission
for reoxygenation
in synaptically
coupled neurons
(Dumanska,
2019)
Table 1: Effects of hypoxia on neuronal activity
4
Developmental spontaneous activity in sensory systems
Electrical activity originating intrinsically during early stages of development in sensory systems,
even before the onset of sensory experience is known as spontaneous activity. This spontaneous
activity in the developing nervous system is required for refinement of synaptic connections,
formation of neuronal maps and establishment of neural circuitry before the onset of stimulus-
induced neural activity (Yin, 2018). Babola et al., in their 2018 study, used wide-field imaging on
P6-P15, SNAP-25-2A-GCaMP-6s positive mice. They showed that in the developing auditory
system, spontaneous activity arose in the cochlea before the period of hearing onset and that such
activity had the potential to influence maturation of sound-processing circuits by propagating to
the central auditory centers. Ackman et al., in their 2012 study demonstrated the presence of
spontaneous neural activity originating in retinal ganglion cells (RGC) in mouse pups of P3-P9
age group using calcium-dye labelling and two-photon imaging. Results of the study showed that
spontaneous waves were present for a week during development and were responsible for
conveying retinal organization information to circuits in the entire visual system through a specific
pattern of activity (Ackman, 2012). It is evident from these studies that the first week of postnatal
development in rodents forms an important period for spontaneously arising neural activity to
refine and prime the sensory circuitry in order to efficiently propagate evoked activity from sensory
organs to the appropriate processing centers in the brain.
Studies aiming to replicate human perinatal hypoxic-ischemic brain damage in rodents require an
appropriate postnatal period that faithfully recapitulates the developmental landmarks. The 7-day
postnatal rodent (P7) is an appropriate model for perinatal hypoxia studies. During this age,
histological similarities between the rodent brain and that of 36 to 40-week gestation human
fetus/newborn infant were observed (Semple, 2013). These similarities included axonal pruning
and apoptosis of overabundant cells (Bockhorst, 2008), heightened rate of birth of glial cells
(Kriegstein, 2009) and completion of layering of neurons in the cerebral region (Weitzdoerfer,
2004).
Although several studies are conducted on brain slices with the aim of understanding the effects
of hypoxia on neuronal activity and comparing them to control conditions, there are no studies
exploring the effects of hypoxia on spontaneous neural activity arising in vivo in unanesthetized
animals at an age corresponding to the perinatal period in human infants (36-40 weeks). Most of
the studies available, have been able to demonstrate different phase changes in a neuron’s response
to hypoxia, hypoxia induced depolarization, heightened excitability and hyperpolarization. Most
of the brain slice studies have employed techniques related to intracellular recording. These
techniques focus mainly on recording electrical activity from single cells, neurons. These studies
also report variations in response to oxygen deprivation among different brain regions, and the
difficulty involved with recording such different responses, under the same field of view
(Hyllienmark, 1999).
In live animals, neural activity occurring in brain tissue is a much-complicated interplay involving
glial cells, local blood flow (forming the basis of neurovascular coupling) and concentration of
5
glucose available in the surrounding extracellular space. Studies have elaborated the roles of
astrocytes and microglial cells as primary responders to neuronal hypoxic insult, by changing their
phenotype and active functions in order to turn them to reactive cells (Mucci, 2017). In 2018,
Karunasinghe et al., in their patch-clamp study demonstrated the response of astrocytes, present in
different brain regions, to acute hypoxic environment. Results of this study concluded that
astrocytes depending on their location in the brain, can either enhance effects of acute hypoxic to
a profound anoxic (complete loss of oxygen) depolarization causing irreversible death of neurons,
or return to normal conditions following reperfusion. Luo et al., in 2019 performed a systematic
review on bibliographic articles to determine if there were changes in the levels of S100B protein
in serum samples taken from neonates with perinatal asphyxia 24 hours after birth. S100B is a
cytosolic calcium-binding protein found concentrated mainly in glial cells. The results of this
literature review concluded that increased serum S100B levels as found in neonates after 24 hours
of birth, can serve as an indicator of brain damage caused by rupture of the blood brain barrier. It
can be inferred from this review that glial cells in live subjects play a role in helping the neurons
combat hypoxic insult.
Significance of the current study
The current study aims to understand the changes in the activity of cortical neurons during
exposure to an acute hypoxic insult for a duration of 10 minutes, using an indirect optical imaging
technique in live and awake animals at postnatal day 7. In this study, neuronal activity is recorded
under baseline conditions (normal air) for a duration of 10 minutes. Under the same experimental
conditions, the study investigates how acute hypoxic insult alters neuronal activity.
This study utilizes in-vivo wide-field epifluorescence imaging in unanesthetized transgenic
(SNAP-25-2A-GCaMP-6s) mice, a calcium-indicator tool strain containing a genetically encoded
calcium indicator (GCaMP-6s). These animals have widespread expression of the slow variant of
GCaMP-6s in neurons. GCaMP-6s has the property of ultrasensitive detection of single neuron
action potentials coupled with slow decay and response kinetics (Chen, 2013). Global cortical
response, in vivo, to hypoxic exposure was optically recorded, overcoming the previously
mentioned limitations as encountered in brain slice studies. Furthermore, this study utilizes
transgenic mouse model at postnatal day 7 (P7) for hypoxic exposure and imaging. P7-P10
corresponds to a period of peak brain growth spurt, peak in gliogenesis, increasing axonal and
dendritic density, oligodendrocyte maturation state changes and consolidation of the immune
system. This period in addition to providing an efficient model to study the impact of hypoxia on
major neurodevelopmental changes also corresponds to human gestation (term infant) of 36-40
weeks (Semple, 2013). This period is known as a perinatal sensitive period, a stage when the
human fetus is highly prone to acute hypoxic insult (Hutter, 2010).
6
HYPOTHESIS
If spontaneous activity in the cortex is affected by hypoxia, then there should exist a quantifiable
difference in neural activity recorded under normal air and hypoxic air.
7
MATERIALS AND METHODS
Animals
Experimental protocol used in this study was approved by the Institutional Animal Care and Use
Committee of the City College of New York. Animals used in this study were housed in a cage
under conditions of 12-hour light/dark cycle and were provided with food and water ad libitum.
Animal cages were kept in an aseptic environment in the City College animal house facility.
SNAP25-2A-GCaMP-6s (Synaptosomal Associated Protein 25kDa) heterozygote females were
purchased from Jackson Laboratory (JAX 025111). One female was paired with a male C57BL6.
The animals were separated after 5 days to a week of pairing. Females were checked for birth after
21 days of gestation every day. The day of birth was recorded as P0. Newborn pups were housed
in the same cage along with their mothers until imaging experiments.
Genotyping
At postnatal day 6 (P6), 2mm of toe tissue was clipped aseptically and immersed in 100 µl of lysis
buffer (200µM EDTA at pH 8.0) taken in a 1.5 ml eppendorf. The tissue along with the lysis buffer
was heated to 97℃ on a hot plate (Thermo Scientific) for 1 hour. Tubes were removed from the
hot plate after 1 hour and vortexed thoroughly. 100 µl of buffer containing 40 mM Tris-HCl at pH
8.0 was added to the tubes and centrifuged at 12,000 rpm for 3 minutes at 4℃. After centrifugation
the tubes were stored at room temperature and DNA template from the solution was used for the
following PCR reaction.
8
Oligonucleotide sequences Concentration
& volume
Source
Primer (Forward): CCCAGTTGAGATTGGAAAGTG
(SNAP25-2A-GCaMP6s-D-Wildtype)
0.5 µl of (10
µM
concentration)
IDT
Primer (Reverse): CTGGTTTTGTTGGAATCAGC
(SNAP25-2A-GCaMP6s-D-Wildtype)
0.5 µl of (10
µM
concentration)
IDT
Primer (Forward, 5’-3’): CCCAGTTGAGATTGGAAAGTG
(SNAP25-2A-GCaMP6s-D-Mutant)
0.5 µl of (10
µM
concentration)
IDT
Primer (Reverse): ACTTCGCACAGGATCCAAGA
(SNAP25-2A-GCaMP6s-D-Mutant)
0.5 µl of (10
µM
concentration)
IDT
Master Mix 10 µl Thermo
Scientific
PCR- grade water 7 µl Thermo
Scientific
Total reaction mixture 20 µl
Table 2: List of reagents and oligonucleotide sequences used in PCR reaction
PCR protocol used in the study was adopted from Jackson Laboratory site (www.jax.org).
Following PCR, samples were run on a 1.5% agarose gel prepared in 1X TBE buffer prepared
from 10X TBE Electrophoresis buffer (89 mM Tris, 89mM boric acid, 2mM EDTA, Thermo
Scientific) in distilled water and ethidium bromide (10 mg/ml, Thermo Scientific). The gel was
allowed to solidify at room temperature. Following gel solidification, 15 µl of the PCR product
was added to each well. A no-template control (water control) was used as a negative control.
Equal volume of 100 bp DNA ladder (50 µg/ml, New England BioLabs) was added to a separate
well. The gel was run at 125 volts for 1 hour or until the DNA bands migrated half the distance
from the starting well position. The gel was viewed under UV light with an exposure duration of
15 seconds.
Anchoring cranial windows
Male and female pups at postnatal day 7 (P7) were separated from their mother and acclimatized
to the surgical room temperature at 25℃ for 15 minutes. Following acclimatization, inhalation
anesthesia was induced using vaporized isoflurane at an initial dose of (4% -5% with pure oxygen)
until the animal became unresponsive to toe pinch. An incision was made starting midline to the
9
ears (lambda suture) and ending at posterior to the eyes (bregma suture). Dorsal surface of the
scalp was removed following subsequent cuts (Babola et al., 2018). The exposed area was cleared
of underlying membranes and other contaminating particles using gelatin sponge soaked in sterile
artificial cerebrospinal fluid (ACSF) and dry sterile gelatin sponge (Gelfoam). Care was taken such
that the imaging field remained free of blood clots that would interrupt capturing reflected
fluorescence signals arising intrinsically. 5mm round glass cover slip (Bellco Glass) was placed
on the exposed skull using super glue (Krazy Glue). Super glue was placed along the edges of the
metal head plate and the plate was anchored such that it surrounded the cover slip. The animal was
weaned from isoflurane supply and placed on a heating pad and allowed to recover from surgery
for an hour (Babola et al., 2018).
Transcranial wide field epifluorescence imaging
Following an hour of post-surgery recovery, the animals were transported to the imaging unit
(Figure 1). The animals were placed on a heating pad to avoid any loss of body temperature
throughout the imaging experiment. Image frames were captured on a digital camera (Thor Labs)
at 8 Hz with an exposure duration of 124 milliseconds at a maximum gain of 24x. Halogen lamp
(HAL 100) was used as the source of white light. The exposed region of the skull was illuminated
with blue light of wavelength (470 nm -490 nm) obtained after filtering through an excitation filter.
The filtered blue light was then directed to a dichroic mirror and after getting reflected from the
mirror, the resulting light was focused on the specimen using a macro lens (SMC Pentax f1.2).
Reflected light from the skull was captured after passing through an emission filter. Image frames
were recorded on Thor camera software as 10-bit monochrome images and saved in 8-bit tiff
format without annotations at a resolution of 968x608 pixels after horizontal 2x and vertical 2x
sub-sampling for an uninterrupted duration of 20 minutes.
10
Figure 1: Wide-field epifluorescence imaging of spontaneous activity in the cortex from lambda to bregma in awake
SNAP-25-2A-GCaMP-6s positive mouse pups at P7.
Imaging session consisted recording GCaMP-6s activity in response to two different conditions
(normal air and hypoxic air). Images were captured from the exposed cortical region starting from
lambda suture until bregma for SNAP25-2A-GCaMP6s- positive and SNAP25-2A-GCaMP6s
negative pups at P7. 20 minutes uninterrupted imaging session started with recording neuronal
activity in response to normal air delivered at a flow rate of 1L/min at a pressure of 1 bar for a
duration of 10 minutes (4800 image frames) followed by hypoxic air (8% O2 and 92% N2) for the
next 10 minutes (4800 image frames) delivered at a pressure of 1 bar.
Immunohistochemistry
One SNAP25-2A-GCaMP-6s positive and one negative animal taken from the imaging experiment
were decapitated to isolate whole brain. As the negative animal lack GFP insertion and do not
exhibit fluorescence on excitation, they were taken as the negative control. Isolated whole brains
were immersed in 4% paraformaldehyde prepared in 0.1M phosphate buffer (pH 7.2). The brains
were subjected to gradient dehydration in 10%, 20% and 30% sucrose solution (prepared in 0.1M
phosphate buffer) until the brains sunk in each of the sucrose solutions. 80µm sections were cut
using a vibratome and the brain slices (from positive and negative animal) were transferred to 24
well dishes for staining.
11
Sliced brain sections were double stained for neuronal nuclei (NeuN, Millipore) and for
fluorescence in neuronal cell bodies. Sections were initially blocked using 0.4% normal goat serum
for 30-45 minutes. This was followed by incubation in primary antibody solutions containing 1%
normal goat serum and permeabilization agent (0.3% Triton-X100). The primary antibody for
staining GFP (present in the neuronal cell body) was raised in chicken and was used at a dilution
of 1:4000 (Stock solution concentration- 10 mg/ml, Abcam, Lot no: 13970). Primary antibody for
staining neuronal nuclei (NeuN) was raised in guinea pig and was used at a dilution of 1:500 (Stock
solution concentration-1 mg/ml). Sections were incubated in primary antibodies overnight at 4℃.
The following day, sections were rinsed twice in washing solution composed of 0.02% Triton-
X100. Secondary antibodies used were Alexa Fluor 647 Goat anti-guinea pig antibody and Alexa
Fluor 594 Goat anti-chicken antibody. Working concentration of secondary antibody was 2 µg/ml
and was prepared in a solution containing 0.02% Triton-X100. Brain sections were incubated in
secondary antibody solution for 2 hours at room temperature. Following secondary antibody
incubation, the sections were washed four times in the washing solution (solution used during the
first wash). Sections were then mounted on a glass slide and covered using a coverslip. The
coverslips and brain sections were sealed to the glass slide using mounting medium (Thermo
Scientific). Images were captured on a confocal microscope (Leiss LSM 800) using a 40X oil-
immersion objective.
Image Processing
Regions of interest in the cortex from GCaMP-6s positive animals were corrected for bleaching
(using bleach correction function on ImageJ) and imported to MATLAB environment. Raw images
were imported on ImageJ platform and each individual collection of image frames were
concatenated on the ImageJ platform. Following this, the images were corrected for
photobleaching using the Bleach Correction function, exponential fit on ImageJ. Region of interest
(ROI) was selected using ROI manager tool on ImageJ manually for every imaging session.
Fluorescence intensity measurements for the selected ROIs were measured and the data was
recorded on an excel sheet.
Data was imported into MATLAB (Mathworks) and fluorescence intensities were normalized
using equation (1)
Normalized fluorescence intensity = ΔF (1)
Fo
ΔF= F – Fo (2)
Where,
F = Amplitude of the recorded fluorescence signals from SNAP-25-2A-GCaMP-6s positive and
negative animals
12
Fo = Minimum computed value of the recorded fluorescence intensity using the min function in
MATLAB.
Following normalization of fluorescence intensities, fluorescence peaks from the signals were
detected using an in-built function on MATLAB (findpeaks) using a fixed value threshold criterion
for all recording sessions. Threshold value was set at 20% by analyzing ΔF/Fo values of SNAP-
25-2A-GCaMP-6s negative animals. Number of peaks computed using findpeaks function was
recorded for baseline condition and for the hypoxic condition. The resulting data was plotted
graphically on MATLAB using the plot function.
Peaks above the set 20% threshold was computed for both conditions (normal air and hypoxic air).
The resulting data was tested for normality using Shaipro-Wilk’s test (using SPSS Software).
Frequency and amplitude distribution of the fluorescence peaks above 20% ΔF/Fo was mapped
using GraphPad Prism software for data obtained from left and right cortex.
Paired Sample t-test
Data collected from five SNAP25-2A-GCaMP6s positive animals under two test conditions
(normal air and hypoxic air) was analyzed using paired t-test function on MATLAB. The null
hypothesis under consideration was that the mean of the differences in intensities between normal
condition and hypoxic condition was zero. The significance level was set at 5% and p-value was
calculated under paired t-test.
MATLAB function: [h,p]= ttest(normal air intensity peaks, hypoxic air intensity peaks)
h value of 1 would be returned if the null hypothesis was rejected and h value of 0 would be
returned if the null hypothesis was failed to be rejected at a significance level of 0.05. p-value
returned by the program was used for computing statistical significance of the data.
Wilcoxon Signed Rank test
Amplitude data collected from all five SNAP25-2A-GCaMP6s positive animals under two test
conditions (normal air and hypoxic air) was analyzed using Wilcoxon Signed-rank test (SPSS
Software). The null hypothesis under consideration was that the median of the differences in
amplitude intensity between normal condition and hypoxic condition was zero. The significance
level was set at 5% and p-value was calculated under Wilcoxon Signed-rank test.
Kurtosis of the amplitude data was measured individually for all five animals (under normoxia and
hypoxia) using MATLAB ‘kurtosis’ function.
13
RESULTS
Genotyping identified GCaMP-6s positive animals
Postnatal day 6 (P6) SNAP-25-2A-GCaMP-6s pups were genotyped to identify GCaMP-6s
positive heterozygous animals. Bands of very low intensity were observed in SNAP-25-2A-
GCaMP-6s negative pups as seen in lane marked pup3 and pup4 in SNAP-25 mutant section. An
exemplar image showing the results of genotyping is given in figure 2.
Figure 2: PCR performed to amplify 498 base pair SNAP-25 wild-type fragments and 230 base pair SNAP-25 mutant
fragments. PCR products were run on a 1.5% TBE agarose gel. L- 100 bp DNA ladder, WC- water control without
template DNA.
As shown in figure 2, DNA bands can found in lanes close to 500 base pair (498 bp) in SNAP-25
wild type primer section, corresponding to DNA ladder and close to 200 base pair (230 bp) in
SNAP-25 mutant primer section, corresponding to DNA ladder for pup 1 and pup 2. Pup 1 and
pup 2 are heterozygous positive for GCaMP-6s as they contain one copy of the calcium indicator
containing the GFP sequence. Heterozygous negative animals will not contain a sequence for GFP
as it is not a knockin line containing calcium indicator. This feature is exhibited by pup 3 and pup
4 with no bright DNA bands in lanes corresponding to 230 bp. Heterozygous positive and negative
male and female pups were selected following genotyping for surgery and imaging.
Wide-field epifluorescence imaging recorded fluorescence peaks in GCaMP-6s positive animals
and not in GCaMP-6s negative animals
Spontaneous activity as indicated by increase in GCaMP-6s fluorescence signals were consistently
present in the cortical region of SNAP-25-2A-GCaMP-6s positive animals at P7 (Figure 3).
Bilaterally spreading spontaneous neural activity as represented by fluorescence signals was found
in all five positive animals that were imaged. The SNAP-25-2A-GCaMP-6s negative animals
14
lacked this fluorescence activity. Image frames were acquired from SNAP-25-2A-GCaMP-6s
positive animals and from one negative animal from every litter. Image frames acquired from a
SNAP-25-2A-GCaMP-6s positive and SNAP-25-2A-GCaMP-6s negative mice pups during
imaging are shown in figure 4 and figure 5.
Figure 3: Representative image of time series of changes in GCaMP-6s fluorescence signal. (A) Changes in GCaMP-
6s signal in a SNAP-25-2A-GCaMP-6s positive animal (B) Changes in signal in SNAP-25-2A-GCaMP-6s negative
animal.
Exemplar traces of fluorescence (indirectly reporting neuronal activity) recorded in GCaMP-6s
positive and negative animals are displayed in figure 4 and figure 5. A decrease in activity can be
observed when the animals were inhaling hypoxic gas (92% N2 & 8% O2) as compared to animals
inhaling normal air at a pressure of 1 bar.
15
Figure 4: Representative image of overlap of GCaMP-6s fluorescence recorded from left and right cortex from SNAP-
25-GCaMP-6s negative animal. (A) GCaMP-6s fluorescence over entire imaging duration (1200 seconds) (B) Zoomed
in image of overlap of left and right cortical fluorescence intensity peaks. ‘o’ represents fluorescence peaks above
.F/Fo under hypoxic conditionߡ F/Fo under normoxic condition. ‘x’ represents fluorescence peaks above 20%ߡ 20%
16
Figure 5: Representative image of overlap of GCaMP-6s fluorescence recorded from left and right cortex from SNAP-
25-GCaMP-6s positive animal. (C) GCaMP-6s fluorescence over entire imaging duration (1200 seconds) (D) Zoomed
in image of overlap of left and right cortical fluorescence intensity peaks. ‘o’ represents fluorescence peaks above
.F/Fo under hypoxic conditionߡ F/Fo under normoxic condition. ‘x’ represents fluorescence peaks above 20%ߡ 20%
Results of the optical recording were categorized as bilateral cortical synchrony of neuronal
activity, frequency and amplitude distribution of fluorescence intensity peaks under normal and
hypoxic conditions.
Presence of bilateral synchrony in neuronal activity
Bilateral synchrony in neuronal activity was found under both normal air conditions and hypoxic
air conditions with the peak intensity being bigger or smaller on one side in comparison with the
contralateral side (Figure 6).
Figure 6: Representative image of bilateral cortical synchrony of intensity peaks under normal air conditions and
hypoxic air conditions for a recording duration of 10 minutes each.
17
Hypoxia causes a reduction in frequency and increase in amplitude of spontaneous neural activity
Analysis of frequency and amplitude distribution of fluorescence peaks revealed the effects of
hypoxia on spontaneous neural activity. A reduction in frequency of fluorescence peaks (above
threshold) is found under the conditions of hypoxia (Figure 7). In five animals imaged under
hypoxic conditions, frequency of fluorescence peaks (above threshold) clearly decreased by an
average of 31± 15.64 as compared to normoxic conditions of 61.3 ± 16.8. Difference in frequency
of fluorescence peaks between control and hypoxia were statistically significant (p<0.01, Paired t-
test).
Figure 7: Plot of frequency distribution of fluorescence peaks (representing neuronal activity) under normoxic and
hypoxic conditions. Results are expressed as the number of peaks above threshold (20% ߡF/Fo) (mean±SEM, n= 10).
**p<0.001.
Amplitude of fluorescence peaks did not follow a normal distribution (n=5 animals) (Figure 8). In
contrast to the reduced frequency of peaks, amplitude of fluorescence peaks under hypoxic
condition had a higher median value of 0.3045±0.1601 as compared to normoxic conditions
0.288±0.0873. Difference in amplitude of fluorescence peaks between control and hypoxia were
statistically significant (p < 0.05, Wilcoxon signed-rank test).
Analysis of kurtosis was performed to understand the distribution of amplitude data. Results of
kurtosis analysis for individual GCaMP-6s positive animals under normoxia and hypoxic is
represented in Figure 8A. Out of five animals recorded, two animals displayed increased kurtosis
value during hypoxic condition as compared to normoxic condition. Two animals displayed
decreased kurtosis value during hypoxic condition as compared to normoxic condition. One animal
did not exhibit any change in kurtosis under normoxic and hypoxic condition.
18
A B
Figure 8: Analysis of amplitude distribution of fluorescence peaks (representing neuronal activity) under normal air
conditions and hypoxic air conditions. (A) Plot of kurtosis of amplitude data (recorded under normoxia and hypoxia)
from five SNAP-25-2A-GCaMP-6s positive animals. (B) Box plot summarizing amplitude distribution recorded under
normoxia and hypoxia from five SNAP-25-2A-GCaMP-6s positive animals. In each box plot, horizontal line crossing
the box is the median, top and bottom of the box are the upper and lower quartiles, whiskers extending up and down
from the periphery of the box are minimum and maximum values. Represents outlier amplitude data points recorded
under normoxia. Represents outlier data points recorded under hypoxia. (Tukey comparison of medians, *p<0.05)
Immunohistochemistry
Coronal slices of the cortex were imaged. Staining was found to be positive for GFP expression in
both SNAP-25-2A-GCaMP-6s positive and negative animals and are represented in red color
(Figure 9). Staining was found to be positive for neuronal nuclei in both SNAP-25-2A-GCaMP-
6s positive and negative animals.
19
Figure 9: GCaMP-6s expression in GCaMP-6s positive and negative animal at P7 on 80 µm thick coronal sections
(A) labeling for neurons expressing green fluorescent protein (GFP) in red (B) labeling for neuronal nuclei (NeuN)
in green color (C) Merge of neuronal cells expressing green fluorescent protein (red) and nucleus present in neurons
(green).
20
DISCUSSION
In the current study, changes in the levels of neuronal calcium were recorded indirectly in
unanesthetized animals using wide-field epifluorescence imaging, facilitating visualization of
neuronal activity under normal and hypoxic conditions on a spatial (over the entire cortical region)
and temporal scale (over time duration of 20 minutes). This study was conducted on animals at
postnatal day 7, an age when the optical properties of the skull are suitable for transcranial imaging
in the presence of an imaging window (Soleimanzad, 2017). At this young age, the animal’s skull
is translucent and therefore, transmits most of the fluorescence signal originating in the brain,
through the skull to reach the sensor (camera) present in the imaging set up. In addition, use of
GCaMP-6s in recording spontaneous resting state activity reveals neuronal activity with a high
spatial accuracy (Vanni, 2014; Mohajerani, 2013). GCaMP-6s rather than any other variant was
suitable for this study as it detects small changes in intracellular calcium concentrations, displays
slower decay kinetics, exhibits higher sensitivity and exhibits lower photobleaching enabling long
term imaging (Garcia, 2017). Long term imaging with low photobleaching is essential to study the
response of neurons to hypoxia over the entire ten minutes of imaging duration.
This study utilized wide-field fluorescence imaging where signal was recorded from neuronal
population over a large surface area (mesoscopic level) rather than recording signals representing
the activity of individual neurons (Homma, 2009). This technique was well suited for this study as
compared to two-photon microscopy as a larger field of view was imaged to understand neuronal
interactions over a large scale without the need for scanning small regions in the cortex. This
facilitated in understanding how various cortical regions (left and right cortex) responded to
hypoxic insult under one imaging field of view.
Bilateral presence of fluorescence activity in the entire cortical area was observed (Figures 4,5 and
7). This activity originated at no defined region in the cortex and spread in the form of short waves
in no specified direction. As shown in figure 7, fluorescence activity in the left and the right cortex
occurred in a pattern where they appear as mirror images of each other in a GCaMP-6s positive
animal at P7. This observation is similar to the results obtained by Babola et al. (2018) where they
recorded spontaneous activity during the pre-hearing phase in SNAP-25-2A-GCaMP-6s positive
animal at P7. In their study, spontaneous activity in two lobes of the inferior colliculus overlapped
on each other and exhibited bilateral synchrony.
Analysis of frequency distribution of fluorescence peaks above the set amplitude threshold
criterion (20% ߡF/Fo) in SNAP-25-2A-GCaMP-6s positive animals revealed an overall reduction
in frequency of intensity peaks under hypoxic condition as compared to normal air conditions.
This reduction in frequency occurred bilaterally under hypoxic condition and it was observed in
all five GCaMP-6s positive animals used in the study (Figure 6). Statistical analysis performed
using paired t-test strengthens the obtained results with a p-value of 0.000572 for data obtained
from both left and right cortex together at a significance level of 0.05. A possible cause for this
21
observed reduction in the fluorescence can be attributed to suppression of neural activity caused
by brief exposure to hypoxic environment. A similar result was observed in a study that
demonstrated pre pathological retinocollicular plasticity facilitated by short-term/acute hypoxia.
Study results demonstrated suppression of AMPA synaptic transmission associated with acute
hypoxia (Dumanska, 2019).
Analysis of amplitude distribution of fluorescence peaks in SNAP-25-2A-GCaMP-6s positive
animals revealed a significant increase in fluorescence peak amplitude under hypoxic condition as
compared to normoxic conditions as revealed by p-value (Figure 8). Kurtosis data revealed two
out of the five animals exhibiting an increased kurtosis during hypoxic condition as compared to
normoxic conditions. Under hypoxia the amplitude distribution did not follow a normal
distribution and fell towards the heavy tailed region. Two animals exhibited decrease in kurtosis
and their amplitude distribution fell towards the lighter tailed region. One animal did not exhibit
change in kurtosis between hypoxia and normoxia and hence the amplitude distribution was not
different between hypoxia and normoxia. The use of SNAP-25-2A-GCaMP-6s positive animals in
this study facilitated an ultrasensitive imaging of calcium ions during neuronal firing. A likely
reason for the increase in amplitude of fluorescence peaks can be attributed to accumulation of
calcium ions near the vesicle and modulating the amount of neurotransmitter released from the
vesicles (Dodge, 1967). However, the difference in mechanisms of calcium influx into the cytosol
of neurons under normoxic conditions and hypoxic conditions remains unknown. As mitochondria
is highly sensitive to hypoxic insult (Lima, 2018) and mitochondrial function and calcium ion
accumulation in the neurons is tightly coupled, it can be hypothesized that calcium ion homeostasis
is imbalanced leading to excessive accumulation of calcium in the cytosol causing an increase
intensity of fluorescence peaks during hypoxia (Duchen, 2012).
Intracellular recordings performed on brain slices obtained from different brain regions indicate
different patterns of response on exposure to hypoxia (Dumanska, 2019; Hyllienmark, 1999).
Mechanisms of response to hypoxia in cortical regions are determined by the type of neurons
present in cortical layers. Such a heterogeneous neuronal population makes the response of
neurons differ widely due to differences in membrane properties. The current study did not identify
any consistent pattern in global cortical response to hypoxia such as, neuronal depolarization
followed by hyperpolarization. Studies conducted in neocortical pyramidal neurons exhibited
hypoxia induced hyperpolarization followed by post-hypoxic hyperpolarization (Nieber, 1999).
Pyramidal cells in the prefrontal cortex showed a membrane depolarization along with decreased
input resistance following hypoxia (Brand, 1998).
Immunohistochemistry staining results in the current study, showed a positive staining for both
SNAP-25-2A-GCaMP-6s positive and negative animals. As GCaMP-6s negative animals lacked
the genes for fluorescence activity, they were used as negative control. Such a negative control is
advantageous as the entire tissue from the negative animal was prepared in the same manner as the
positive sample, minimizing experimental variability arising between positive and negative
experimental samples (Burry, 2011). The most likely reason for observing a positive staining result
22
in GCaMP-6s negative animal can be attributed to the reason that immunohistochemistry protocol
did not work properly leading to false positive result for GCaMP-6s negative animal.
Few limitations of this study are as follows: (1) using the current imaging set up, fluorescence
intensity is measured entirely from the cortical surface of the brain. It is unknown if sub-cortical
neurons also contribute to the recorded fluorescence signal. This is essential as the transgenic
mouse model used in this study has a pan-neuronal GCaMP-6s expression (2) spatial and temporal
information about the point of origin of fluorescence (in the right and left cortex) was not measured
in the study. It is unknown if the region of origin for this fluorescence activity is fixed or variable
among animals (3) owing to the ultrasensitive nature of GCaMP-6s, in addition to detecting rapid
changes in intracellular free calcium caused by neuronal activity, intracellular reserves of calcium
may also be detected by GCaMP-6s in this study. This could overestimate neuronal activity as
recorded by fluorescence signals.
Directions for future research aligned with this study: (1) introducing normoxia (normal air)
following hypoxic air exposure and investigating whether reduction associated with frequency of
neuronal firing under hypoxia is a reversible or an irreversible manifestation; (2) investigating the
effects of acute hypoxia on the ability to perform behavioral tasks on adult animals, that were
exposed to acute hypoxic exposure during their first week of postnatal development; (3)
investigating suppressive effect of hypoxia on neuronal activity with increasing age (from P8-
P15); (4) experiment to quantitatively determine the concentration of calcium present in the
neuron’s cytosol, during the firing of an action potential can be performed. Using this experiment,
a comparison between the concentration of calcium ions during neuronal firing under normoxic
and hypoxic conditions can be performed. Such an analysis of concentration would clarify the
theory of accumulated calcium ions causing greater amplitude fluorescence peaks under hypoxic
conditions. Pivovarova et al., in their 2010 review paper list the various techniques that are
available for estimating the concentration of intracellular calcium ions; (5) analysis to trace the
spread of fluorescence activity (representing neuronal activity) over the cortical area, indicating
origin and direction of spread of fluorescence signals.
CONCLUSION
In the current study, a reduction in the number of intensity peaks immediately following exposure
to a hypoxic environment of 10 minutes as compared to baseline (normal air) was observed. To
the best of knowledge this study is the first of its kind to perform an in-vivo recording of response
of an unanesthetized and awake animal to an acute-hypoxic environment at P7. An observed
reduction in the frequency and increase in amplitude of neuronal activity under hypoxic conditions
was observed. Synchrony in neuronal firing between the left and right cortex was observed. The
results of this study are in alignment with the previous studies performed using brain slices-
presence of suppression of neural activity in response to exposure to hypoxic air.
23
REFERENCES
Ackman, J. B., Burbridge, T. J., & Crair, M. C. (2012). Retinal waves coordinate patterned
activity throughout the developing visual system. Nature, 490(7419), 219–225.
https://doi.org/10.1038/nature11529
Babola, T. A., Li, S., Gribizis, A., Lee, B. J., Issa, J. B., Wang, H. C., … Bergles, D. E. (2018).
Homeostatic Control of Spontaneous Activity in the Developing Auditory System. Neuron,
99(3), 511-524.e5. https://doi.org/10.1016/j.neuron.2018.07.004
Balestrino, M. (1995). Pathophysiology of anoxic depolarization: new findings and a working
hypothesis. Journal of Neuroscience Methods, 59(1), 99–103. https://doi.org/10.1016/0165-
0270(94)00199-Q
Bayer, C., Shi, K., Astner, S. T., Maftei, C. A., & Vaupel, P. (2011). Acute Versus Chronic
Hypoxia: Why a Simplified Classification is Simply Not Enough. International Journal of
Radiation Oncology, 80(4), 965–968. https://doi.org/10.1016/j.ijrobp.2011.02.049
Bockhorst, K. H., Narayana, P. A., Liu, R., Ahobila-Vijjula, P., Ramu, J., Kamel, M., … Perez-
Polo, J. R. (2008). Early postnatal development of rat brain: In vivo diffusion tensor
imaging. Journal of Neuroscience Research, 86(7), 1520–1528.
https://doi.org/10.1002/jnr.21607
Burry, R. W. (2011). Controls for immunocytochemistry: An update. Journal of Histochemistry
and Cytochemistry, 59(1), 6–12. https://doi.org/10.1369/jhc.2010.956920
Chen, T.W., Wardill, T. J., Sun, Y., Pulver, S. R., Renninger, S. L., Baohan, A., … Kim, D. S.
(2013). Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature, 499, 295-
300. https://doi.org/10.1038/nature12354
Choi, D. W. (1990). Cerebral hypoxia: some new approaches and unanswered questions. The
Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 10(8),
2493–2501.
De Haan, M., Wyatt, J. S., Roth, S., Vargha-Khadem, F., Gadian, D., & Mishkin, M. (2006).
Brain and cognitive-behavioural development after asphyxia at term birth. In
Developmental Science, 9, 350-8.
Dodge, F. A., & Rahamimoff, R. (1967). Co‐operative action of calcium ions in transmitter
release at the neuromuscular junction. The Journal of Physiology, 193(2), 419-32.
https://doi.org/10.1113/jphysiol.1967.sp008367
Duchen, M. R. (2012). Mitochondria, calcium-dependent neuronal death and neurodegenerative
disease. Pflugers Archiv European Journal of Physiology, 464(1), 111-21.
https://doi.org/10.1007/s00424-012-1112-0
Dumanska, H., & Veselovsky, N. (2019). Short-term hypoxia induces bidirectional pathological
long-term plasticity of neurotransmission in visual retinocollicular pathway. Experimental
Eye Research, 179, 25–31. https://doi.org/10.1016/j.exer.2018.10.014
Erecińska, M., & Silver, I. A. (2001). Tissue oxygen tension and brain sensitivity to hypoxia.
Respiration Physiology, 128(3), 263–276. https://doi.org/10.1016/S0034-5687(01)00306-1
Funke, F., Kron, M., Dutschmann, M., & Müller, M. (2009). Infant Brain Stem Is Prone to the
Generation of Spreading Depression During Severe Hypoxia. Journal of Neurophysiology,
101(5), 2395–2410. https://doi.org/10.1152/jn.91260.2008
Garcia, M. I., Chen, J. J., & Boehning, D. (2017). Genetically encoded calcium indicators for
studying long-term calcium dynamics during apoptosis. Cell Calcium, 61, 44–49.
https://doi.org/10.1016/j.ceca.2016.12.010
24
Homma, R., Baker, B. J., Jin, L., Garaschuk, O., Konnerth, A., Cohen, L. B., … Zecevic, D.
(2009). Wide-field and two-photon imaging of brain activity with voltage- and calcium-
sensitive dyes. Methods in Molecular Biology, 489, 43-79. https://doi.org/10.1007/978-1-
59745-543-5_3
Hutter, D., Kingdom, J., & Jaeggi, E. (2010). Causes and Mechanisms of Intrauterine Hypoxia
and Its Impact on the Fetal Cardiovascular System: A Review. International Journal of
Pediatrics, 2010, 1–9. https://doi.org/10.1155/2010/401323
Hyllienmark, L., & Brismar, T. (1999). Effect of hypoxia on membrane potential and resting
conductance in rat hippocampal neurons. Neuroscience, 91(2), 511–517.
https://doi.org/10.1016/S0306-4522(98)00650-2
Karunasinghe, R. N., Dean, J. M., & Lipski, J. (2018). Acute sensitivity of astrocytes in the
Substantia Nigra to oxygen and glucose deprivation (OGD) compared with hippocampal
astrocytes in brain slices. Neuroscience Letters, 685, 137–143.
https://doi.org/10.1016/j.neulet.2018.08.033
Kriegstein, A., & Alvarez-Buylla, A. (2009). The Glial Nature of Embryonic and Adult Neural
Stem Cells. Annual Review of Neuroscience, 32(1), 149–184.
https://doi.org/10.1146/annurev.neuro.051508.135600
Lawn, E., Cousens, J. Z. (2005). MDGs and newborn babies. Lancet, 365, 891–900.
https://doi.org/10.1016/S0140-6736(05)71048-5
Lima, M. J. P., Rayee, D., Silva-Rodrigues Thaiaand Paes Pereira, P. R., Miranda Mendonca, A.
P., Rodrigues-Ferreira, C., Szczupak, D., … Uziel, D. (2018). Perinatal Asphyxia and Brain
Development: Mitochondrial Damage WithoutAnatomical or Cellular Losses. Molecular
Neurobiology, 55(11), 8668–8679. https://doi.org/10.1007/s12035-018-1019-7
Lewis, S. W., & Murray, R. M. (1987). Obstetric complications, neurodevelopmental deviance,
and risk of schizophrenia. Journal of Psychiatric Research, 21(4), 413-21.
https://doi.org/10.1016/0022-3956(87)90088-4
Lipton, S. A., & Rosenberg, P. A. (1994). Excitatory amino acids as a final common pathway for
neurologic disorders. The New England Journal of Medicine, 330(9), 613-22.
https://doi.org/10.1056/NEJM199403033300907
Lunyak, V. V. (2016). Corepressor-Dependent Silencing of Chromosomal Regions Encoding
Neuronal Genes. 298(5599), 1742-52.
Luo, Q., Pin, T., Dai, L., Chen, G., Chen, Y., Tian, F., & Zhang, M. (2019). The Role of S100B
Protein at 24 Hours of Postnatal Age as Early Indicator of Brain Damage and Prognostic
Parameter of Perinatal Asphyxia. Global Pediatric Health, 6, 2333794X1983372.
https://doi.org/10.1177/2333794x19833729
Maiti, P., Singh, S. B., Sharma, A. K., Muthuraju, S., Banerjee, P. K., & Ilavazhagan, G. (2006).
Hypobaric hypoxia induces oxidative stress in rat brain. Neurochemistry International,
49(8), 709–716. https://doi.org/10.1016/j.neuint.2006.06.002
Mucci, S., Herrera, M. I., Barreto, G. E., Kolliker-Frers, R., & Capani, F. (2017).
Neuroprotection in Hypoxic-Ischemic Brain Injury Targeting Glial Cells. Current
Pharmaceutical Design, 23(26), 3899-906.
https://doi.org/10.2174/1381612823666170727145422
Nalivaeva, N. N., Turner, A. J., & Zhuravin, I. A. (2018). Role of prenatal hypoxia in brain
development, cognitive functions, and neurodegeneration. Frontiers in Neuroscience, 12, 1–
21. https://doi.org/10.3389/fnins.2018.00825
Nieber, K. (1999). Hypoxia and neuronal function under in vitro conditions. Pharmacology and
25
Therapeutics, 82(1), 71–86. https://doi.org/10.1016/S0163-7258(98)00061-8
Nho, K., Kim, S., Risacher, S. L., Shen, L., Corneveaux, J. J., Swaminathan, S., … Saykin, A. J.
(2015). Protective variant for hippocampal atrophy identified by whole exome sequencing.
Annals of Neurology, 77(3), 547–552. https://doi.org/10.1002/ana.24349
Peacock, A. J. (1998). Oxygen at high altitude. Bmj, 317(7165), 1063.
https://doi.org/10.1136/bmj.317.7165.1063
Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., & Noble-Haeusslein, L. J. (2013).
Brain development in rodents and humans: Identifying benchmarks of maturation and
vulnerability to injury across species. Progress in Neurobiology, 106–107, 1–16.
https://doi.org/10.1016/j.pneurobio.2013.04.001
Shimoda, L. A., & Polak, J. (2010). Hypoxia. 4. Hypoxia and ion channel function. American
Journal of Physiology-Cell Physiology, 300(5), C951–C967.
https://doi.org/10.1152/ajpcell.00512.2010
Soleimanzad, H., Gurden, H., & Pain, F. (2017). Errata: Optical properties of mice skull bone in
the 455- to 705-nm range. Journal of Biomedical Optics, 22(4), 049802.
https://doi.org/10.1117/1.jbo.22.4.049802
Van De Berg, W. D. J., Blokland, A., Cuello, A. C., Schmitz, C., Vreuls, W., Steinbusch, H. W.
M., & Blanco, C. E. (2000). Perinatal asphyxia results in changes in presynaptic bouton
number in striatum and cerebral cortex - A stereological and behavioral analysis. Journal of
Chemical Neuroanatomy, 20(1), 71–82. https://doi.org/10.1016/S0891-0618(00)00078-8
Vanni, M. P., & Murphy, T. H. (2014). Mesoscale Transcranial Spontaneous Activity Mapping
in GCaMP3 Transgenic Mice Reveals Extensive Reciprocal Connections between Areas of
Somatomotor Cortex. Journal of Neuroscience, 34(48), 15931–15946.
https://doi.org/10.1523/jneurosci.1818-14.2014
Weitzdoerfer, R., Gerstl, N., Pollak, D., Hoeger, H., Dreher, W., & Lubec, G. (2004). Long-term
influence of perinatal asphyxia on the social behavior in aging rats. Gerontology, 50(4),
200-5. https://doi.org/10.1159/000078348
Yin, X.-L., Jie, H.-Q., Liang, M., Gong, L.-N., Liu, H.-W., Pan, H.-L., … Yin, S.-K. (2018).
Accelerated Development of the First-Order Central Auditory Neurons With Spontaneous
Activity. Frontiers in Molecular Neuroscience, 11, 1–17.
https://doi.org/10.3389/fnmol.2018.00183
Zimna, A., & Kurpisz, M. (2015). Hypoxia-Inducible Factor-1 in Physiological and
Pathophysiological Angiogenesis: Applications and Therapies. BioMed Research
International, 2015, 1–13. https://doi.org/10.1155/2015/549412