Ii a COMPARISON of INTRINSIC and TRANSPLANTED

A COMPARISON OF INTRINSIC AND TRANSPLANTED CHANDELIER CELLS DURING CORTICAL DEVELOPMENT IN MICE

Michael Leary

A Thesis Submitted to the Faculty of

The Wilkes Honors College

in Partial Fulfillment of the Requirements for the Degree of

Bachelor of Arts in Liberal Arts and Sciences

with a Concentration in Biological Chemistry

H. L. Wilkes Honors College of

Florida Atlantic University

Jupiter, Florida

December 2016

A COMPARISON OF INTRINSIC AND TRANSPLANTED CHANDELIER CELLS DURING CORTICAL DEVELOPMENT IN MICE

Michael Leary

This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Chitra Chandrasekhar, and has been approved by the members of her/his supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences.

SUPERVISORY COMMITTEE:

______

[Dr. Andre Steinecke]

______

[Dr. Chitra Chandrasekhar]

______

Dean Ellen Goldey, Wilkes Honors College

______

Date

iii

ABSTRACT

Author: Michael Leary

Title: A comparison of Intrinsic and Transplanted Chandelier Cells during Cortical Development in Mice

Institution: Wilkes Honors College of Florida Atlantic University

Thesis Advisor: Dr. Chitra Chandrasekhar

Degree: Bachelor of Arts in Liberal Arts and Sciences

Concentration: Biological Chemistry

Year: 2016

Recent findings show it is possible to genetically target Chandelier Cells (ChC) by using transgenic animals expressing a CRE recombinase under the control of a transient transcription factor. This targeting allows for continuous expression of a Green Fluorescent Protein (GFP) to study ChCs intrinsic cellular processes. However, the expression of GFP in these mice is weak and the transient transcription factor limits the ability to achieve cell type specific expression of genes. To overcome these problems a technique of transplanting transfected ChC-progenitors into the cortex of developing mice, using single cell electroporation, was created. To compare both methods an analysis of the axon arborization and innervation of ChCs on post-natal day 16 and 21 in mice brains was done. The results show there is not a significant difference between the transplanted and intrinsic signal brains, and that the process of transplantation is a viable method for studying the ChC development.

Table of Contents Introduction ...... 1 Methods ...... 8 Results ...... 15 Conclusion ...... 19 Discussion ...... 21 References ...... 23

INTRODUCTION

Immanuel Kant postulated “all our knowledge begins with the senses, proceeds then to the understanding, and ends with reason. There is nothing higher than reason.” If the apex of human understanding is reason, then the brain is the location for our reasoning capabilities. The multitude of cells which allow our brain to operate are still being classified in an attempt to understand how reason is created.

The study of the brain begins with its structure. Korbinian Brodmann studied the brain and elucidated the structural differentiation in the cortex. He labelled the cerebral cortex into 52 zones called Brodmann areas. They are defined by their cytoarchitecture, or histological structure and organization of cells. Brodmann published his cytoarchitectonic maps of the cortex of Homo sapiens and eight other mammals and found that some cortical areas are present in almost all species examined (Geyer et al., 2011).

His ideas and mapping provided the basis for brain function being localized to specific areas, opposed to holistic idea that cognitive functions involve the brain in its entirety.

The creation of cell theory established by Rudolf Virchow, allowed scientists to understand the cell was the basic unit of structure and function for all living organisms; not scientists studying the structure and function of the nervous tissue discovered it did not fit the parameters of the cell theory. This led to the idea that the nervous system was an exception to the theory. Camillo Golgi proposed the Reticular theory which postulated the nervous system is composed of a continuous reticular network of cells. This theory would be challenged by

Santiago Ramón y Cajal after his own research into the structure of nervous tissue. He first

1 learned the silver chromate staining method introduced by Camilo Golgi and improved upon it by creating the double impregnation procedure. This method helped him study the cerebellum during the embryonic development of animals. His research created the neuron doctrine which states nervous cells are independent not continuous. Nerve cells always remain free, independent, and individual, and are the fundamental unit of the nervous system as a whole.

Nerve impulse transmission from neuron to neuron is by contact rather than continuity” (Andres-

Barquin 2002).

The neuron doctrine allowed Ramón y Cajal to categorize cell types. He classified them as pyramidal cells and stellate cells with pyramidal cells projecting throughout the brain and stellate cells remain within distinct cortical regions. The stellate cells have two subsets: spiny and smooth. Spiny have many dendritic spines while smooth have none (Dowling 2011).

Pyramidal and spiny stellate cells have further been classified as excitatory neurons while the smooth stellate cells are inhibitory neurons. These neurons use specific neurotransmitters.

The types of neurotransmitters can be classified as either inhibitory or excitatory. The main excitatory neurotransmitter is the amino acid Glutamate, and the main inhibitory neurotransmitter is gama-aminobutyric acid (GABA) (Purves et al., 2012).

A cell type which uses glutamate (glutamatergic) are Pyramidal cells (PC). PC are found in every layer of the neocortex, except layer 1. They are known as the primary excitation neurons of a human’s prefrontal cortex. A cell type which uses GABA (GABAergic), are local circuit neurons, or cortical interneurons. Interneurons help mediate overall neural networks of excitability and synaptic integration.

Inhibitory local circuit neurons, or interneurons, use the neurotransmitter GABA and make up around 20% of the cortical neurons in the brain. Their roles in the brain include the

2 regulation of excitatory neurons and the synchronous activity of projection neuron ensembles

(Chu and Anderson 2014).

The regulation of excitatory neurons like PC, cause them to be shut down or their excitation limited in a downstream neuron in a neural circuit. It has been postulated the dysfunctions of interneurons can cause autism and psychiatric diseases.

There is a large diversity of interneurons. The attempt to classify these neurons have been based on morphology, connectivity, synaptic properties, genetic markers, and intrinsic firing properties. These properties of classification lead to the idea of interneurons not having exact group to where a type may belong, but more of a spectrum. However difficult it is to classify an interneuron based on these qualities, the similarities between the different types allow for better understanding and grouping. The similarities which scientists look at include the genetics and connectivity of each cell.

GABAergic Interneurons can be classified into three major subgroups according to biochemical markers. These subgroup are interneurons which express the neuropeptide somatostatin, interneurons which express the ionotropic serotonin receptor 5HT3aR and interneurons which express the calcium binding protein paravalbumin (Chu and Anderson 2014).

Somatostatin is a neuropeptide found in 30% of all cortical inhibitory interneurons. They are axodendritic, innervating the dendrites of PC, and are located in all layers of the neocortex.

(Taniguchi 2014). These interneurons have delayed spiking intrinsic properties, which may modulate later arriving inputs on the dendrites of principal neurons (Ma et al., 2010). Martinotti cells are a type of somatostatin-expressing interneuron.

5HT3aR is an ionotropic serotonergic receptor which makes up 30% of all cortical interneurons, and is expressed in neither the parvalbumin or somatostatin interneurons.

(Taniguchi 2012). These neurons are very heterogeneous physiologically, anatomically, and biochemically (tamiguchi 2014). 5HT3aR interneurons can be divided into vasoactive intestinal peptide neurons (VIP) and non-vasoactive intestinal peptide neurons (Non-VIP). VIP interneurons comprise 40% of the 5HT3aR group. They are irregular-spiking cells found primarily in layer 2/3 of the neocortex, forming connections with PC and other interneurons classifying them as axodendritic (Taniguchi 2014). Their purpose is to disinhibit PC (Taniguchi

2014). Non-VIP interneurons comprise 60% of the 5HT3aR group. Reelin-positive cells are a type of interneuron is found in layer 1 of the neocortex and are classified as late-spiking neurogliaform cells (Lee et al., 2010). They form gap junctions with Reelin interneurons, and other GABAergic interneurons (Taniguchi 2014).

Parvalbumin is a calcium binding protein and make up 40% of all cortical inhibitory interneurons (Taniguchi 2012). These interneurons are fast spiking and can innervate the axon, soma or dendrites of PC. Most parvalbumin interneurons are Basket cell which innervate the soma and proximal dendrites of PC (Taniguchi 2014). A smaller amount of parvalbumin expressing cells are Chandelier Cells (ChC). ChC specifically innervate on the axon initial segment of excitatory PC.

The ChC is a fast-spiking cell, found in layers 2/3 and 5 of the cerebral cortex. It specifically innervates, or distributes its axons to the AIS of excitatory neurons: the site of the pyramidal neurons generating their output in form of action potentials.

The basic morphology of a Figure 1. (A) Diagram of Chandelier Cell. (B) Image of P21 Chandelier Cell in Medial Prefrontal Cortex mature ChC can be seen in figure 1. The figure shows the origin of the name of the interneurons as the shape looks like a chandelier. ChC form boutons on the axons of pyramidal cells. A bouton is the location of where the presynaptic and post synaptic neuron forms a synapse. The axonal projections of the ChC form distinct arrays called cartridges. Each cartridge establishes large number of links with a pyramidal neuron.

The origin of interneurons arise from two embryonic subcortical progenitor zones: the medial ganglionic eminence (MGE) or the caudal ganglionic eminence (CGE) (Kepecs and

Fishell 2014). The different interneuron subtypes arise from either of these two zones. The interneurons which form from either of these two areas will migrate to their determined location in the layers of the neocortex.

Parvalbumin and somatostatin expressing interneurons arise from the MGE, whereas the

CGE gives rise to more rare interneurons such as vasoactive intestinal peptide neurons (Kepecs

5 and Fishell 2014). The ChC originates within the ventral-most region of the MGE at the end of neurogenesis (Chu and Anderson 2014).

The separation of interneurons from either the MGE or the CGE can be better understood through gene expression. The gene expressed specifically in MGE progenitors is the transcription factor Nkx2.1. This transcription factor helps regulate the specification of the cortical interneurons which arise from this zone. (Taniguchi 2012).

The extraction of detailed structural and molecular information from intact biological systems has long been a fundamental challenge across fields of investigation, and has spurred considerable technological innovation (Chung et al., 2013). As shown, neuronal cells can be classified in a myriad of ways based on morphology, biochemistry, physiology, place of origin and where they innervate. The use of these classifications can give rise to ambiguous labeling of neuron cell types. ChC are a specific type of interneuron whose classification is not complete. A more precise method is needed to accurately define the neuronal cell types. The use of transgenic animals is one such method which has allowed scientists to control specific gene functions and reporter genes within neuronal cells. The problem with this method is the transient expression of control. The expression of a desired gene in mice is very weak and the transient expression of transcription factors limits the ability to achieve cell type specific expression of genes. To overcome these problems we developed a technique to transplant transfected ChC-progenitors into the cortex of developing mice using single cell electroporation. The heterochronous transplantation of cells could lead to alterations in the development of ChC and ultimately a more accurate method for cell type classification.

Three variables will be used to determine similar neuronal development between the intrinsic and transplanted ChC: bouton number, axon length and axonal branching. We

6 hypothesize he number of non-AIS boutons, should decrease as the ChC becomes more developed from p16 to p21. Therefore the number of AIS boutons should increase and the total number of boutons should have a net decrease. Similarly the number of branch points and axonal length should decrease from p16 to p21.

METHODS

Mouse Preparations

The mouse model has played a vital role in science because it allows for researchers to study specific cell types with an animal with similar genomics. Mice are also valuable research tools because of the ability to manipulate their genome.

The mouse genome has a specific area called the ROSA26 locus which is constantly transcriptionally active. This property makes it a preferred site for the integration and constant expression of transgenes and reporter genes (Bouabe and Okkenhaug 2013). Genes of interest can be introduced into the Roas26 locus by homologous recombination. Scientists can conditionally express a specific gene of interest within the ROSA26 locus of the mouse by using

Cre/loxP system.

The Cre/loxP system is a tool used for genetic manipulations. The system utilizes the Cre recombinase enzyme and loxP sites flanking a specific DNA sequence to either turn on or off a gene of interest. The orientation of the loxP sites determine the effect when the Cre recombinase cuts them. Two loxP sequences in opposite orientation mediate the inversion of the intervening

DNA, two sites in the same orientation mediate excision of the intervening DNA between the sites after which only one loxP site remains, and if the loxP sites are located on different chromosomes, Cre recombinase can mediate a chromosomal translocationally (Bouabe and

Okkenhaug 2013). Generally, any DNA sequence of interest can be deleted by flanking it with loxP sites. The loxP sites can be introduced into the genomic locus of interest by homologous recombination.

The Cre/loxP system can be used effectively by creating two mouse lines: a driver and reporter line. By cross breeding these two mouse lines the gene of interest can be turned off or on

8 through specific controls. The reporter line mice have DNA sequence of interest which are in between two loxP sites. The driver line is Cre recombinase transgenic mice, where the Cre is expressed under the control of a promoter that is active in specific cell types or tissues. When the two mice lines are crossed, the loxP site mouse with a Cre transgenic mouse, the DNA sequence is subsequently deleted in the cell types or tissues, where Cre is expressed (Bouabe and

Okkenhaug 2013).

This research experiment used a temporal control system in ChC where the Cre recombinase has been engineered to fuse with a modified estrogen-binding domain of the estrogen receptor creating a CreER bacterial artificial chromosome (BAC) (Taniguchi 2014).

The BAC is under the regulation of the Nkx2.1 transcription factor which is present in formation of the MGE interneuron: the ChC. The reporter protein in the ChC, GFP, is induced by tamoxifen, an estrogen analog, on embryonic day 17 in the embryos of the Nkx2.1-CreER knocking mice (Taniguchi 2012). The estrogen receptor is found in the cytoplasm of the cells and can translocate into the nucleus to cause recombination in the ROSA26 locus causing the expression of the GFP in ChC. This method limits the recombination activity to specific times during cell maturation (Taniguchi 2012).

The experiments done on these mice were in accordance with the Max Planck Florida

Institute’s animal use protocol as set by the International Animal Care and Use Committee. To compare the development of transplanted and intrinsic ChC development, 10 mice were sacrificed on post-natal day 16 and 21 for each mouse type.

A pregnant mother, carrying Nkx2.1-CRE transgenic mice, was given 300μL of 2mg/mL concentration of tamoxifen via a gavage feeding method on embryonic day 17. When the mice were born, fluorescent light is used to check which mice have received the Nkx2.1-CreER, and

9 floxed GFP strands. This result is usually 50% of all mice when the transgenic mice are crossed with a Swiss Webster mouse. The positive mice are labeled and grown until either post-natal day

16 or 21, where they are subsequently sacrificed.

Figure 3. (A) Diagram of intrinsic mouse time line. (B) Diagram of transplanted mouse time line

To prepare the transplanted mice, non-transgenic mice are sacrificed on embryonic day

17, where ChC progenitor cells are collected from the ventral MGE. These cells are electroporated with a plasmid containing the fluorescent protein Zs Green, another type of green fluorescent protein. These electroporated cells are injected into non-transgenic mice on post-natal day 1. Incongruous to the intrinsic mice, the transplanted mice are sacrificed on post-natal days

19 and 24 because the development of the progenitor ChC is three days behind the growth of the host mouse.

Mice that are sacrificed undergo a transcardial perfusion fixation procedure. The preparation occurs on a perfusion table, to allow for waste to be removed. The mouse must first be anesthetized before the preparation can begin. The mouse is injected with an analgesic: 300

μL of a ketamine and xylazine solution. After waiting for 10-15 minutes, checking the lack of responsiveness of the mouse through pinching its feet the brain perfusion can occur. First a

20mL solution of 0.9% saline was pumped throughout the mouse body to remove the blood.

Next 20 mL of a 4% solution of paraformaldehyde (PFA) in 7.4 phosphate buffered saline solution was pumped through the mouse fixing all proteins in its body. The mouse brain is removed and placed in a 2% PFA in 7.4 phosphate buffered saline solution overnight at 4 °C to completely fix the brain.

Immunohistochemistry Labeling

The creation of polyclonal and monoclonal antibodies (Ab) have led to their use in immunohistochemistry. Immunohistochemistry is a process which uses antibody binding to specific antigens for in situ cell and tissue typing. This technique is used with light microscopy for research and diagnostic purposes. There are two methods which use the antibody antigen binding to identify cellular structures: the direct and indirect. The direct method is one step and the indirect method is two steps.

The direct method is the simplest and requires a one-step process with a primary Ab conjugated with a reporter molecule (Ramos-Vara 2005).

The indirect method requires two steps having the same first layer of antibodies unlabeled, but the second layer, raised against the primary Ab, is labeled. The benefit of the indirect over the direct staining method is due to the higher sensitivity and fluorescence the indirect method yields than the direct method. The reason for this is because the primary Ab, in the indirect method, is not labeled, which allows for a large number of labeled Ab to bind to the primary Ab, increasing the intensity of the fluorescence, such as green fluorescent protein (GFP).

In this experiment there are two structures we are looking at: the AIS and the ChC. The ChC will either be observed with the fluorescent protein Zs

Green or GFP, in the transplanted and intrinsic mice brains respectively. The AIS in both mice type will be observed staining the protein Ankyrin G, which is a scaffolding protein that recruits ion channels and cytoskeletons, responsible for the internal structure of the AIS. The ChC will be stained green and the

AIS will be stained red.

The brains left overnight were washed with phosphate buffered saline solution and prepared to Figure 2. Chandelier cell innervates the axon initial segment of excitatory neurons: the site of the pyramidal be sliced using a vibratome. The brains are first neurons generating their output in form of action potentials. looked at under a fluorescent microscope to locate a single isolated ChC. The brain is mounted on block and 60 μm coronal sections are obtained.

These slices were stored in phosphate buffered saline solutions until stained.

The staining process begins by washing the sections in a 0.5% Triton X-100 solution for ten minutes. The 5% solution is removed from the sections and washed with phosphate buffered saline. The samples are next blocked for 2 hours with rotation in a phosphate buffered saline solution of 10% donkey serum and 0.1% Triton X-100. A primary Ab solution for the transplanted coronal sections was made having a 1:1000 dilution of Rabbit anti-ZsGrn, and a

1:500 dilution of Mouse anti-AnkG. The primary Ab solution for the intrinsic coronal sections was a 1:500 dilution of Chicken anti-GFP and a 1:500 dilution of Mouse anti-AnkG. The blocking solutions was removed from the sections and the primary Ab solution is washed over

12 the sections overnight at 4 °C with rotation. The sections were taken out of the freezer and washed in a phosphate buffered saline solution with rotation 4 times every 15 minutes. A secondary Ab solution for the transplanted coronal sections was made having a 1:1000 dilution of Donkey anti-Mouse Cy3 and a 1:1000 dilution of Donkey anti-Rabbit 488. The secondary Ab solution for the intrinsic coronal sections was made having a 1:1000 dilution of Donkey anti-

Mouse Cy3, and a 1:1000 dilution of Donkey anti-Chicken Alexa 488. Finally the secondary Ab solution was washed over the sections for 2 hours at room temperature.

The coronal brain sections were labeled and put on slides to be observed under a confocal microscope.

Imaging and Analysis

The advent of the light microscope has given scientists the ability to observe the morphology of different cell types and helped them understand the context of what these cells do in the systems they are a part of.

Confocal microscopy, invented by Marvin Minsky, uses a pinhole in the front of the sample for optical imaging. This pinhole gives the confocal microscope an advantage over regular light microscopes because it acts as a filter to only allow the in-focus portion of the light to be imaged. The light from above and below the plane of focus of the object is eliminated from the final image. This technique allows for better 3D renderings of samples. The most common type of confocal microscopy is laser scanning confocal microscopy (LSCM), and it allows for more complex 3D renderings. LSCM works by using a laser light directed at the sample and an objective focuses the light to a diffraction limited spot in the sample. Emission light from the sample is directed to a photomultiplier, a light sensing detector, through a pinhole that is in the

13 conjugate image plane to the point of focus in the sample. Once the light goes through the pinhole it is sensed by detectors, and the image is displayed.

The combination of LSCM and fluorescent labeling, a detailed z-stack outline of a sample can be rendered.

The stained samples from the transplanted and intrinsic mice brains were observed under a Zeiss 780 LSCM. Z-stacks were taken of each 60 μm section at 100 μm by 100 μm. The images next were uploaded to Imaris 3D imaging software to reconstruct the axonal boutons, located on and off the AIS of pyramidal cells, as well as the arborization of the ChC axons.

Imaris is a software product created by Bitplane. This software allows data management, visualization, analysis, segmentation and interpretation of 3D and 4D microscopy datasets. The benefit of using this product means a neurons Distribution of bouton varicosities, axon branch points, and axon length can be quantified. The 3D rendering had to be checked and labeled by hand to include any boutons or incorrect arborizations the program did not accurately create.

These renderings allowed us to determine whether there was a significant difference in the development of the ChC in the intrinsic to the transplanted mice. Comparison t-tests were done with all variables to obtain a p value. Any value less than 0.05 meant a significant difference between the means of the variable tested.

RESULTS Figure 4. Results showing the comparison of the transplanted and intrinsic ChC development. All results for Transplanted ChC p16 n=9, p21 n=10 for all Intrinsic ChC p16 n=10, p21 n=10. Comparisons: Transplanted p16 and p21, Intrinsic p16 and p21, Transplanted and Intrinsic p16, Transplanted and Intrinsic p21. All error bars represent SEM. (A) Overall trend of bouton growth confirmed as the number of non-AIS boutons decrease as the number of AIS boutons increase resulting in a net decrease in total number of boutons. (B) Percent of boutons seen on the AIS of the PC between the transplanted and intrinsic ChC development: p16 and p21 transplanted ChC P< 0.05, p16 and p21 intrinsic ChC P< 0.05, p16 transplanted and intrinsic ChC P>0.05, p21 transplanted and intrinsic ChC P< 0.05.

Figure 5. Results showing the comparison of the transplanted and intrinsic ChC development. All results for Transplanted ChC p16 n=9, p21 n=10 for all Intrinsic ChC p16 n=10, p21 n=10. Comparisons: Transplanted p16 and p21, Intrinsic p16 and p21, Transplanted and Intrinsic p16, Transplanted and Intrinsic p21. All error bars represent SEM. (A) Number of branch points between the transplanted and intrinsic ChC: p16 and p21 transplanted ChC P<0.05, p16 and p21 intrinsic ChC P<0.05, p16 transplanted and intrinsic ChC P>0.05, p21 transplanted and intrinsic ChC P>0.05 (B) Length of axons between the transplanted and intrinsic ChC: p16 and p21 transplanted ChC P<0.05, p16 and p21 intrinsic ChC P>0.05, p16 transplanted and intrinsic ChC P<0.05, p21 transplanted and intrinsic ChC P<0.05

The development of the transplanted and intrinsic ChC were compared to each other to determine developmental patterns. Both the transplanted and intrinsic mice were perfused on either p16 or p21 and then stained. Using Imaris the mean values were taken for each of the three variables: bouton number, axon length and axonal branching. A comparative t-test was done to compare the means for both the intrinsic and transplanted mice for both age groups. Our null hypothesis for the comparison t-tests done was that the means of the different mouse lines for the same age was not the same. This would show the development of the intrinsic and transplanted

ChC follow the same developmental pattern. The means were considered significantly different if a calculated P value was less than 0.05

Figure 4A shows the trend in the number of boutons located on and off the AIS for transplanted and intrinsic ChC for age’s p16 and p21. As can be observed from the figure there is an overall trend in intrinsic ChC development where the number of non-AIS boutons decrease and the amount of AIS boutons increases. The net result is an overall decrease in the number of boutons during the development of a ChC. The transplanted ChC also exhibits this bouton trend seen in the intrinsic ChC. This implies a similar bouton developmental pattern, however a more accurate comparison must be made for the number of boutons on the AIS to determine if there is a significant difference.

Figure 4B shows the results of comparing the percent of boutons found on PC AIS. Using

Imaris the mean amount of AIS boutons were recorded and comparative t-tests were between the transplanted and intrinsic ChC AIS bouton means. The first a comparison t-test was done to determine significance between different ages of the same ChC. The results show a significant difference in the amount of boutons on the AIS between p16 and p21 for both types of ChC. This shows a developmental difference between ages which was expected. The next step was to

17 determine significance between the same ages of different ChC. The results of this comparison t- test show no significant difference the AIS boutons for both ages between the transplanted and intrinsic ChC.

The next measurement made was comparing branch points as shown in figure 5A. The mean number of branch points were collected from the axonal arborization model rendered using

Imaris. Comparison t-tests were done to between the intrinsic and transplanted axonal branch points means. The first comparison t-test was done between different ages of the same ChC type.

This result showed a significant difference in the mean value of branch points, again this indicates an expected difference between developmental ages. A comparison t-test was next done between the same ages for different ChC type. These results show no significant difference between the mean values of axonal branch points.

Lastly we compared the axonal length between the intrinsic and transplanted ChC as shown in figure 5B. Using Imaris the average axonal length was obtained for the intrinsic and transplanted ChC. A comparison t-test was done between different ages of the same ChC type.

The results show a significant difference in the means for the transplanted ChC, but not between p16 and p21 of intrinsic ChC. Lastly a comparison test was done between the same ages of different ChC types and both showed significant differences in the mean of axonal length.

CONCLUSION

Our hypothesis was proven correct, the overall trend for the three variables follows an increase for the number of AIS bouton, and a decrease in the number of branch points and axonal length. The transplantation method which was compared IS boutons, axonal branch points and axonal length in intrinsic ChC shows only an overall similar trend between the intrinsic and the transplanted ChC for the three variable

Through Imaris 3D rendering and comparison t-tests the results showed significant differences in development between ages of the same type of ChC except when looking at axonal length for p16 and p21 intrinsic ChC. This difference was expected because of previous data showing this developmental feature of ChC. The comparison of p16 and p21 intrinsic ChC did not show a significant difference when comparing their mean axonal length, however both the transplanted and intrinsic ChC show a decreasing trend in axon length indicating a similar developmental trajectory.

The results show no significant difference between the same ages of different ChC when observing their mean AIS boutons and axonal branch points, but there is a significant difference when looking at the axonal length. What can be concluded from this data is though not all of the variables tested can be statistically shown to be significantly similar between the development of the intrinsic and transplanted ChC, during p16 and p21, there is still a similar overall trend seen in figures 4B, 5A and 5B. Both the intrinsic and transplanted ChC increase the mean amount of

AIS boutons from p16 to p21, and decrease in the mean amount of axonal branch points and length from p16 to p21.

for both It is hypothesized this result is because of axonal which did not show a significant difference still showed a trend The results obtained when using the comparison t-tests

19 results show a significant difference for the development of means we could not disprove the null hypothesis, the transplanted and intrinsic ChC follow the same developmental pattern for axonal branching points for p16 and p21.

The experiment shows the transplantation of ChC as a viable research method to overcome the difficulties of cell type identification. More research must be done using this transplantation method to determine if progenitor ChC develop with no significant difference to intrinsic ChC.

DISCUSSION

The problems surrounding cell type classification are due to the myriad of variables a scientists can use to study them. This experiment showed it is viable to transplant ChC from the progenitor region of an embryonic brain into the neocortex of the brain of a normal mouse, which then follows the same developmental trajectory as an intrinsic ChC.

Progenitor ChC were successfully electroporated with ZS Green and injected into mice at p1. Mice were successfully perfused and sacrificed on either p16 or p21. Brains were fixed and sliced. The GFP found in intrinsic ChC, the Zs Green in transplanted ChC and the Ankyrin G found in the AIS of both ChC types were labeled using immunohistochemistry techniques. Z stack images and Imaris 3D analysis allowed the means of AIS boutons, axonal branch points and length to be determined. This was used to show statistical similarities ChC development.

Additionally, this experiment showed the results of neuronal pruning between the p16 and p21 ages. ChC lose branch points, become shorter and change their AIS/non-AIS bouton distributions.

The results of this technique have two major implications for research and the study of humans in pathology.

This method allows more access to the study of chandelier cells through specific selection of candidate genes. The innervation patterns of ChC is not fully understood and this method offers a way to study these interneurons wiring specificities with other interneurons and

PC.

The study of ChC can be augmented because this method would allow researchers not just to study candidate genes in ChC but other neurons. Genes selected need only to be labeled and transplanted into mice and then their development studied. This would be a new and novel

21 technique to allow researchers to brightly label ChC, or other interneurons, to study single cell development in vivo using a 2-Photon microscope. Any transplanted cell can now be observed in an animal model to determine their morphology and place of origin.

This study has a major implication in human cell development. Since it has been shown transplanted progenitor cells can follow the same developmental pattern as intrinsic cells, viable studies into progenitor transplantations for humans could be a possibility for future physicians to consider when treating genetic disorders.

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