CALCIUM IMAGING OF DEVELOPING PROPRIOCEPTIVE DORSAL ROOT

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

By

KAITLYN LOUISE PARKES B.A., University of Alabama at Birmingham, 2014

2019 Wright State University

WRIGHT STATE UNIVERSITY

GRADUATE SCHOOL

April 24, 2019

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Kaitlyn Louise Parkes ENTITLED Calcium Imaging of Developing Proprioceptive Neurons BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science.

David R. Ladle, Ph.D. Thesis Director

Eric S. Bennett, Ph.D Department Chair Department of Neuroscience, Cell Biology and Physiology

Committee on Final Examination

David R. Ladle, Ph.D.

Patrick M. Sonner, Ph.D.

Gary L. Nieder, Ph.D.

Barry Milligan, Ph.D. Interim Dean of the Graduate School

ABSTRACT

Parkes, Kaitlyn Louise. M.S. Department of Neuroscience, Cell Biology and Physiology, Wright State University, 2019. Calcium Imaging in Developing Proprioceptive Dorsal Root Ganglion Neurons.

Proprioception is an important sensation capable due to proprioceptive sensory afferents found in muscles in the periphery being processed by the central . The nervous system is a complex system that continues to develop and mature as an animal ages. Much is not known of proprioceptive neurons and how they develop with time. Calcium is an important molecule in maintaining action potentials and homeostasis in neurons which can be studied to understand a variety of things about a neuron. This

study uses genetically encoded calcium indicators to tag parvalbumin positive cells in the dorsal root ganglion (DRG) in order to image the calcium handling in these cells. Five parameters were measured across three time points to provide quantifiable results as to how calcium handling changes as mice age. We found that of the five parameters investigated, peak amplitude was the only significant difference between P5/6 and adult mice. There was a downward trend in the peak amplitude as the mice aged.

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TABLE OF CONTENTS

Page I. Introduction……………………………………………………….………..1 Proprioceptive Neuron Subtypes……………………..……….…...1

Cellular and Behavioral Maturation During Postnatal

Development……………………………………………………….5

Calcium Regulation in Neurons…………..………………………..6

II. Materials and Methods……………………………………………….…….8

Animals………………………………………………………….…8

Ex Vivo Quadriceps Nerve and L2-L4 DRG Preparation…………9

Calcium Imaging………………………………………………….11

Data processing…………………………………………………...12

Statistics…………………………………………………………..13

III. Results……………………………………………………………….…....14

Calcium Imaging Data Acquisition……………………………....14

Description of Proprioceptive Calcium Transients………….…...14

IV. Discussion……………………………………………………….….…….22

V. References ………………………………………………………….….…25

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LIST OF FIGURES

Figure Page

1. Calcium imaging of proprioceptive DRG neurons………………….……..17

2. Box and whisker plots of the five parameters studied………………….….20

3. Representative calcium transients for each age group evaluated…….…....21

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LIST OF TABLES

Table Page

1. Gender count for each age group used for calcium imaging………………….16

2. Descriptive statistics for calcium transient variables for each studied

age group……………………………………………………………………...19

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I. Introduction

Proprioceptive neurons are special sensory afferents that provide feedback to the (CNS) on the stretch and contraction of muscles. The central nervous system integrates this feedback which allows us to sense our body’s position in space. Together with the CNS, proprioceptive neurons are responsible for the coordination of movement and the ability to complete coordinated tasks without visual aid. A study showing the difficulty of slicing a piece of bread after upper limb nerve injury is a perfect demonstration of proprioceptive input needed for daily life activities. In this study, patients with neuropathy in large-fiber sensory nerves were compared to

controls in a repeat back and forward cutting motion with blocked vision. It was shown

that those with neuropathy had non-linear, jagged movement demonstrating the lack of

coordination of the shoulder and elbow that normally aids with (Sainburg,

Ghilardi, Poizner, & Ghez, 1995).

Proprioceptive Neuron Subtypes

There are two classes of proprioceptive sensory neurons (PSNs) known as muscle

spindle and Golgi organ afferents. The cell bodies of both of these PSNs are

located in the dorsal root ganglia (DRG) along with other somatosensory neurons. These

PSNs differ in terms of function, peripheral location, morphology, and patterns of

in the . Very early in the studies of these afferent fibers, it was

apparent that, based on function and form, muscle spindles are subdivided into two

categories (Hunt & Kuffler, 1951). These two subcategories were defined as type Ia and

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II fibers. Both of these afferent fibers sense the stretch on muscle fibers, but they have

unique functions that subcategorize them. On the other hand, Golgi tendon organs are

innervated by type Ib afferent fibers that are located at the musculotendon junction and

provide feedback on muscle tension.

When looking at the function of the three special sensory afferents that comprise

the proprioceptive sensory neurons, differences are apparent. Muscle spindles are stretch

receptors which provide feedback on the rate and degree of elongation of muscle fibers

(Zelená, 1994). Golgi tendon organs are which are sensitive to

contraction and responsible for feedback regarding muscle tension (Jami, 1992; Zelená,

1994). Further functional differences between Ia and II afferents have also

been identified. Ia fibers are activated by brief stretches and small amplitude vibration

(Baldissera, Hultborn, & Illert, 1981). They are statically and dynamically responsive to

both length changes in muscle fibers and the velocity of the length changes (De-Doncker,

Picquet, Petit, & Falempin, 2003). Type II afferents are also responsive to static

stimulation and muscle stretch, but do not respond to muscle vibration (Baldissera et al.,

1981; Brown, 1981).

The pattern of connections with neurons in the spinal cord also differs between

muscle spindle and Golgi tendon organ neurons. When looking at the spinal cord, neuron

cell bodies are found in the . The grey matter is divided into ten different

domains, called laminae, based on the size and type of neurons located there (Rexed,

1952). Terminal fibers from both muscle spindles and Golgi tendon organs project into

the same laminae of V, VI, and VII, but each specific PSN class (Ia, II, Ib) have nuanced

differences in their connections. Ia fibers also project into laminae VIII, and IX. The most

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dense population are in in laminae V and VI (Jankowska, 1992; Vincent et al., 2017).

Entering at the dorsomedial portion of the dorsal horn, they travel directly to laminae V

and VI where they further branch. Synapses in lamina VII are with inhibitory

, but many fibers travel to in lamina IX at the motor nuclei. Here they synapse with homonymous muscle motoneurons (those that innervate the same

muscle) as well as heteronymous muscle motoneurons (innervating muscles that share a

similar action of a muscle) (Brown, 1981). Group II afferents have connections that

resemble that of the Ia afferents, but compared to other PNS fibers their branching once in the spinal cord is more variable. One main difference is that their presence in lamina

IX is sparse (Brown, 1981; Jankowska, 1992; Vincent et al., 2017). Ib afferents remain

contained in laminae V, VI, and VII with the majority being located in the medial part of

laminae VI (Jankowska, 1992; Vincent et al., 2017). The afferent fiber typically enters at

the dorsomedial portion of the dorsal horn with a direct path to laminae V where it then

branches widely between laminas V, VI, and VII (Brown, 1981).

Peripheral projections of muscle spindles (Ia and II) and Golgi tendon organs

differ not only in location but in morphology. Golgi tendon organs are found in the

muscular connection of a tendon or aponeurosis. GTOs are encapsulated bundles of large

afferent fibers that have a spindle shape with tapered ends. These can often be bifid, with

two heads at both ends, or trifid, with three heads. Their size and abundance in a muscle

depends on function and size of the muscle it is innervating. For example, small muscles

such as the masseter have far less GTOs than a large muscle like semitendinosus.

Muscles of mastication also seem to have a lower number of GTOs than limb muscles

(Jami, 1992). Muscle spindles are found in parallel with extrafusal muscle fibers, the

3 normal muscle fibers that make up the bulk of a muscle (McCloskey, 1978). They are comprised of three different intrafusal fibers (specialized muscle fibers) known as bag1, bag2, and chain fibers, encapsulated in a spiral shape. These muscle spindles are supplied by one Ia fiber and often times supplied by one or more type II fiber, but there are some spindles that receive no type II connection (Zelená, 1994).

While proprioceptive neurons differ in function, location and morphology, identifying a molecular marker that is unique to any one PSN has yet to happen (Poliak,

Norovich, Yamagata, Sanes, & Jessell, 2016). There are, however, known molecular markers for proprioceptors as a whole. One key distinguishing factor of the DRG cell population is the dependency on neurotropin-3 (NT-3). The proprioceptive neurons are known to express TrkC, which acts as the receptor for NT-3, which is proven to be vital to survival of these cells. The other DRG cells express TrkA and TrkB, setting them apart for these proprioceptive neurons (Inoue et al., 2002; W. D. Snider, 1994). Ia afferents have a known dependency on ER81 for development of their synapses on motor neurons in lamina IX (Arber, Ladle, Lin, Frank, & Jessell, 2000). While no single gene appears to be uniquely expressed by PSNs, they can be identified by combinatorial expression of

Runx3, Etv1, and TrkC. No other sensory neurons in the DRG express this exact combination of genes (de Nooij, Doobar, & Jessell, 2013). Parvalbumin(Pv) is a calcium binding protein that is found to be expressed in a subset of dorsal root ganglia cells

(Copray, Mantingh-Otter, & Brouwer, 1994; Honda, 1995). Honda (1995) revealed in his particular study that many cells were immunopositive for parvalbumin seen within motor afferent neurons, but few to none were seen in visceral sensory neurons. It was later demonstrated that more than 85% of the Pv positive neurons in the fifth lumbar dorsal

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root ganglia are proprioceptors with the other remaining neurons being low-threshold

cutaneous mechanoreceptors (de Nooij et al., 2013). This means that there is a high likelihood a Pv positive cell in the DRG will be a proprioceptor. This molecular feature of PSNs is leveraged in this study using a mouse genetic strategy described below.

Cellular and Behavioral Maturation During Postnatal Development

Development and maturation of the nervous system in mice continues postnatally, as in other organisms. One aspect of development that has visible changes in the first weeks of life is myelination in the central and peripheral nervous system. In the brain and spinal cord, from postnatal day one to three, mature and

myelination begins. Between postnatal day seven and 10 glial cell production peaks and

by day 21 myelination rate is at its peak. The cortex and gray matter in the brain reach its

max volume by day 35 (Semple, Blomgren, Gimlin, Ferriero, & Noble-Haeusslein,

2013). Myelination in the periphery reaches its max rate in the first three weeks of

postnatal life (Garbay, Heape, Sargueil, & Cassagne, 2000). When specifically looking at

development of proprioceptive neurons in the nervous system, Ia fibers form boutons with their counterparts before birth (E17), but redistribution does occur in the weeks following birth(W. Snider, Zhang, Yusoof, Gorukanti, & Tsering, 1992). The number of muscle spindles is established before birth, but growth of the muscle spindle is significant after birth (Kozeka & Ontell, 1981). Golgi tendon organs (Ib fibers) reach their musculotendon junctions by E18 and the capsule is formed by postnatal day two.

Golgi tendon organs reach maturation by day 21 (Zelená, 1994).

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Not only can these changes be seen by visual observation of myelination and growth, but also by developmental milestones in behavior. When looking at developmental milestones of mice, there are several that directly reflect the development of the nervous system and proprioception. The righting reflex, or turning from supine to dorsal position, utilizes the vestibular system as well as feedback by proprioceptive afferents. On a flat surface, righting is achieved in 30 seconds by postnatal day five in the mouse (Hill, 2008). When comparing to other murine species such as the , righting on a surface occurs within 30 seconds by day three (Altman & Sudarshan, 1975). Righting can also be achieved midair when dropped in the supine (belly up) position. This is achieved in the mouse by day 11 when dropped form 10.5 cm (Hill, 2008). In the rat, air righting when dropped from 60 cm was completed by 100% of the animals by day 17

(Altman & Sudarshan, 1975). Murine species open their eyes between postnatal days 10-

15(Semple et al., 2013). When the eyes are not open in mice, proprioception is the key factor that helps orientation and coordination of movement. Cliff avoidance before the eyes are open utilizes many sensory sensations in order to avoid falling off a ledge. Mice are successful 100% of the time by postnatal day 5 whereas are not 100% successful until day 7 (Altman & Sudarshan, 1975; Hill, 2008). When compiling the visual changes in the neurons and their , connections in the spinal cord and receptors in the periphery, and the developmental milestones in behavior, it is obvious that changes in the nervous system continue weeks after birth.

Calcium Regulation in Neurons

In the neuron, calcium plays a major role in depolarization as well as synaptic activity. During depolarization, calcium enters the cell through plasma membrane

6 channels and intracellular calcium concentrations can be augmented by the release of internally stored calcium from organelles such as the endoplasmic reticulum. Maintaining low resting calcium levels is important for neuron function and neurons use several mechanisms to return quickly to resting state levels. One mechanism is via pumps that send calcium to internal storage and out of the plasma membrane. An additional mechanism utilizes calcium binding proteins which act to bind any free calcium in the cytosol. Studying and understanding the handling of calcium in the neuron is useful in understanding neuronal functionality and activity (Brini, Calì, & Ottolini, 2014).

Calcium binding proteins can be leveraged to bind fluorescent sensors to measure calcium transients in cells. There are two techniques used, synthetic dyes or genetically encoded calcium indicators (GECIs). While synthetic dyes can be injected directly into a specific cell, a general limitation of these dyes is non-specific uptake in neurons when added to the bath or in tissue. Where synthetic dyes fall short with specificity, GECIs offer an advantage. Utilizing unique molecular features of a set of neurons to control expression, GECIs are able to be expressed in only a subset of neurons, such as parvalbumin expressing neurons in these experiments (Grienberger & Konnerth, 2012).

GECIs first surfaced in the late 1990s utilizing fluorescence resonance energy transfer

(FRET) between green florescent proteins (GFP) bound to a calcium binding protein

(calmodulin). This allowed for robust and repeatable localized recording of calcium fluctuations. Initial GECI variants were challenged by limited sensitivity, or how brightly the cell fluoresced when calcium was bound, as well as relatively slow kinetics, or how quickly changes in calcium were reported. However, in the last ten years, great advances have taken place making GECIs one of the best tools in monitoring calcium handling in a

7 cell (Rose, Goltstein, Portugues, & Griesbeck, 2014). GCaMP, a single fluorophore sensor, has become the most popular sensor. Multiple rounds of optimization without compromising important aspects of this sensor have brought about GCaMP6, the latest series of variants. GCaMP6 has the best response sensitivity and highest calcium affinity of any current GECI (Chen et al., 2013). Utilizing the specificity of GECIs and improvements made with GCaMP, calcium transients of a molecularly unique group of cells can be quantifiably studied.

In the context of visual and behavioral changes seen in the developing mouse, it is reasonable to assume calcium regulation mechanisms may also be maturing during postnatal development. It has been demonstrated that PSN response to stretch changes, especially in the first postnatal week (Vejsada, Hník, Payne, Ujec, & Paleĉek, 1985). This implies that there are different frequencies and therefore different influxes of calcium. The experiments to be explained below will test the hypothesis that calcium transients in early postnatal PSN will be distinct from signals seen in adults.

Using the GCaMP6s model, three time points will be investigated for five parameters of the calcium transient.

II. Materials and Methods

Animals

All animal experimental procedures were conducted under the approval of the

Wright State University Institutional Animal Care and Use Committee. The transgenic mice utilized in this study were PVcre/+;GCaMP/+ off-spring of a PV-Cre/Cre and a

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GCaMP/GCaMP parent. The PV-Cre/Cre knock-in mouse expresses Cre recombinase

from the parvalbumin (PV) locus, JAX Stock 008069. (Hippenmeyer, Sigrist, & Laengle,

2005) The GCaMP/GCaMP mouse expresses the genetically encoded calcium indicator

GCaMP6s (PV-cre/+;GCaMP/+; JAX Stock 024106) from the ROSA26 locus. This

pairing allows for optical imaging of cells that express parvalbumin (PV) due to

expression of GCaMP6s, a calcium indicator, in their protein. Cells that express

parvalbumin include proprioceptors and low-threshold mechanoreceptors. Stimulation of

the quadriceps nerve, which is a purely , allows for confidence that the PV

cells responding are proprioceptors. An even number of male and female transgenic mice

aging from P0 to P11 were used in this study. Five adult mice were also used as a time

point comparison.

Ex Vivo Quadriceps Nerve and L2-L4 DRG Preparation

Prior to dissection, animals were terminally anesthetized in an ice bath if they

were in the P5 and P6 time point. P10, P11, and adult animals were anesthetized with an

intraperitoneal injection of Euthasol (≥270mg/kg IP; Virbac). After removing the anterior

portion of the rib cage, animals were perfused with 5 mL artificial cerebral spinal fluid

(ACSF) into the apex of the heart followed by decapitation. The ACSF solution

contained the following compounds: 127 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 1 mM MgSO4•7H2O, 26 mM NaHCO3, 16.9 mM D(+)-glucose monohydrate, and 2 mM

CaCl2. Skin, upper limbs and left hindlimb were removed. The isolated vertebral column with right hindlimb still intact was placed, dorsal side up, into chamber filled with recirculating, cold (14-15C) ACSF. All further dissection steps were completed from the dorsal aspect of the remaining tissue. First, a dorsal laminectomy was completed to

9 expose the spinal cord to ACSF. The sciatic nerve was located on the posterior thigh and dissected out and transected at the mid-thigh. The remaining stump of the sciatic nerve was used later as a landmark to locate the quadriceps nerve. The coccyx bone was pulled laterally and separated at the articulation point of the sacrum. Lifting the coccyx and femur vertically, the muscle tissue on the anterior thigh was cut away to remove the femur and coccus completely. The femoral nerve was located deep to the sciatic nerve. It was then traced distal to the bifurcation of the saphenous nerve and quadriceps nerve.

The saphenous nerve was transected first, leaving only a short stump branching from the femoral nerve. The quadriceps nerve was then transected at the point where it enters the muscle, preserving a long muscle nerve for stimulation. The remainder of the right hindlimb tissue was then removed. To free up the femoral nerve proximal to the quadriceps branch point, any remaining muscle, , and portions of the vertebral column were removed, thus exposing the junctions of the femoral nerve with lumbar (L) 2 through L4 dorsal root ganglia (DRG). A digestion solution (12.5% 10 mg/mL collagenase type I; Sigma C0130, 12.5% thermolysin; Sigma P1512 1000 units/mL, 75% H20) was applied to the DRG using a small glass pipette and allowed to sit for 5 minutes. The dura mater was then carefully removed to reduce light scatter in imaging. The L2-L4 dorsal and ventral roots were transected close to the spinal cord, leaving long roots that could be used for electrical stimulation and recording. When complete, the final preparation contained the quadriceps nerve motor branch from the femoral nerve attached to the dorsal root ganglia of L2, L3 and L4 with their dorsal and ventral roots. Fire polished glass pipettes were fitted to the quadriceps nerve and L3

10 dorsal root for stimulation during imaging. ACSF was removed from ice and allowed to return to room temperature (22-24C) for approximately one hour before imaging.

Calcium Imaging

Calcium imaging and data analysis were performed according to protocols described in Walters, Sonner, Myers, & Ladle (2019). The preparation was moved to the imaging room. Under a dissecting scope, the quadriceps nerve was suctioned up into the stimulating pipette. The L3 dorsal root was suctioned up into the recording pipette.

Although L4 is the DRG imaging occurs on, L3 dorsal root was used to allow room for the objective to move around on L4. The recording pipette probe was connected to the head stage of an EX4-400 Quad differential amplifier (low cut filter: 2Hz, high cut filter:

10k Hz, gain: 1,000X; Dagan Corp.) to record the health and viability of the preparation.

The stimulating probe was connected to the WPI A360 stimulation unit. This was controlled by Clampex (Version 10.7) to provide electrical stimulation to the nerve and create action potentials and therefore calcium transients. Once the probes were secure and nerves were in the pipettes, the preparation was transitioned under the two-photon microscope (Olympus FV1000-MPE; 25X 1.05NA objective, 920 nm excitation wavelength). The L4 DRG was aligned under the objective. An initial stimulus at each of the currents (0.01mA and 0.6 mA) utilized during imaging was conducted to establish a baseline viability of the preparation. Next, using the epifluorescence, proprioceptive neurons were identified for imaging.

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The software used to manipulate the two-photon microscope and image cells was

FluoView (Version 4.0b). A 0.1ms pulse at 50 Hz and a current of 0.01 mA for 1 second followed by 2 second rest intervals was given to the quadriceps nerve and visual observation under epifluorescence was made. The most superficial (shallow) plane of L4

DRG was observed for responding cells. All responding cells on a single plane were imaged, moving left to right, before moving to the next focal plane to image. If needed, imaging of cells in L3 was also utilized after all responding cells in L4 were imaged. An image of the cell was captured first at the 0.01 mA stimulation current. Then a 9.3 µm line scan was drawn across the brightest section of the cell, avoiding the nucleus, and scanned 20,000 times (28.16 seconds at a frequency > 700Hz). The initial 5 seconds measured baseline fluorescence, followed by a 0.5 second pulse train (0.1 ms pulses at 50

Hz, current 0.6 mA). The line scan was completed three times on each cell to obtain an average. Electrophysiology recordings were also taken in synchrony to monitor consistency of the stimulation.

Data processing

Data from the line scans were imported into MATLAB (R2015a). Using the

‘designfit’ function on MATLAB a Butterworth filter was used to process transients. An iterative smoothing process was applied to remove any low-amplitude fluctuations in turn reducing noise (Yaksi & Friedrich, 2006). When looking at the transient, the rising phase as calcium influxes into the cell fit a polynomial function. The decay best fit a second- order exponential decay function. Utilizing these functions, the slope of the rising phase and decay coefficient (decay time 1, DT1) were calculated for each transient. DT1 is the time it takes to decay to 63% of the peak. Resting baseline fluorescence is calculated with

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the first five seconds of the transient before stimulation. Rise time (RT) as well as the point of peak amplitude was calculated using the slope. Decay time 2 (DT2) was calculated by measuring the time it took the transient to return to 10% of the resting fluorescence from DT1.

Statistics

The aim of the statistical analysis was to determine if there was a significant difference between age groups when looking at peak, rise time, DT1, DT2, and raw baseline fluorescence. The age groups were P5/6, P10/11, and adults (greater than two months). A two-way mixed effects ANOVA was conducted utilizing peak, rise time,

DT1, DT2, and raw baseline fluorescence as dependent variables and Age and Sex as independent variables. In order to account for any possible correlation between multiple

measurements for a single mouse, a random effect for mouse was applied. A Bonferroni

correction was used to adjust the probability (p-value). This prevents a type I error that can occur if the null hypothesis is rejected when it is true (Armstrong, 2014). It is also

called alpha and does not exceed 0.05(Banerjee, Amitav, Chitnis, U.B., Jadhav, 2009).

This alpha was used to calculate the Bonferroni correction, taking the alpha (0.05)

divided by the number of models being run (five in this experiment). Bonferroni’s

multiple comparison procedure was utilized for all pair-wise comparisons of factor levels

of any significant independent variables. The model-wise type I error rate was alpha=

0.01. SAS version 9.4 (SAS Institute, Inc., Cary, NC) was used for all analyses.

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III. Results

Calcium Imaging Data Acquisition

In order to visualize the calcium transients of proprioceptive dorsal root ganglion

(DRG) neurons, GCAMP6s transgenic mice were used. This was done via an ex vivo preparation of the 4th lumbar DRG as explained in the methods. There were 246 cells imaged across 25 mice averaging 10  1 (mean standard deviation) cells per animal.

The number of cells imaged ranged from 5 cells to 13 cells. Five males and five females from the P5/P6 and P10/P11 time point were investigated and five total adults (2 males 3 females). See table 1. As explained in the methods, cells were captured in a standardized way for each animal ensuring a random array of cells were imaged, not relying on brightness of fluorescence. This allows the results to account for the dynamic ranges seen in the intensities of fluorescence seen. Utilizing the line scan approach (Figure 1) and data analysis software, calcium transients provided quantifiable information for each cell imaged.

Description of Proprioceptive Calcium Transients

The calcium transient from each cell provided resting fluorescence, peak amplitude and rise time. Calculation of DT1 and DT2 were attainable for all but one cell for DT1 and 5 cells for DT2 due to not fitting the second-order exponential decay function or the decay exceeding the 23 second recording time following stimulation.

Table 2 has a summary of the data collected for each parameter and age group. Figure 2 displays box and whisker plots of each parameter. When looking at the two-way comparison between age group and sex, no evidence of a significant two-way interaction.

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This means each parameter could be analyzed using a mixed effects ANOVA. There

were no significant differences in sex when looking at the results of the mixed effects.

The p-value for each parameter for sex are as followed: resting fluorescence p= 0.37, peak amplitude p= 0.31, rise time p=0.19, DT1 p= 0.12, DT2 p= 0.27.

When looking at the mixed results regarding age group for the parameters, there were no significant results for resting fluorescence, rise time, DT1, or DT2. The age

group two-way mixed ANOVA for resting fluorescence was F(2, 20.9) = 0.71, p= 0.50.

Rise time results were F(2,21) =0.39, p=0.68. DT1 and DT2 results were F(2, 20.8)

=0.48, p=0.62 and F(2,20.9) =3.79, p=0.0395. The p-value for age group regarding DT2

was close to significant at 0.0395, but the Bonferroni-corrected threshold is 0.01. Peak

amplitude had a significant difference with a p-value of 0.0038 (F(2,17.4) =7.79). Due to

this significance, a pair-wise comparison was run between each age group for peak

amplitude. The only significant result was between P5/6 and adult mice. The average difference in peak amplitude was 0.59 (p=0.0037 95% confidence interval (0.07,1.11)).

Figure 3 shows example calcium transients for each age group. The peak visibly decreases as age increases.

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Tables and Figures

Table 1. Animals Used for Calcium Imaging Males Females Totals P5/6 5 5 10 P10/11 5 5 10

Adult 2 3 5

Table 1. Gender count for each age group used for calcium imaging. Animals were chosen at random from different litters. Equal numbers of males and females from P5/6 and P10/11 were used. Random adults (greater than 2 months old) were chosen as controls.

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Figure 1. Calcium imaging of proprioceptive DRG neurons. A) Setup of preparation

under 2-photon microscope. Entire preparation included quadriceps nerve attached to L3

and L4 DRG with L3 dorsal root still intact. Quadriceps nerve was stimulated electrically

evoking action potentials and excitation of L4 DRG neurons. Recording of action

potentials achieved by dorsal root of neighboring L3 DRG. Proprioceptive neurons

expressing GCAMP6s fluoresced for imaging by 2-photon microscopy. B) of

responding proprioceptive neuron in L4 DRG of P10 mouse. Yellow line indicates the

region chosen for line scan analysis (at ~700Hz scanning rate). C) Example calcium

transient collected from averaging fluorescence across line scan for a 28 second period.

D) A smoothing algorithm was used to remove fluctuations leaving the final transient

evaluated for the following parameters: RF= Resting Fluorescence, Peak= peak

17 amplitude, RT= rise time, DT1= decay time 1, DT2= decay time 2. Figure adapted from

Walters, Sonner, Myers, & Ladle (2019).

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Table 2. Age Group Descriptive Statistics P5/6 (10) P10/11 (10) Adult (5) Resting F (a.u.) 251 ± 37.3 233 ± 36.7 251 ± 37 Peak (%∆F/F) 105 ± 28 75 ± 27 46.1 ± 28 Rise Time (s) 1.37 ± 0.14 1.31 ± 0.13 1.35 ± 0.14 DT 1 (s) 3.59 ± 0.5 3.37 ± 0.48 3.49 ± 0.49 DT 2 (s) 11.1 ± 0.53 10.9 ± 0.52 10.3 ± 0.52 Mean ± SD. Number of animals in parentheses.

Table 2. Descriptive statistics for calcium transient variables for each studied age

group. Peak was the only parameter with a significant result between P5/6 and adult

mice.

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Figure 2. Box and whisker plots of the five parameters studied. Light blue is P5/6, blue is P10/11 and dark blue is adult. A) Resting fluorescence B) Peak amplitude C) Rise time D) DT1 (decay time 1) E) DT2 (decay time 2).

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Figure 3. Representative calcium transients for each age group evaluated. Light blue is P5/6, blue is P10/11 and dark blue is adult. Note the downward trend in the peak amplitude.

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IV. Discussion

This study investigated the difference in calcium handling in proprioceptive dorsal root

ganglion neurons at three different age points. A PVcre/+;GCaMP/+ mouse was utilized in

all experiments to ensure only parvalbumin positive cells responded to stimulation. Use of

this animal model and our experimental design, allowed us to eliminate challenges previously

encountered as to whether responding cells were truly proprioceptive neurons or also

included the small subset of cutaneous mechanoreceptors that express parvalbumin (de Nooij

et al., 2013). In order to account for this challenge, the quadriceps nerve was used for

stimulation. This nerve branches from the femoral nerve which divides into the muscular

quadriceps nerve branch and a cutaneous saphenous branch. When the quadriceps nerve is

stimulated, there are no cutaneous axons activated therefore any cell responding in the DRG

must be a proprioceptive neuron. When looking at the spinal nerves leading into the spinal

cord from the femoral nerve, we observed that a majority of the fibers lead to the fourth

lumbar DRG, therefore that was the DRG that was isolated for imaging. The L4 DRG had

approximately 10 cells imaged for their calcium transient per animal, when possible. Five

parameters were measured across the three age groups which included resting fluorescence,

peak amplitude, rise time, and DT1, DT2.

First a two-way comparison of gender for each age group was completed and there were

no significant differences. A mixed effects ANOVA was then run on each parameter for age

group. All variables showed no significant results except peak amplitude. DT2 was close to

being significant but did not cross the needed threshold. These results were somewhat

surprising and did not support the initial hypothesis. As explained in the introduction, the

nervous system is developing and changing postnatally. Even by eye, progressive increases in

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the myelination of the peripheral nerves could be observed when comparing older and

younger animals. There are also examples in the literature of functional changes in

proprioceptive neurons during postnatal development (Vejsada et al., 1985). The

morphological development of proprioceptive endings in muscle also occurs during the first

three postnatal weeks, with some changes even happening in adulthood. For these reasons, it

was hypothesized that changes in the parameters of action potential-evoked calcium transient

would be more visible.

Peak amplitude was the only parameter with a significant result. There was a downward

trend in peak amplitude values from P5/6 to P10/11 to adults. The peak amplitude for P5/6

animals was 44% brighter than adults. Although this was the only significant result, there

were non-quantifiable, but visible differences in the calcium handling in the younger animals.

Often times the P5/6 age group needed 10-15 minute periods of rest from stimulation to get

back to a normal resting fluorescence. Thus, it appeared these cells were only able to

maintain normal calcium handling for a shorter period of stimulation than cells in adult

animals. Another observation was that in P5/6 and P10/11 animals, initial stimulation at the

beginning of an experiment would result in dozens of responding cells. However, after two to

three rounds of stimulation, only half the number of cells would respond.

Rise time and DT1 were very stable variables across all age groups. Rise time did not

vary more than a few hundredths of second with a standard deviation of 0.14 seconds. DT1

did not vary more than two tenths of a second with a 0.5 second standard deviation. This

observation demonstrates that calcium handing is something that is closely regulated,

regardless of the age of the animal. Consistent rise time values for all ages studied, suggest

proprioceptive DRG cells have established a regulated rate at which calcium comes into the

cell. Likewise, consistent DT1 values suggest they are also efficient at all ages at excreting

excess calcium from the cell. As stated, DT2 was close to being significant. As the animal

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ages from P5/6 to P10/11 to adult, they become more efficient at getting the cell’s calcium

back to a resting state. Between P5/6 and adult mice, there is a 0.8 second difference. As an

animal ages and proprioceptive neuron firing rates increase, it is more important for a neuron

to quickly return to resting and be ready to transmit another action potential.

Although the results were not as expected when looking at all the other changes occurring

in the nervous system as an animal is maturing, the results were very stable across each age

group and variable. The descriptive statistics (Table 2) display that the standard deviation is

almost identical for each age group. This demonstrates that even if the number of animals

investigated was increased, there would likely be no differences in the results. Increasing the

number of adult mice investigated would be an improvement made in this experiment, but as

just stated, it would likely not change the results. Another possible change for future

experiments would be counting how many cells initially fluoresce with the first waves of

stimulation and how that number drops off as the experiment continues. The current

experiments did not provide quantifiable information on this interesting observation. This

study was important in explaining that some parameters of calcium handling are highly

regulated from birth. There were interesting visible differences in the calcium handling in the

younger mice, but more parameters would need to be investigated and studies would need to

be conducted to understand why these observations are seen.

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