The Effects of Developmental Nicotine Exposureon Hypoglossal Motoneuron Primary Dendrite

and Soma Development in the Neonatal Rat

Item Type text; Electronic Thesis

Authors Gaddy, Joshua L.

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 07/05/2018 23:15:47

Link to Item http://hdl.handle.net/10150/621005

1

THE EFFECTS OF DEVELOPMENTAL NICOTINE EXPOSURE ON HYPOGLOSSAL

MOTONEURON PRIMARY DENDRITE AND SOMA DEVELOPMENT IN THE

NEONATAL RAT

By

JOSHUA GADDY

_________________________

A Thesis Submitted to the Faculty of the

DEPARTMENT OF CELLULAR AND MOLECULAR MEDICINE

In Partial Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

2016

2

STATEMENT BY AUTHOR

The thesis titled THE EFFECTS OF DEVELOPMENTAL NICOTINE EXPOSURE ON

HYPOGLOSSAL MOTONEURON PRIMARY DENDRITE AND SOMA DEVELOPMENT

IN THE NEONATAL RAT prepared by JOSHUA GADDY has been submitted in partial

fulfillment of requirements for a master’s degree at the University of Arizona and is deposited in

the University Library to be made available to borrowers under rules of the Library. Brief

quotations from this thesis are allowable without special permission, provided that an accurate

acknowledgement of the source is made. Requests for permission for extended quotation from or

reproduction of this manuscript in whole or in part may be granted by the head of the major

department or the Dean of the Graduate College when in his or her judgment the proposed use of

the material is in the interests of scholarship. In all other instances, however, permission must be

obtained from the author.

SIGNED:_______ _____ _____________

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

______________________________ __________________

Ralph Fregosi Date

Professor of Physiology

3

ACKNOWLEDGEMENT

To start I would like to thank Dr. Ralph Fregosi and Dr. Richard Levine, for their

patience and guidance throughout my graduate education. It was an honor to work with them on

my thesis project. Additionally, I would like to thank my committee members Dr. Paul St. John

and Dr. Helen Amerongen for supporting my education. I would like to acknowledge, Drs.

Marina Cholanian and Andrew Hill, Lila Wollman for helpful critics and feedback on my

practice presentations. I would like to thank Sowmiya Murali and Drs. Gregory Powell and Fei

Xu for allowing me to contribute to their work and for their help. Thank you to Seres Cross for

outstanding technical assistance. I would like to acknowledge Dr. Naomi Rance’s lab for their

help and equipment in analyzing our motoneuron tracings. I would like to thank the Cellular and

Molecular Medicine graduate program, including faculty, staff, and students.

I would like to thank Dr. Maria Velez for funding and always being there when I or any

student needed help. I would also like to thank Donna Treloar for her help and hard work. To my

family, thank you for your support and positive encouragement throughout my education.

Lastly I would like to thank my wonderful wife, Valerisa. You have always supported

me, helped me study and encouraged me to go beyond what I thought I was capable of. You

always believed in me when I doubted myself. You are the hardest working, kindest and the most

altruistic person I know and I am beyond grateful to have you in my life.

4

DEDICATION

I would like to dedicate this thesis to my wife, who provided me with her love and

extraordinary support throughout my educational endeavors.

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TABLE OF CONTENTS

LIST OF FIGURES ......................................................................................................... 6

LIST OF TABLES ........................................................................................................... 7

ABSTRACT ...................................................................................................................... 8

INTRODUCTION ............................................................................................................ 9

MATERIALS AND METHODS .................................................................................... 13

RESULTS .......................................................................................................................... 20

DISCUSSION ................................................................................................................... 25

CONCLUSION ................................................................................................................ 30

REFERENCES ................................................................................................................. 32

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LIST OF FIGURES

MATERIALS AND METHODS:

FIGURE 1: FILLED AND TRACED CONTROL AND DNE HYPOGLOSSAL

MOTONEURONS .............................................................................................................. 15

FIGURE 2: BRAINSTEM SLICES CONTAINING THE HYPOGLOSSAL

NUCLEUS (RED ARROWHEAD) AND THE FOURTH VENTRICLE (WHITE

ARROWHEAD) ................................................................................................................. 16

RESULTS:

FIGURE 3: COMPARING TREATMENT AND LOCATION EFFECTS ON

MAXIMUM AND MINIMUM SOMA DIMETER AND THE NUMBER OF PRIMARY

NODES ............................................................................................................................... 24

DISCUSSION:

FIGURE 4: ILLUSTRATION OF XIIMN ORGANIZATION WITHIN THE NUCLEUS

AND THE INNERVATION OF TONGUE MUSCLES.................................................... 28

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LIST OF TABLES

RESULTS:

TABLE 1: THE EFFECTS OF DNE ON XIIMN MORPHOLOGY PROPERTIES ........ 22

TABLE 2: THE EFFECTS OF DNE ON XIIMN MORPHOLOGY PROPERTIES IN

CAUDAL AND ROSTRAL LOCATIONS ....................................................................... 23

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Abstract

Nicotine from smoking or from other products containing nicotine has adverse effects on

the fetus during pregnancy, such as respiratory problems. Our laboratory has previously shown

that exposure to nicotine during development (DNE) alters hypoglossal motor neuron (XII MN)

function, including decreased excitatory synaptic input, desensitized nicotinic acetylcholine

receptors, increased input resistance, and differences in the precision and reliability of spike

timing in XIIMNs. Evidence of DNE effects on XIIMN function prompted us to test the

hypothesis that DNE will affect the development of primary dendrites and the soma. Brainstem

slices were collected from neonates and motoneurons were filled with neurobiotin via whole-cell

patch clamp. Filled cells were visualized with heavy metal intensified-3,3'-Diaminobenzidine

(DAB) reaction. DAB-stained cells were analyzed using Neurolucida hardware and software. On

average, the maximum soma diameter of more rostral XIIMNs was larger than that in more

caudal cells. Also, caudal XIIMNs had more primary nodes than rostral XIIMNs, and there was

a significant treatment effect on minimum soma diameter (Control, 13.76 ± 0.71 µm; DNE,

18.09 ± 1.22 µm). The results from this study uncovered potential effects of nicotine on XIIMNs

found in rostral and caudal regions of the hypoglossal nucleus.

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Introduction

Worldwide, approximately 5 million deaths occur per year due to direct use of tobacco,

with smoking being the most common method (WHO, 2015), with approximately 480,000

deaths per year in the United States (U.S. Department of Health and Human Services, 2014). The

number of women who smoke while they are pregnant and/or immediately after child birth is a

cause for major public health concern. According to Tong et al., (2013) 23% of women smoked

before pregnancy, 54% quit during pregnancy, 16% began smoking again after delivery, and

11% of women smoked throughout their pregnancy in 2010. Looking at these percentages it is up

to clinicians to provide information on smoking cessation practices. According to one study one-

fifth of pregnant women received information about smoking cessation methods from clinicians,

while one-fourth of pregnant women did not (Kapaya, et al., 2015). These numbers are a cause

for concern not only for the mother, but also for the unborn child.

Out of the thousands of chemicals found in a cigarette, nicotine is among the most

clinically relevant. Pauly and Slotkin (2008) found the effects of nicotine exposure and cigarette

smoke exposure in utero had the same effect on neurogenesis in hippocampal neurons which

exemplifies the effects of nicotine alone. The focus on nicotine is because nicotine replacement

therapy (NRT), such as nicotine patches and nicotine gum, is prescribed to pregnant smokers;

also electronic nicotine delivery systems (ENDS), such as e-cigarettes, are readily available in

the market (Kapaya, et al., 2015; Berlin, et al., 2014; Kralikova, et al., 2013). One of the most

devastating attributes of nicotine is that it can cross the placental barrier in pregnant women and

can cross the fetal blood brain barrier (Benowitz et al., 2009; Slotkin, 1998). Within the fetus

nicotine can bind to acetylcholine (ACh) receptors, which are expressed early in development,

but normally bind endogenous ACh rather than nicotine (Gotti et al., 2006; Zoli et al., 1995).

10

Acetylcholine receptors (AChR) are crucial in regulating fetal brain development, and

overstimulation by nicotine can lead to critical periods in development being disrupted

(Muhammad et al., 2012; Dwyer et al., 2009). Increased risk for sudden infant death syndrome

(SIDS), neurocognitive defects, breathing problems, underweight births and an increased risk of

stillbirth have been attributed to nicotine exposure in utero (Aoyama et al., 2015; England et al.,

2015; U.S. Department of Health and Human Services, 2014; Lavezzi et al, 2010; Button et al.,

2007; Raymond et al., 2005; Alm et al., 1998). Animal models have shown that developmental

nicotine exposure (DNE) is associated with cell damage, including reduced cell number and size,

impaired synaptic activity, and a premature change from cell replication to differentiation

(Aoyama et al., 2015; England, et al., 2015; Pilarski et al., 2011; Dwyer et al., 2009, 2008).

Among the most devastating effects of smoking are those related to respiratory function.

Many neuronal structures in the brainstem contribute to the normal respiration. For example the

preBӧtzinger Complex (preBӧtC) generates the inspiratory rhythm during breathing and

influences the respiratory final motor output to the muscles of breathing, including the tongue

muscles, which are innervated by hypoglossal motoneurons (XIIMNs). The tongue muscles

participate in essential motor functions such as breathing, mastication, swallowing, and

vocalization (Fregosi and Ludlow, 2014; Fregosi, 2011; Bailey and Fregosi, 2004; Berger et al.

1996; Lowe et al., 1980). Previous studies have shown DNE effects on brainstem neurons that

control respiration, including desensitization of nicotinic AChRs on XIIMNs (Pilarski et al.,

2012), reduced excitatory synaptic input and increased excitability (Jaiswal et al., 2013; Pilarski

et al., 2011), increased expression of glutamatergic receptor subunits in preBӧtC and XIIMNs

(Jaiswal et al., 2013), and altered the precision and reliability of spike timing in XIIMNs (Powell

et al., 2015). Continuous bombardment of nicotine and ACh on AChRs at the postsynaptic,

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presynaptic and axon terminal locations during development can lead to motoneurons being

hyperexcitable (Paterson and Norberg, 2000). This hyperexcitability may be affecting the way

these motoneurons develop, and how they receive and transmit information (Pilarski et al.,

2011).

The current study stemmed from a previous study that demonstrated nicotine exposure

stunted the dendritic growth of higher branch orders in DNE XIIMNs compared to control

XIIMN dendrites (Powell, et al., 2016). Other DNE neuronal morphology studies demonstrated

effects on potentially important features of the XIIMNs, such as the branching points (node), the

base diameter of dendrites, and where these XIIMNs are located in the nucleus (Jaiswal et al.,

2013; Muhammad et al., 2012; Pilarski et al., 2011; Núñez-Abades, 1995; Roy and Sabherwal,

1994). Obtaining a complete image of XIIMN morphology may shed light on how nicotine may

affect the development of the motoneurons and dendrites in the hypoglossal nucleus of neonatal

rats. In this study we focused on how DNE affects primary dendrite and soma development, but

also how DNE affects XIIMN development in the rostral and caudal ends of the hypoglossal

nucleus. There are many studies looking at how nicotine affects the overall XIIMN population,

but no studies have investigated the effects of nicotine on XIIMNs located in different regions of

the hypoglossal nucleus, such as rostral and caudal ends. Expanding the knowledge of DNE

effects on XIIMNs in various regions of the hypoglossal nucleus is crucial because the different

areas of the nucleus innervate specific muscles of the tongue, for example the ventrolateral

subnucleus in the caudal end of the nucleus innervates the geniohyoid muscle (Slotkin, 1993).

Dendrites are important cellular processes that help the neuron receive and integrate the

signals from other neurons. Dendrites are essential features of neurons due to the fact that the

majority of a neuron’s surface area exists in the dendrites (Sholl, 1955, 1953). A failure in

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normal dendritic development may compromise the normal synaptic connectivity and the overall

function of these motoneurons (Powell et al., 2016; Powell et al., 2015; Pilarski et al., 2011;

Jaiswal et al., 2013; Puram et al., 2011). Multiple studies have demonstrated effects of DNE on

the dendritic morphology in various brain regions, including the hypoglossal nucleus (Powell, et

al., 2016; Powell et al., 2015; Jalili et al., 2014; Jaiswal et al., 2013; Mychasiuk et al., 2013;

Muhammad et al., 2012; Pilarski et al., 2011; Roy and Sabherwal, 1994). Investigating the

effects of teratogens, such as nicotine, on XIIMN development can potentially help understand

respiratory diseases associated with smoking, for example sleep apnea (Kahn et al., 1994). The

influence of DNE on soma size, morphology, and other cellular features in other regions of the

brain prompts us to hypothesize that DNE will have a negative effect on XIIMN primary

dendrite and soma development, and that the influences of DNE in rostral and caudal XIIMNs

will differ.

Our hypothesis was tested using a dark stain to obtain detailed morphologic

characteristics of XIIMNs. XIIMNs used in the study came from neonatal rats with an age range

of postnatal day one (P1) to postnatal day four (P4). In an in vivo study, Huang et al. (2010,

2004) demonstrated that perinatal nicotine exposure affected breathing in the days just after

birth. XIIMN soma area and primary dendrite morphology (number of primary dendrites,

dendritic length and surface area) was previously investigated by Powell at al. (2016). For this

study we analyzed these features, but used different analytical methods to analyze the XIIMN

morphology. We measured (1) the number of primary nodes, (2) maximum and minimum soma

diameter, (3) dendritic base diameter, and (4) average branch diameter. Our results found no

treatment and developmental effects on the basic features of XIIMN primary dendrite and soma

maximum and minimum diameter. Treatment and hypoglossal nucleus location had no effect on

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primary dendrite features, except on the number of branch points on dendrites originating

directly from the soma (primary dendrite) and on maximum soma diameter. The minimum soma

diameter in DNE caudal XIIMNs was larger on average than control caudal XIIMN (DNE, 18.09

± 1.22 µm; Control, 13.76 ± 0.71 µm).

Materials and Methods

Animal Usage and Treatments

All animal care/use and procedures were approved by the Institutional Animal Care and

Use Committee at the University of Arizona. Data was collected from 71 neonatal Sprague-

Dawley rats, ranging in age from postnatal day (P) 1 to P4. The 71 motoneurons that were traced

and measured were separated into two age groups (P1+P2 and P3+P4) and two treatment groups

(control and DNE), as follows: 20 control/P1+P2 individuals; 23 DNE/P1+P2 individuals; and

14 P3+P4/control individuals; 14 P3+P4/DNE individuals. In another analysis we separated

hypoglossal motoneurons (XIIMNs) into rostral versus caudal positions (33 rostral XIIMNs and

34 caudal XIIMNs). Rostral-caudal separation was accomplished by confirming the transition of

the spinal central canal into the 4th

ventricle using images and tracings of the XIIMNs and the

Atlas of the Newborn Rat Medulla oblongata for reference (Figure 2; Ruangkittisakul, et al.).

Pregnant Sprague-Dawley rats were implanted with an osmotic mini-pump (Alzet,

Cupertino, CA) subcutaneously between the shoulder blades on gestational day 5 (Jaiswal et al.

2013; Pilarski et al. 2011; Huang et al. 2004, 2010; Pilarski and Fregosi 2009; Luo et al. 2004,

2007). Rat mothers and pups were exposed to nicotine bitartrate (6 mg/kg/day) or physiologic

saline for 28 days after implantation. After birth the neonatal rats continued to receive nicotine

through the mother’s milk. Treated animals included an unexposed treatment group that did not

14

undergo surgery or pump implantation. There were no electrophysiological differences between

the unexposed and saline-exposed animals, the data collected from both of these treatment

groups were combined into a single control group, and will be referred to as such throughout the

thesis.

15

DNE

A. B.

Control

C. D.

Figure 1. Filled and traced control and DNE hypoglossal motoneurons. (A) An example of a DNE

motoneuron that was filled with neurobiotin (magnification 40x), underwent DAB staining, and (B)

traced with the Neurolucida tracing system. (C) A filled control and (D) reconstructed XIIMN. Scale bars

represent 25 µm.

40x

40x

16

A.

B.

Figure 2. Brainstem slices containing the

hypoglossal nucleus (red arrowhead) and

the fourth ventricle (white arrowhead). These brainstem slices were used as a

reference to divide the XIIMNs into rostral

versus caudal, where: (A) caudal region of

the hypoglossal nucleus, (B) central canal is

opening into the fourth ventricle, marking

the obex, which we used to demarcate the

transition from rostral to caudal regions of

the hypoglossal nucleus, (C) shows the fully

opened fourth ventricle (Ruangkittisakul et

al.). All XIIMNs located below the obex,

are considered caudal, while those above the

obex are defined as rostral. The other

arrowheads label various structures in the

medulla, for example the dark green

arrowhead labels the medial inferior olive,

gray arrowhead labels the dorsal motor

nucleus of the vagus nerve, dark blue

arrowhead labels the dorsal inferior olive,

light blue arrowhead labels principal inferior

olive.

C.

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Brainstem Slice Preparation and Electrophysiology

Rat pups of either sex were randomly collected from their litters and weighed. The rat

pups were anesthetized with ice until they were unresponsive to paw pinch, and quickly

decerebrated at the coronal suture. Evisceration ensued to expose the surrounding vertebral

column and ribcage and transferred to a dish containing chilled (4°C–8°C) and oxygenated

(95%O2/5%CO2) artificial cerebrospinal fluid (aCSF) solution (in mM: 120 NaCl, 26 NaHCO3,

30 glucose, 1 MgSO4, 3 KCl, 1.25 NaH2PO4, 1.2 CaCl2, pH 7. 4). The medulla and upper

spinal cord were extracted and pinned to a cutting block, with the rostral surface up, for serial

microsection slicing using a Vibratome™ (VT1000, Leica), as described previously (Jaiswal et

al. 2013). Transverse medullary slices were taken until the rostral inferior olive and the most

rostral hypoglossal nerve rootlet were near the surface. 400 µm slices were collected to capture

the majority of the hypoglossal motor nucleus. Preparations were then transferred to a recording

chamber, which was continuously perfused with oxygenated and modified room temperature

aCSF (in mM: 120 NaCl, 26 NaHCO3, 30 glucose, 1 MgSO4, 9 KCl, 1.25 NaH2PO4, 1.2

CaCl2, pH 7. 4).

Staining Protocol

Electrophysiological recordings were conducted on XIIMNs from the brain stem slices

that were collected, as described previously (Pilarski et al., 2011). After electrophysiological

experiments, hypoglossal motoneurons (XIIMNs) were slowly filled with the tracer Neurobiotin

(Vector Labs, Burlingame, CA) to acquire detailed morphology. Visualization of filled XIIMNs

was obtained using a protocol developed by McMullen and deVenecia (1993). Briefly,

Neurobiotin-filled cells were incubated with a primary goat anti-biotin antibody (1:10,000,

Sigma), a secondary biotinylated rabbit antigoat IgG (1:250; Vector Labs, Burlingame, CA), the

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Elite ABC reagent (Avidin-Biotin Complex; Vector Labs, Burlingame, CA), and heavy metal

intensified 3,3’-diaminobenzidine (DAB; Sigma). This staining protocol produced a dark brown

reaction product and clearly stained the cell body and all its processes. Stained medullary slices

were mounted on brass rings with dimethyl sulfoxide solution (DMSO, Sigma-Aldrich, St.

Louis, MO).

Neuron Tracing

A detailed method of XIIMN tracing was previously described in Powell et al. (2016). A

light microscope (Nikon) and Neurolucida® software/equipment (MBF Bioscience, Williston,

VT) were used to capture and measure stained motoneurons. Briefly, Neurolucida was used to

trace the medullary slice and the edges of the fourth ventricle at 5x magnification. Stained cells

were located and magnified to 63x magnification with an oil immersion lens (Zeiss). The soma

(cell body) was labeled and its edges were traced. Dendrites and their branches were traced to

completion and labelled as “normal” or “incomplete”. Normal endings were those dendritic

branches that had a clear ending, whereas incomplete endings were those dendritic branches that

disappeared into the tissue or were cut at the surface of the tissue. Dendrite tracing was repeated

for all dendritic trees that sprouted off the soma.

Complete dendrites (dendrites labeled as “normal” ending during the Neurolucida

tracing) were selected for analysis to avoid errors that would result from including cut or faded

processes in the quantification. Some spines were traced and labeled, but not all XIIMNs had

spines. Dendritic length, surface area, volume, number of primary dendrites, number of branch

points (nodes) and soma size (maximum and minimum diameter) were the measurements

selected for further analysis (Table 1-3).

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Neurolucida Explorer®

Branched Structure Analysis

Traced XIIMNs were analyzed using the Neurolucida Explorer (MBF Bisoscience,

Williston, VT), a companion software to the Neurolucida tracing software. Cells underwent an

analysis that quantified basic cellular structures, such as number of branches, branch endings,

branch order, branch length, branch surface area, soma maximum/minimum diameter, and other

properties. Examples of filled and reconstructed XIIMNs for each treatment condition can be

seen in Figure 1.

Statistical Analysis

Cellular properties that were measured in the Branched Structure Analysis included the

number of primary dendrites, dendritic total length and surface area, soma maximum/minimum

diameter, mean base diameter, mean average branch diameter and the number of branching

points. Neurolucida measures the maximum and minimum soma diameter as two independent

measurements, and the two diameter measures do not have to be at 90o angles to one another.

Any unbranched primary dendrites with a total length less than 10 µm were not used in this

study. Excluding short unbranched primary dendrites was implemented to control for potential

spines on the cell body. Spines originating from the cell body were excluded because the

majority of XIIMNs traced did not possess spine-like features.

Prism 6.05 (Graphpad, San Diego, CA, USA) was used to conduct a 2-way ANOVA with

age (P1+P2 and P3+P4) and treatment (Control and DNE) as the two main factors, and the

dependent variable being each of the cellular properties measured. A univariate 3-way ANOVA

was conducted using SPSS version 23 (IBM, Corp., Armonk, NY, USA) to further investigate

the interactions between all three main factors (age x treatment x position) on XIIMN maximum

20

and minimum soma diameter, and the number of nodes. After each significant ANOVA test, post

hoc analyses were done with the Tukey’s multiple comparison test to compare mean values , for

example DNE:P1+P2 and control:P1+P2 or DNE:P3+P4.

The data included in this report were derived from staining and analysis that I performed

on 68 motoneurons that were recorded in separate experiments by Drs. Fei Xu and Gregory

Powell.

Results

Branched Structure Analysis of XIIMNs

The number of primary dendrites, complete primary dendritic length and surface area,

average base diameter, mean average branch diameter, and soma maximum/minimum diameter

were quantified in Branched Structure analysis using Neurolucida. The standard error of the

means and P-values were analyzed with a 2-way ANOVA, where age and treatment or XIIMN

location and treatment were the two factors and each morphological property was the dependent

variable. There were no significant treatment or age effects on XIIMN morphology (Table 1; P >

0.05). Interestingly, there were significant treatment and location effects on the number of

primary nodes (P = 0.04), maximum and minimum soma diameter (P = 0.04 and P = 0.02

respectively; Table 2). Tukey’s multiple comparisons test found smaller soma minimum

diameter in Control:Caudal (Mean = 13.76 ± 0.71 µm) compared to Nicotine:Caudal (Mean =

18.09 ± 1.22 µm). Tukey’s multiple comparisons test could not find exactly where the

significant differences occurred in the number of primary nodes and maximum soma diameter,

which indicates that the data are too variable, or the differences are too small to provide

significant statistical power. Cellular features with significant differences are graphed in Figure

3.

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Maximum and minimum soma diameter and number of nodes in primary dendrites were

further investigated using a 3-way ANOVA, where age, treatment, and location were the 3

factors. The 3-way ANOVA found no significant treatment, age, or location effects on maximum

or minimum soma diameter. The location factor had significant effects on the number of nodes

(P = 0.02), but Tukey’s multiple comparisons test could not find exactly where the differences

occurred because the interaction terms were not statistically significant.

22

P1+P2 P3+P4 P-

Value

N

(Control/DNE) Control DNE Control DNE

Number of

Primary

dendrites

per neuron

4.1±0.37 4.8±0.43 3.92±0.37 4.0±0.33 P >

0.05 33/34

Complete

primary

total

dendritic

length (µm)

213.14±31.5

4

265.86±44.4

9 203±50.71

171.71±29.9

1

P >

0.05 33/34

Complete

primary

total

dendritic

surface

area (µm2)

732.14±112.

96

1273.32±232

.48

832.65±170.

57

749.91±156.

94

P >

0.05 33/34

Average

Primary

Base

Diameter

(µm)

1.86±0.16 1.77±0.18 2.36±0.36 2.80±0.96 P >

0.05 33/34

Mean

Primary

Average

Branch

Diameter

(µm)

1.56±0.13 1.66±0.13 2.08±0.27 2.32±0.68 P >

0.05 33/34

Total

number of

Primary

Nodes

2.90±0.25 3.10±0.36 2.61±0.37 2.79±0.38 P >

0.05 33/34

Soma

maximum

diameter

(µm)

24.16±1.17 29.40±1.97 27.43±2.60 27.09±1.88 P >

0.05 33/34

Soma

minimum

diameter

(µm)

14.08±0.74 17.24±0.93 14.68±0.93 15.89±1.75 P >

0.05 33/34

Table 1. The effects of DNE on XIIMN morphology properties. Developmental nicotine exposure

(DNE) had no treatment or age effect on the morphological properties. Values represent the mean ± the

standard error of the mean (SEM). P-values were obtained using a 2-way ANOVA, with P ≤ 0.05

considered statistically significant. Units, μm = micron and μm2 = microns squared.

23

Rostral Caudal

P-Value N

(Rostral/Caudal) Control DNE Control DNE

Number of

Primary

dendrites

per neuron

4.31±0.44 4.71±0.47 3.82±0.30 4.24±0.35 P > 0.05 33/34

Complete

primary

total

dendritic

length (µm)

253.71±44.8

2

235.69±40.8

4

167.37±29.4

7

218.49±44.2

2 P > 0.05 33/34

Complete

primary

total

dendritic

surface

area (µm2)

889.01±166.

18

1035.09±22

5.52

661.37±94.1

7

1080.52±22

2.17 P > 0.05 33/34

Average

Primary

Base

Diameter

(µm)

2.05±0.26 1.54±0.20 2.07±0.24 2.85±0.78 P > 0.05 33/34

Mean

Primary

Average

Branch

Diameter

(µm)

1.74±0.19 1.45±0.15 1.81±0.20 2.42±0.54 P > 0.05 33/34

Total

number of

Primary

Nodes

2.69±0.31 2.41±0.37 2.88±0.28 3.53±0.32 P = 0.04 33/34

Soma

maximum

diameter

(µm)

26.69±2.19 30.86±0.2.0

3 24.24±1.26 25.96±0.78 P = 0.04 33/34

Soma

minimum

diameter

(µm)

14.91±0.91 15.4±1.22 13.77±0.71 18.09±1.22 P = 0.02 33/34

Table 2. The effects of DNE on XIIMN morphology properties in caudal and rostral locations.

Developmental nicotine exposure (DNE) had no treatment or location effect on the morphological

properties, except on the number of nodes, max and min soma diameter. Values represent the mean ± the

standard error of the mean (SEM). P-values were obtained using a 2-way ANOVA, with P ≤ 0.05

considered statistically significant. Units, μm = micron and μm2 = microns squared.

24

A.

B.

.

B.

C.

.

B.

Figure 3. Comparing

treatment and location

effects on maximum and

minimum soma dimeter and

the number of primary

nodes. A.) 2-way ANOVA

found locational effects on

maximum soma diameter; on

average rostral max soma

diameters were larger than

caudal max soma diameters,

but no DNE effect. B.) 2-way

ANOVA found significant

treatment effect on minimum

soma diameter; on average the

soma minimum diameter in

Control:Caudal (Mean = 13.76

µm) was smaller compared to

Nicotine:Caudal (Mean =

18.09 µm). C.) 2-way

ANOVA found location

effects on number of primary

nodes; on average caudal

XIIMNs had more primary

nodes than rostral primary

nodes, but no statistical

significant DNE effect.

25

Discussion

Tracing programs like Neurolucida collect and provide a wave of information about

complex morphologic structures like those found on neurons. Morphology analysis can consist

of counting the total number of dendrites, number of branching points, measuring total dendritic

length, conducting Sholl analysis, or convex hull analysis (Kawa, et al., 1998; Sholl, 1953).

These morphometrics alone cannot provide a full understanding of the dendritic field, but

collectively can provide ample information about dendritic arborization (Langhammer et al.,

2010). The data collected and presented here show that overall treatment and age had no effect

on the total number of XIIMN primary dendrites, primary dendritic length, primary dendritic

surface area, average base diameter, mean average branch diameter, and soma maximum and

minimum diameter. Treatment and location affected XIIMN maximum and minimum soma

diameter and the number of primary nodes. Statistical analyses found that maximum soma

diameter and number of nodes varied with rostral and caudal locations (Figure 3). Treatment

affected caudal XIIMN minimum soma diameter, where DNE caudal XIIMNs had larger

minimum soma diameters compared to control minimum soma diameter (Figure 3).

This study was conducted to test the hypothesis that DNE would affect primary dendrite

and soma morphometric development in XIIMNs and have an effect on motoneurons located in

rostral and caudal ends of the hypoglossal nucleus. Prenatal nicotine exposure studies have found

disturbances to neuronal growth and proliferation, axonogenesis and synaptogenesis, apoptosis

and migration, which are all important events in nervous system development (Slotkin, 2008).

The cell body has important functions, such as protein synthesis, and if DNE affected the size in

anyway, this may alter the overall functionality of the neuron. Results from previous studies

found dendritic branching and soma size development were affected by DNE (Mychasiuk et al.,

26

2013; Muhammad et al., 2012; Roy and Sabherwal, 1994). The Sholl analysis used in the Powell

et al. (2016) study found changes in dendritic arborization patterns as a function of distance from

the soma. For example, DNE hypoglossal motoneuron dendrite length and surface area

decreased, but control hypoglossal motoneuron dendrite length and surface area increased as the

animal aged. The Powell et al. (2016) study also looked at primary dendritic length and surface

area, and soma area, but did not examine the effects of location of the cell in the nucleus or the

density of somatic spines.

Investigating where these XIIMNs are located in the nucleus is predicated on the fact that

XIIMNs found in specific regions of the nucleus innervate specific muscles of the tongue

(Kanjhan, et al., 2015; Sokoloff et al., 1993). Significant DNE effects on neuron morphology on

multiple regions of the central nervous system, including the hypoglossal nucleus, and on

XIIMN electrophysiology warranted an in-depth investigation into XIIMN primary dendrite and

soma development (Jalili et al., 2014; Jaiswal et al., 2013; Pilarski et al., 2011; Núñez-Abades,

1995; Roy and Sabherwal, 1994). The primary dendrite features investigated here showed no

significant effects of DNE or age on XIIMN development (Table 1). However, these results do

not counter the results found in Powell et al. (2016), where DNE affected soma size when

comparing treatment and age factors. Utilizing different analytical methods, such as (identifying

potential soma spines), looking at other potentially important dendritic features (base diameter,

average branch diameter, number of nodes), and the multiple ways to measure the soma

(maximum and minimum diameter versus soma area) adds to what was found in Powell et al

(2016). Soma spines were not included in this study due to the majority of the XIIMNs traced not

having spines on the soma, though spines due exist on soma surfaces (Nimchinsky et al., 2002).

Other measured features of the XIIMNs, such as base diameter and average branch diameter,

27

described dendrite morphology. If any changes were detected in base diameter and average

branch diameter it could imply potential effects on the dendrite membrane conductance and

synapses (Powell et al., 2016). Effects on branch points can indicate how complex a neuron is

because branch points (nodes) lead to higher branch orders, which potentially affects the entire

dendritic field.

We analyzed XIIMN cellular structures based on hypoglossal nucleus regions and found

that soma max diameter and the number of primary branch points (nodes) differed in rostral and

caudal regions (Table 2). The hypoglossal nucleus is a complex structure that consists of

subdivisions dedicated to controlling specific muscles in the tongue. The hypoglossal nucleus

can be divided into three subdivisions; the ventral subdivision is found in the caudal 2/3, the

dorsal subdivision in the rostral 2/3 of the nucleus, with overlap in the middle of the nuclear

structure (Sokoloff, 1993; Krammer, et al., 1979). Motoneurons in the dorsal subdivision

innervate the tongue retrusor muscles (hyoglossus and styloglossus); the ventral subdivision

innervates the tongue protrusor muscle (genioglossus); and the ventrolateral subdivision

innervates the suprahyoid muscle and geniohyoid (Figure 4; Kanjhan, et al., 2015; Sokoloff,

1993). Krammer et al (1979) found that XIIMNs innervating the suprahyoid and geniohyoid

muscles were located in the most caudal side of the hypoglossal nucleus, while XIIMNs

innervating the genioglossus are located throughout the length of the hypoglossal nucleus (Figure

4; Krammer et al., 1979).

28

Figure 4. Illustration of XIIMN organization within the nucleus and the innervation of tongue

muscles. (Left) Transverse sections illustrate the differences between subnucleus regions as you move

from the caudal (level 2) to the rostral (level 6) ends of the hypoglossal nucleus. (Right) Diagram shows

what regions of the nucleus innervate which muscles of the tongue. Right diagram also shows what

proportion of the nucleus contributes to what tongue muscle, for example the dorsal subnucleus can be

29

found in the 2/3 rostral end and innervates the styloglossus and hyoglossus muscles. Diagram from

Krammer et al., 1979.

The effects of DNE on XIIMN electrical properties, such as reduced excitatory synaptic

input and increased excitability (Jaiswal et al., 2013; Pilarski et al., 2011), and differences in the

precision and reliability of spike timing in XIIMNs (Powell et al., 2015) may be attributed to the

changes in soma morphology (Jaiswal et al., 2013; Pilarski et al., 2011). Maximum soma

diameter varied with nuclear location, and the minimum soma diameter of XIIMNs in the caudal

region of the hypoglossal nucleus showed DNE effects (Table 2). Our results and previous

evidence show that rostral and caudal XIIMNs show differences in morphology and function

(Kanjhan, et al., 2015; Krammer et al., 1979). In early nervous system development the AChRs

are among the first types of receptors expressed and are expressed first in the spinal cord, and

then continue in the rostral direction (Zoli et al., 1995). The way AChRs develop in the nervous

system (in a spinal cord to rostral direction) can be affected by nicotine exposure. Potentially,

XIIMNs in the caudal regions may be affected by nicotine or exposed to nicotine earlier than the

XIIMNs rostral regions within the hypoglossal nucleus. This mean that the caudal XIIMNs

innervating the suprahyoid and geniohyoid muscles of the tongue, which assist in respiration, are

affected by nicotine exposure during development (Takahashi et al., 2002; Sokoloff, 1993;

Krammer et al., 1979).

Future Methodological Suggestions

Our present study was a step in the right direction for a complete study of the entire XII

motor nucleus, and the effects nicotine may have on XIIMN development and function. Primary

dendrites were the only branch order analyzed because there is high probability of higher branch

orders being truncated during brainstem acquisition. Obviously, XIIMNs have higher branch

30

orders and capturing these branch orders is ideal for complete morphometric analysis of XIIMNs

(Powell et al., 2016; Kanjhan, et al., 2015; Nunez-Abades et al., 1995 and 1994). Obtaining the

majority of neuron tracings can be accomplished by serial sectioning by reducing the number of

higher branch orders being lost, but can still introduce some pieces of dendrites being lost

(Kanjhan, et al., 2015). The maximum and minimum soma diameters were interesting

measurements of the soma, but exactly how the maximum and minimum soma diameters were

calculated in Neurolucida is still unclear. The aspect ratio (how flat the soma can be) and form

factor (a measurement that combines how smooth the perimeter and how compact a cell body is)

could further characterize the XIIMN soma by Neurolucida. In addition to rostral versus caudal

regions the dorsal subdivision, ventral subdivision, and ventrolateral subdivision may be of

interest due to the tongue muscle that they innervate and its contribution to respiratory function

(England et al., 2015; Kanjhan, et al., 2015; Sokoloff, 1993; Krammer et al., 1979). Postnatal day

one to four was used in this study, but investigation of nicotine on days and weeks beyond

postnatal day four, or the influence of stopping nicotine exposure after birth may be interesting to

observe (Carrascal et al., 2005). The addition of these methods in a DNE study may provide

more information on nicotine effects on XIIMNs in all regions of the nucleus, including the long-

term effects on XIIMNs.

Conclusions

In the present study we found the hypoglossal motoneuron features, maximum and

minimum soma diameter and the number of nodes varied based on nuclear locations. The caudal

DNE XIIMNs soma exhibited larger minimum soma diameter compared to caudal control

XIIMNs. Prenatal nicotine exposure studies continue to find nervous system disruptions, for

example respiration in infants is critical for life and infants born to smoking mothers have a

31

higher incidence of sudden infant death syndrome (SIDS) and central and obstructive apneas

(England, et al., 2015). Nicotine replacement therapy continues to be prescribed to pregnant

smokers and exposure to other nicotine sources warrants an investigation into how nicotine

affects the development and function of hypoglossal motoneurons.

32

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