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The Role of NKCC2A on the Nervous System Activity The Role of NKCC2A on the Nervous System Activity

Kavya Annu Wright State University

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The Role of NKCC2A on the nervous system activity

A Thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science

By

Annu Kavya

Bachelor of Pharmacy, Osmania University, 2013

2015

Wright State University

WRIGHT STATE UNIVERSITY

GRADUATE SCHOOL

Date: May 8, 2015

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY

SUPERVISION BY Annu Kavya ENTITLED The Role of NKCC2A on the nervous

system activity BE ACCEPTED IN PARTIAL FUFILLMENT OF THE

REQUIRMENTS FOR THE DEGREE OF Master of Science.

________________________

James B. Lucot, Ph.D.

Thesis Director

_______________________

Jeffrey B. Travers, M.D.,

Ph.D.

Chair, Department of

Pharmacology and

Toxicology

Committee on Final Examination

_______________________

James B. Lucot, Ph.D.

_______________________

Mauricio Di Fulvio, Ph.D.

_______________________

Nadja Grobe, Ph.D.

_______________________

Robert E. W. Fyffe, Ph.D.

Vice President for

Research and Dean of the

Graduate School

iii

ABSTRACT

Annu Kavya, M.S. Department of Pharmacology and Toxicology, Wright State

University, 2015. The Role of NKCC2A co-transporter in the nervous system activity.

The NKCC2A is a splice variant of the Na+K+2Cl- co-transporter 2 (Slc12a1), which

is abundantly expressed in macula densa and in the apical membrane of the tubular

cells in the kidney. Most of our knowledge regarding NKCC2 function is limited to

the kidney, the organ where NKCC2 is abundantly expressed. However, recent studies

have demonstrated that NKCC2 is also expressed in extra-renal tissues. This study

was designed to determine if NKCC2A, a splice variant of NKCC2 has an impact on

behavior and nervous system activity of mice. To these ends, we used mice (wild

type, WT), lacking a single or both alleles of NKCC2A (heterozygous, HE and

homozygous, HO, respectively) which were subjected to battery of standardized

behavioral tests. NKCC2A-HO mice exhibited a significant gating impairment when

compared to the WT or HE genotypes. Interestingly, NKCC2A-HO mice also

exhibited a significantly lower immobility time compared to the WT and HE mice in

the forced swim test (FST), a finding that could not be reproduced in the tail

suspension test (TST). Nevertheless, the caudate nucleus of NKCC2A-HO mice

exhibited low content of serotonin (5-HT) and dopamine metabolites, which might

account for the reduced immobility time assayed by the FST. NKCC2A-HO mice had

significantly lower levels of epinephrine (EPI) and norepinephrine (NE) in the

adrenals, with the HE mice having intermediate levels suggesting a role of NKCC2A

iv

in sympathetic activity, a conclusion suggested by the finding that these mice excreted

low urinary EPI and NE. Together, the results of this study indicate that NKCC2A

impacts behavior and neurochemistry in specific regions of the brain and the

periphery.

v

Table of Contents

1. Introduction ................................................................................................................ 1

1.1 SLC12A Gene family ......................................................................................... 1

1.2 NKCC1 ............................................................................................................... 1

1.3 NKCC2 ............................................................................................................... 3

1.4 NKCC2 Isoforms ................................................................................................ 3

1.5 NKCC2A ............................................................................................................ 4

1.6 Expression and role of NKCC2 in extra-renal tissues ........................................ 4

1.7 Behavior.............................................................................................................. 6

Table 1 - Summary of behavioral tests................................................................ 7

1.8 Neuronal systems ................................................................................................ 9

1.9 Brain - monoamines.......................................................................................... 15

1.10 Role and Metabolism of DA and 5-HT .......................................................... 15

1.11 Kidney Dopaminergic Systems ...................................................................... 16

1.12 NKCC2 and Dopamine in the Kidney ............................................................ 17

2. Development of Hypothesis ..................................................................................... 18

2.1 Specific Aims ................................................................................................... 18

3. Experimental Design ................................................................................................ 20

4. Materials and Methods ............................................................................................. 21

4.1 Animals ............................................................................................................. 21

4.2 NKCC2A-HO mice generation ........................................................................ 21

4.3 NKCC2A-HO, HE and WT genetic analysis ................................................... 21

4.4 Behavioral Testing ............................................................................................ 22

4.5 Brain dissection ................................................................................................ 29

vi

4.6 Neurochemical studies ...................................................................................... 31

4.7 Spot Urine Collection ....................................................................................... 33

4.8 Urine catecholamine extraction ........................................................................ 33

4.9 Determination of catecholamines by High Performance Liquid

Chromatography (HPLC) ....................................................................................... 34

4.10 Adrenal isolation and preparation of the tissue sample .................................. 34

5. Data Analysis ........................................................................................................... 35

6. Results ...................................................................................................................... 35

6.1 Behavioral tests................................................................................................. 35

6.2 Neurochemical Analysis ................................................................................... 36

6.3 Urine ................................................................................................................. 37

6.4 Adrenals ............................................................................................................ 37

7. Results ...................................................................................................................... 38

7.1 Behavior Results ............................................................................................... 38

7.2 Neurochemistry Results .................................................................................... 40

7.3 Spot Urine Results ............................................................................................ 42

7.4 Adrenal Results................................................................................................. 43

Table 2 - Summary of Behavioral results.......................................................... 45

Table 3 - Summary of Neurochemistry results ................................................. 46

8. Discussion ................................................................................................................ 47

9. Acknowledgements .................................................................................................. 53

10. References .............................................................................................................. 54

vii

List of Figures

Figure 1 - Diagrammatic representation of NKCC2 functional role in the kidney ....... 2

Figure 2 - Dopaminergic cell bodies (A8, A9, and A10) projects to the ventral

tegmental area (VTA), substantia nigra (SNc) and the retrorubral field (RRF). ......... 10

Figure 3 - Dopamine pathways in the brain ................................................................. 13

Figure 4 - 5-HT pathways in the brain ......................................................................... 14

Figure 5 - DA metabolism in the dopaminergic nerve terminal and synapse. ............. 16

Figure 6 - Experimental design .................................................................................... 20

Figure 7 - Brain blocker – used to dissect the brain into 1 mm slabs. ......................... 30

Figure 8 - Sagittal section of the mouse brain. The vertical line rostral to the intraural

line indicates the slots where razor blades were placed for dissection. ....................... 31

Figure 9 - Functional Observational Battery ............................................................... 38

Figure 10 - Forced swim test ....................................................................................... 38

Figure 11 - Tail suspension test ................................................................................... 39

Figure 12 - Open field test - resting time ..................................................................... 39

Figure 13 - Open field test - rearing ............................................................................. 40

Figure 14a - 5-HIAA/5-HT ratios in caudate ............................................................... 40

Figure 14b - DOPAC/DA ratios in caudate ................................................................. 41

Figure 14c - DOPAC/DA ratios in septum .................................................................. 41

Figure 15a - Norepinephrine levels in spot urine ......................................................... 42

Figure 15b - Epinephrine levels in spot urine .............................................................. 42

Figure 15c - Dopamine levels in spot urine ................................................................. 43

Figure 16a - Norepinephrine levels in adrenals ........................................................... 43

Figure 16b - Epinephrine levels in adrenals ................................................................ 44

viii

List of Tables

Table 1 - Summary of Behavioral tests .......................................................................... 7

Table 2 - Summary of Behavioral results .................................................................... 45

Table 3 - Summary of Neurochemistry results ............................................................ 46

1

1. Introduction

1.1 SLC12A Gene family

The electroneutral inorganic cation-chloride co-transporters (CCCs) belong to the

solute carrier 12 gene family (SLC12) which consist of nine homologous members:

SLC12A1-9. They play several important roles in human physiology, including cell

volume regulation, hydro electrolytic balance, fluid secretion, neurotransmission and

hormone secretion. This family of genes encode plasma membrane proteins that

mediate the movement of inorganic sodium (Na+) and/or potassium (K+) cations

coupled to the movement of chloride (Cl-) anions. For instance, the SLC12A1 and

SLC12A2 genes encode Na-dependent K-Cl co-transporters widely known as

NKCCs, whereas SLC12A3 encode several Na-Cl co-transporters. These proteins

upload Cl- into the cells by using the thermodynamic energy produced by the inward

movement of Na+ (Gagnon and Delpire. 2013). All members of the SLC12A family

are structurally closely related but differ in functional properties, transcriptional

regulation and post-transcriptional modulation of activity which may contribute to

their relative role in different functions (Hubner et al. 2001).

1.2 NKCC1

The SLC12A2 gene encodes the Na+/K+/2Cl- co-transporters-1 (NKCC1) which are

ubiquitous and secretory in nature (Gamba et al. 1994). Multiple studies on NKCC1

knock out mice confirmed that it is not essential for the survival. Head bobbing and

circling behavior is the most confounding phenotype of NKCC1 knockout mice

(Delpire et al. 1999). They had defects in hearing and balance suggesting both

cochlear and vestibular defects (Gagnon and Delpire. 2013; Hubner et al. 2001).

NKCC1 was found to be involved in a variety of adult organ functions including the

2

central nervous system (CNS). Active Cl- accumulation via NKCC1 activated the

GABAA receptor mediated depolarization in immature neurons (Clayton et al. 1998).

As the neurons mature, the intracellular Cl- concentration decreases which result in a

shift towards GABAA–mediated inhibition. It was also shown that GABA was

excitatory in arginine vasopressin (AVP) neurons which are located in the

paraventricular nucleus of the hypothalamus that produce and release oxytocin and

vasopressin into the blood circulation and so, this effect was increased by chronic

hyperosmotic stress of salt loading. In this study, these effects were attributed to

NKCC1. However, NKCC2 may also be important in this regard. Indeed, it has been

recently demonstrated that NKCC2 is expressed in AVP neurons and that its

expression is increased in response to chronic dehydration (Konopacka et al. 2015).

Figure 1 - Diagrammatic representation of NKCC2 functional role in the kidney

3

1.3 NKCC2

The SLC12A1 gene encodes several Na+/K+/2Cl- co-transporters-2 (NKCC2) which

are expressed in the apical membrane of the tubular cells of the thick ascending limb

(TAL) of Henle and in the macula densa (Gamba et al. 1994; Gagnon and Delpire.

2013; Hubner et al. 2001). NKCC2s are considered absorptive isoforms of the

NKCCs. They are responsible for 25-30% of body’s total renal salt retrieval and

therefore, they participate in the concentration capacity of the kidney (Castagné et al.

2001). Indeed, approximately 80% of the NaCl uptake across the apical membrane of

the TAL cells is mediated by NKCC2 (Castrop and Schnermann. 2008). NKCC2s as

well as NKCC1 are sensitive to furosemide and bumetanide the most commonly

prescribed diuretics. Inactivating mutations in the SLC12A1 gene cause Bartter’s

syndrome type 1, an autosomal recessive salt-wasting disorder characterized by

polyuria, renal tubular hypokalemic alkalosis, and hypercalciuria (Simon et al. 1996;

Gagnon and Delpire. 2013).

1.4 NKCC2 Isoforms

The SLC12A2 gene produce at least three variants named NKCC2A, NKCC2B and

NKCC2F. They are the result of alternative selection of three mutually exclusive

exons (Gamba et al. 1994; Carota et al., 2010; Payne and Forbush, 1994; Vargas-

Poussou et al., 1998). These isoforms differ in their localization and transport

characteristics along the TAL. The renal localization of the NKCC2 isoforms follows

a general pattern despite a few interspecies differences. They are found in the apical

membrane and in subapical vesicles along the TAL and in the macula densa segment.

NKCC2A is expressed in the outer stripe of the outer medulla and in the cortical

portions of the TAL in rodents (Oppermann et al., 2006; Oppermann et al., 2007).

NKCC2A is the dominant isoform in the human cells and is found along the entire

4

kidney. The expression of different isoforms in humans is

NKCC2A>>NKCC2F>NKCC2B whereas in the mice it is

NKCC2F>>NKCC2A>NKCC2B (Castrop and Schnermann. 2008).

1.5 NKCC2A

NKCC2A exhibits a markedly higher Cl⁻ affinity than NKCC2F whereas NKCC2B

has highest Cl⁻ affinity but lower transport capacity (Plata, Meade, Vazquez, Hebert,

and Gamba, 2002). At low tubular Cl⁻ concentrations, Cl⁻ reabsorption was

significantly reduced in NKCC2B-deficient mice when compared to WT mice,

whereas no differences were observed for NKCC2A-HO mice (Oppermann et al.,

2007). However, the lack of NKCC2A in mice resulted in reduced Cl⁻ absorption at

high Cl⁻ concentrations. Under low Cl⁻ load, TAL reabsorption becomes dependent

on the activity of the high- Cl⁻-affinity NKCC2B isoform. Conversely, absence of

NKCC2A function becomes obvious in mice subjected to a high load of NaCl

including that high tubular Cl⁻ concentrations modulate the transport activity

NKCC2A. This suggests that NKCC2A transport activity exceeds that of NKCC2B,

which might be related to both the higher transport capacity of NKCC2A essentially

and its higher expression levels (Castrop and Schnermann, 2008).

1.6 Expression and role of NKCC2 in extra-renal tissues

NKCC2 has been typically regarded as a kidney-specific ion transporter. However,

recent findings suggest that it is also expressed in the endolymphatic sac (ES)

epithelium (Nishimura, Kakigi, Takeda, Takeda, and Doi, 2009). There was also

evidence for NKCC2 mRNA transcripts, and protein expression in the rat

gastrointestinal tract (Xue et al. 2009). In fact, NKCC2 appears to be widely

distributed and expressed in the enteric neurons in the myenteric plexus and it is

5

considered to play a role in the ion transport and neuronal excitability in the gut. Its

expression in other nerve tissues has never been reported previously but there is a

recent study showing the expression of NKCC2 in the brain hypothalamo-

neurohypophyseal system (HNS), in particular the supraoptic and paraventricular

nuclei (Konopacka et al. 2015). HNS is a specialized brain region which contain the

neurons that produce peptide hormones, arginine vasopressin (AVP) and oxytocin

(OXT). AVP modulates the ability of the kidney to retain water free of solutes

whereas OXT has an important role in sexual reproduction, social behavior, lactation

or parturition (Konopacka et al. 2015).

The hypothalamus is a fundamental structure involved in the integration of the

nervous and endocrine systems, and a key regulator in the whole body homeostasis

including glucose homeostasis (Lam et al. 2009). Although the impact of NKCC2 in

glucose homeostasis remains undefined, a potential role of NKCC2 in insulin

secretion has been suggested. Indeed, NKCC1 and NKCC2 are co-expressed in

insulin-secreting beta-cells (Alshahrani and Di Fulvio. 2012a; Alshahrani and Di

Fulvio. 2012b) and elimination of NKCC1 does not preclude secretion of the hormone

in response to bumetanide.

If a mutation or deletion of a gene is thought to modify a physiological response or a

single function, there could be other unexpected alterations in the whole animal.

However, this reasoning cannot exclude the possibility that the gene of interest may

produce its proteins in previously undescribed locations exhibiting different functions.

This may be the case of mice lacking a single variant of NKCC2, where its “kidney-

specific” location and very well-known function cannot represent its potential

function in extra-renal tissues including brain, where our preliminary data suggested

NKCC2 expression (unpublished data) and a recent report confirmed (Konopacka et

6

al. 2015). The influence of a given gene on a specific behavior can be evaluated by

conducting behavioral studies in mice lacking expression of such gene (Komada et al.

2008). NKCC1 knockout mice had behavioral alterations relating this effects to its

presence in the central and peripheral neurons. So, a quick way to evaluate neural

functions and activity alterations is behavior. Behavioral effects reflect the changes in

nerve cell communication and integration as well as morphological alterations (Kulig

et al. 1996). Our preliminary studies indicating presence of NKCC2 in some brain

areas which lead us to investigate effects of deletion of NKCC2A in the mice.

1.7 Behavior

The work of the present thesis sprouted upon casual noticing of uncommon behavior

in mice with functional deficiency in a single or in both alleles of the SLC12A1 gene.

As mentioned, this gene is responsible for one of the three main splice variants of the

Na+/K+/2Cl- co-transporter-2 (NKCC2), named NKCC2A. These observations were

originally made in the laboratory of Dr. Mauricio Di Fulvio and included: unusual

aggressiveness and defensive postures toward the corner of cages, absence of

withdrawal reflexes, head bobbing, seizure-like behavior or abnormal weight gain in

some but not all of these mice. Hence, a battery of standardized behavioral tests was

requested and thoroughly conducted by Teresa Garrett in the laboratory of Dr. James

Lucot in the Department of Pharmacology and Toxicology at Wright State University,

Boonshoft School of Medicine.

Several tests to analyze behavior in mice have been developed. Some of them are

more specific than others and represent the functional interaction of particular regions

within the brain. Several of these tests are summarized in Table 1.

7

Table 1 - Summary of Behavioral tests

Visually observed deficits in the gait of NKCC2A-HO mice led us to design

experiments to evaluate the behavioral abnormalities of this animal model according

to the established tests (Table1). Their unusual overall aggressiveness (unpublished

observation) suggests there might be increased sympathetic activity which can be

Behavioral test

Implicated brain regions

Use

Functional operational

battery (FOB)

CNS To assess and quantify the behavioral and

physiologic state of the mouse

Open field test (OFT) Amygdala

Hippocampus

To assess locomotor activity and anxiety in

rodents

Elevated plus maze (EPM) Amygdala

Right prefrontal cortex

To assess anxiety in rodents

Y maze test Hippocampus To assess exploratory activity

Prepulse inhibition (PPI) Pons

Prefrontal cortex

Forebrain

Measure of sensory motor gating

Rotarod Cerebellum To evaluate motor coordination and balance

Forced swim test (FST) Rostral anterior cingulate

cortex (amygdala, nucleus

accumbens, hypothalamus

To evaluate anti-depressant-like effect

Tail suspension test (TST) Anterior cingulate cortex

(prefrontal cortex, amygdala,

nucleus accumbens,

hypothalamus)

To evaluate anti-depressant-like effects

8

evaluated by testing urinary catecholamines or by analyzing changes in urine

catecholamine output rate.

The behavior of mice is usually assessed by subjecting them to a battery of

standardized tests. Collectively known as the functional observational battery (FOB),

this systematic observational method is applied to comprehensively assess and

quantify the behavioral and physiologic state of the mouse. For instance, locomotor

activity and thigmotactic (tolerance to less stressful environment) behavior of mice

can be assessed by using the Open field test (OFT) (Castagné, Moser, Roux, and

Porsolt, 2001). It can also be used as a surrogate to assess anxiety in rodents. The

elevated plus maze (EPM), however, exploits the natural aversion of mice for open

and elevated areas, as well as on their normal exploratory behavior towards novel

environments (Komada et al. 2008). The EPM is sensitive to anxiolytic effects in

neurotoxic lesions of serotonergic neurons and other effects caused by anxiolytic

drugs. The prepulse inhibition of startle (PPI) test is an operational measure of

sensory gating, usually disrupted in schizophrenia and some other mental disorders

and neurodegenerative diseases. The rotarod test is used to evaluate motor

coordination and balance. It assesses the animal’s ability to maintain balance and

walk on a rotating rod. The forced swim test (FST) is a rodent behavioral test used for

evaluation of the antidepressant efficacy of new compounds and experimental

manipulations that are aimed at generating or preventing depressive-like states

(Castagné et al. 2001). The tail suspension test (TST) is used to screen potential

antidepressant drugs, and to assess other manipulations expected to affect depression-

related behaviors (50 Can, Adem 2012).

Forced swimming or exposure to high intensity acoustic stimuli or any other stress

activates the hypothalamo-pituitary axis and has an anxiogenic effect on behavior.

9

This activation is dependent on hypothalamic centers including the paraventricular

and supraoptic nuclei and the release of hormones which regulate the secretion of

adrenocorticotropic hormone from the pituitary as well as sympatho-adrenal outflow.

Stressors that disturb physiological homeostasis activates pituitary-adrenal and

sympatho-adrenal axis via extended amygdala inputs to the hypothalamus and brain

stem, and exert anxiogenic-like behavioral consequences via actions on a variety of

mid and forebrain nuclei. Therefore, changes in the mice behavior as analyzed by

using FOB may indicate a disturbance in the physiological homeostasis and involves

many brain areas such as the amygdala, hypothalamus, brain stem, mid and forebrain.

1.8 Neuronal systems

The neuronal systems involved in the mice behavior can be evaluated by measuring

the neurotransmitter concentrations while correlating them to a particular behavior

change. As many major theories suggest, neurobehavioral disorders may result from

changes in the dopamine (DA) usage in specific brain areas such as frontal cortex,

caudate, brain stem, nucleus accumbens, and hypothalamus (Howes and Kapur. 2009;

Mehler-Wex et al. 2006). Therefore these mouse brain areas are usually selected to

analyze changes in DA or the turnover of the neurotransmitter serotonin/5-

hydroxytryptamine (5-HT).

10

Figure 2 - Dopaminergic cell bodies (A8, A9, and A10) projects to the ventral

tegmental area (VTA), substantia nigra (SNc) and the retrorubral field (RRF).

The dopaminergic and noradrenergic cell groups are designated with letter A, where

A8-A15 are dopaminergic groups. The serotonergic cell groups are designated with

letter B and adrenergic with letter C. Fig 2 illustrates different dopaminergic cell

groups such as olfactory bulb (OB) dentritic periglomerular (A16) neurons, the

hypothalamic (Hyp; A12, A14 and A15) cell groups. Among this A12 provides

11

tuberoinfundibular and tuberohypophysial projections involved in the neuroendocrine

regulation. A8-A10 are the mesodiencephalic tegmental cell groups comprising SNc

(A9 neurons), the VTA (A10 neurons) and the retrorubral fields (RRF; A8 neurons).

Brain areas receive DA neuronal projections from distinct dopamine pathways which

have different physiological and pathophysiological significance e.g. mesolimbic-

mesocortical and nigrostriatal projections (Fuxe et al. 1974; Inglis and Moghaddam.

1999; Oades and Halliday. 1987; Feldman. 1997). Figure 2 illustrates different DA

neuron bodies as mentioned above.

Ventral tegmental area (VTA; A10 cell groups) is the origin for the dopaminergic cell

bodies of mesocortical system and mainly innervated frontal cortex of the brain (Fuxe

et al. 1974; Oades and Halliday. 1987; Feldman. 1997). It is mainly involved in

cognition and behavior. It contains neurons projecting from prefrontal cortex to the

caudate and brain stem (Bjorklund et al. 1975; Bjorklund and Dunnett. 2007).

In the nigrostriatal pathway, as illustrated in figures 2 and 3, dopaminergic pathways

run from the zona compacta of the substantia nigra (A9), and nigrostriatal fibers

which produce very dense innervation project into the caudate nucleus, putamen and

the globus pallidus (Fig 3). It is especially involved in the production of movement

which is a part of basal ganglia motor loop. Loss of dopamine neurons in this pathway

is implicated in several disorders like Parkinson’s disease, tardiv dyskinesia (Fahn and

Mayeux. 1980; Feldman. 1997).

In the mesolimbic pathway, the dopamine is transmitted from the VTA (A10) to the

limbic system along nucleus accumbens. Mesolimbic and nigrostriatal dopamine

axons arising from midbrain nuclear groups retrorubral nucleus (A8), substantia nigra

(A9) and VTA (A10) forms a bundle lateral to hypothalamus and projects into

12

amygdala, hippocampus and some brain regions of the medial prefrontal cortex

(Feldman. 1997, Bjorklund and Dunnett. 2007). Different brain regions receiving

dopaminergic neurons are involved in different functions.

The amygdala, a component of mesolimbic DA system, participates in conditioned

fear (Fadok et al. 2009). Hippocampus is a primary component of the mesolimbic

dopaminergic system involved in cognitive functions and dopaminergic projections to

the hypothalamus regulates neuroendocrine functions (Bjorklund et al. 1975).

13

Figure 3 – Dopamine pathways in the brain

5-HT is also a monoamine neurotransmitter with an important role in the regulation of

sleep, mood, appetite and cognitive functions. 5-HT pathways are named from B1-B9

where B1, B2, and B3 are called the caudal group and as illustrated in figure 3, caudal

group gives rise to descending pathways into the dorsal, intermediate and ventral

horns of the spinal cord. B4-B9 makes up the ascending 5-HT pathways where B5

14

and B6 pathways innervate the limbic structures, especially from raphe medianus,

hypothalamus and the preoptic area. B7, B8 and B9 gives rise to lateral pathway

which innervates cingulate cortex. There is another pathway that originates from B7-

B9 and primarily innervates the extrapyramidal motor pathways which arise from the

caudate nucleus. The cerebellum is also innervated by 5-HT. Very fine 5-HT neurons

were observed in septum, hippocampus and into the neo- and mesocortex (Figure 4:

Feldman. 1997).

Figure 4 - 5-HT pathways in the brain

15

1.9 Brain – monoamines

Behavioral deficits are usually correlated with neurochemical changes in specific

regions of the brain. The monoamine neurotransmitters norepinephrine (NE),

dopamine (DA), and serotonin (5-HT) are important to normal functioning of the

brain and periphery. Current theories of the basis for major neurobehavioral disorders

involve abnormalities in the use/metabolism of NE, 5-HT and DA in specific brain

regions. To correlate potential changes in these monoamines with observed behavior,

the concentration of NE, DA and their metabolites homovanillic acid (HVA) and

dihydroxyphenylacetic acid (DOPAC) as well as, 5-HT and its metabolite 5-

hydroxyindole acetic acid (5-HIAA) are usually measured in the frontal cortex,

amygdala, hippocampus, hypothalamus, brainstem, accumbens, caudate and

hypothalamus by using reverse phase high performance liquid chromatography.

1.10 Role and Metabolism of DA and 5-HT

DA is the precursor to norepinephrine (NE) and epinephrine (EPI). Apart from this, it

has many other important functions in the brain.

Dopaminergic neurotransmission in different brain regions such as the frontal cortex

(FC), amygdala and the caudate plays an essential role in cognition, attention,

motivation and reward, learning, creativity and motor activity. Catechol-O-

methyltransferase (COMT) and monoamine oxidase (MAO) metabolizes DA to HVA

and DOPAC. These are the most significant DA metabolites. The neurotransmitter

rate of usage can be determined from the ratio of the metabolite to the parent

compound e.g. HVA/DA or DOPAC/DA. HVA is the extra-neuronal (metabolized

outside the neurons) metabolite whereas DOPAC is the intra-neuronal (metabolized in

the neurons) metabolite of DA (Figure 5: (Feldman. 1997)).

16

Figure 5 - DA metabolism in the dopaminergic nerve terminal and synapse.

In addition to nervous tissue, adrenal glands produce a NE and E as sympathetic

nervous system transmitters. Thus to further extend behavioral studies, it is

convenient to determine the levels of these catecholamines in adrenal glands as well

as in the kidney, the major organ involved in excretion.

1.11 Kidney Dopaminergic Systems

The kidney’s intrarenal dopaminergic system is distinct from other neural

dopaminergic input. Dopamine acts as an intrarenal natriuretic hormone (Jose et al.

1992). Kidney dopamine levels can reach high nano molar concentrations whereas

circulating dopamine levels are in the pico molar range (Zeng and Jose, 2011). The

dopamine precursor L-DOPA (L-dihydroxyphenylalanine) is taken up from the

circulation by the proximal tubule or through filtration via the glomerulus. It is then

converted to dopamine by aromatic amino acid decarboxylase. This dopamine

produced in the proximal tubule serves as an autocrine/paracrine agent that inhibits

17

the Na+/K+-ATPase activity in the medullary TAL and in the cortical collecting duct

mediated by D1 receptors. Renal proximal tubules produce dopamine which serves as

an intrarenal natriuretic factor by direct tubular action independent of hemodynamic

mechanisms. Approximately 60% of the sodium excretion is mediated by D1

receptors which in turn is mediated by increased renal dopamine production and

increased sensitivity of sodium transporters to dopaminergic inhibition.

1.12 NKCC2 and Dopamine in the Kidney

In the kidney, 25-30% of the body’s total renal salt retrieval is through the

reabsorption of NaCl in the TAL, a phenomenon mediated by NKCC2. Dietary salt

modulates intrarenal dopamine production (Nishimura et al., 2009). In the mammalian

kidney, dopamine receptor activation due to increased dopamine release decreases

NaCl and water reabsorption through inhibition of specific tubule transporter activity

along the nephron including the Na+/K+-ATPase in the proximal tubule, and NKCC2

in the TAL (Corrigenda for vol. 283, p. F221.2003; Bacic et al., 2005; Grider, 2003).

Therefore, dopamine receptor activation indirectly inhibits Na/K-ATPase and NKCC2

function. This is due to increased renal dopamine release and increased sensitivity of

sodium transporters to dopaminergic inhibition. Therefore, absence of NKCC2A in

mice might impact dopamine utilization and this can be tested by analyzing dopamine

concentrations in the urine samples.

In the TAL, NaCl reabsorption is stimulated by vasopressin, parathyroid, glucagon,

epinephrine and norepinephrine. This mechanism is mediated, either directly or

indirectly, by altering NKCC2 activity. Sodium transport via NKCC2A or NKCC2B

at the macula densa initiates the tuberoglomerular feedback mechanism. Dopamine in

the kidney is known to stimulate NKCC2 activity, but controversially, dopamine

18

inhibits the tubuloglomerular pathway. Interestingly NKCC2A-HO mice exhibited

decreased maximum tubuloglomerular feedback responses (Oppermann et al. 2007).

Therefore, this study is designed to determine the effect of NKCC2A on the E, NE

and DA levels in the urine a reflex of the basal sympathetic activity of the animal.

2. Development of Hypothesis

The hypothesis is that elimination of NKCC2A in vivo alters behavior and in

neurochemical usage in the central and peripheral nervous systems.

NKCC2A has different level of expression in WT, HE and HO mice tissues. HE mice

have NKCC2A expression intermediate to HO and WT. WT had much higher

expression than HE whereas the HO did not have any expression (unpublished data).

Therefore, to find the effect of NKCC2A, we have included HE mice group along

with WT and HO in our studies.

2.1 Specific Aims

Aim 1 -

To test the hypothesis that NKCC2A-HO mice have neurobehavioral deficits.

NKCC2A-HO mice were used as a model to evaluate the behavioral phenotypes.

NKCC2A-HO, HE and WT mice were screened for gross functional deficits,

locomotion, psychosis, working memory, anxiety, and depression.

Aim 2 -

To test the hypothesis that NKCC2A-HO mice have altered DA, 5-HT metabolism

and changes is NE levels in selective brain regions.

19

Brain regions such as frontal cortex, amygdala, hippocampus, brainstem, accumbens,

caudate and hypothalamus were evaluated for DA, 5-HT usage. Reverse-phase high

performance liquid chromatography (RP-HPLC) was used to measure concentrations

of NE, DA and its metabolites DOPAC and HVA, 5-HT and its metabolite 5-HIAA in

selected brain regions with DA and 5-HT neuronal projections.

Aim 3 -

To evaluate the impact of NKCC2A-HO on kidney DA levels as well as on basal

sympathetic activity by measuring urinary DA, NE and EPI.

Aim 4 -

To test the hypothesis that NKCC2A-HO might show changes in total NE and EPI in

the adrenal glands. HPLC was used to measure them in the adrenal glands.

20

3. Experimental Design

Figure 6 - Experimental design

21

4. Materials and Methods

4.1 Animals

Mice subjected to behavioral studies were generated from mice lacking a single

functional allele of NKCC2A (heterozygous) kindly provided by Dr. Hayo Castrop

(University of Regensburg) to Dr. Di Fulvio. Mice were used according to approved

animal protocols and the material transfer agreement signed by both parties. Mice

were used at the age of 8 weeks and both female and male mice were used in all

experiments.

4.2 NKCC2A-HO mice generation

NKCC2A-HO mice were generated by Dr. Hayo Castrop at the University of

Regensburg and all the procedures were approved by the Animal Use and Care

Committee of the National Institute of Diabetes and Digestive and Kidney Diseases

and by the Animal Care Committee of the University of Regensburg. NKCC2A-HO

mice were generated according to a similar strategy which was used to generate

NKCC2B-HO mice earlier (Oppermann et al. 2006). They were generated by altering

the exon 4A of the SLC12A1 gene by the introduction of in-frame stop codons which

result in the premature termination of the translation. Homologous arms were

generated by long-distance PCR (Roche, Indianapolis, IN). Progeny lacking the neo

gene was intercrossed to generate NKCC2A-HO, HE and WT littermates (Oppermann

et al. 2007).

4.3 NKCC2A-HO, HE and WT genetic analysis

To identify WT, HE or HO mice, we took advantage of the genetic strategy followed

to create these mice to design primer sets for genotyping. We used the polymerase

22

chain reaction (PCR) to determine them. The genotyping primers recognizing

flanking intronic sequences 5’ and 3’ exon A were: 5’-TGC AGG AGT GGG TAG

TCC AA-3’ AND 5’-CCG TAG CAT GTG ACG TGG AT-3’. This primer set

amplifies a single DNA fragment of 206 bp or 302 bp from genomic DNA obtained

from WT or HO mice, respectively, to the two fragments simultaneously in the case

of HE mice. The procedure for genotyping mice was developed and set up in our

laboratory by Victor Otano-Rivera (Undergraduate STREAMS student) and Amma

Boayke (Undergraduate HONORS student) and was based on the original protocol

written by Glenn Travis in Laboratory of Molecular Systematics, Smithsonian

Institution. Washington, DC 20560, and the protocols of Walsh and collaborators and

the one described by Morin and Woodruff in Paternity exclusion using multiple

hypervariable microsatellite loci amplified from nuclear DNA of hair cells. Pages 63-

81 of (Martin R.D. (Zürich) Dixson A.F. (Franceville) Wickings E.J. (Franceville).

1992). When genotypes were not clearly defined by the PCR, we troubleshooted this

by blindly testing the animals and then regenotyping them from the tissues. All

animals were fed on standard chow diet and water and were housed under 12h light:

12h darkness cycles.

4.4 Behavioral Testing

a. General Handling Procedures:

Animal handling was performed first. The animals were removed from home cages,

placed in clean clothes and handled approximately 30seconds each. All animals were

handled before individual tests in order to minimize handling-related stress. All

animals were acclimatized for at least 30minutes in the testing room before each test.

The behavioral tests were spaced by a week to minimize interference by previous

23

procedures. Tests were administered in ascending order of novelty in order to reduce

carry over effects. All observational scoring was checked for inter-rater reliability.

The behavioral studies are described below in the order in which they were

conducted.

b. Functional Observational Battery:

The functional observational battery is a noninvasive method used to detect gross

functional deficits prior to more specific neurobehavioral evaluation. It is used to

comprehensively assess and quantify the behavioral and physiological state of the

mouse. Due to the visually observed deficits in ambulation, a functional observational

battery was developed from a published industry version (Irwin, 1968) for use with

this group of animals. The body position, locomotor activity, bizarre behavior,

tremors, finger-withdrawal, touch escape, ataxic gait, and hypotonic gait are observed

and scored in these tests (Irwin. 1968).

First the animals were subjected to the least provoking stimuli which involved an

initial phase of undisturbed observation and a later manipulative phase. The

assessment in this phase and the undisturbed behavior i.e., body position, locomotor

activity, bizarre behavior, exophthalmos, respiration, tremors, twitches and

convulsions were assessed. The animals were then transferred and were briskly

dropped onto the floor of the viewing arena for testing the transfer arousal and spatial

locomotion. Observations were then made if tremors were present. Observations for

gait and limb rotation were also made. The categories measured included general

arousal, gait, reflexes, and motor behavior. Scoring of 0 to 8 range was used

throughout FOB, with values 0, 2, 4, 6, and 8 which represents none, slight, moderate,

24

marked and extreme magnitudes of behavior. The scores were summed for each

category and then for over all categories (Irwin, 1968).

c. Open field test (OFT):

The locomotor activity and thigmotactic behavior can be evaluated using an open

field chamber in which infrared photo-beams (Hamilton Kinder, Motor Monitor

Version 3.11; Poway, CA, USA) are employed to measure locomotor activity in real

time. The open field arena has dimensions of 40 X 40 cm and is divided into central

(20X20 cm) and peripheral (10 cm wide) zones. The IR beams are interrupted by

movements and sorted into different measures based on algorithms in the Motor

Monitor Software (Lad et al. 2010; Tatem et al. 2014). Each mouse was placed in the

center of the open field arena under the red light and allowed to explore it for 10

minutes. Measurements such as horizontal beam interruptions, vertical beam

interruptions (rearing), distance traveled, time ambulating, spatial distribution of

behaviors, and fine movements (repeated activation of one horizontal beam only)

were measured. If mice spend more time in the area close to the wall then it is an

indicator of increased stress level, whereas if they explore the center of the arena then

it indicates that they can tolerate a mildly stressful environment. The open field arena

was cleaned with 70% ethanol solution and let dry after testing each mouse.

d. Elevated Plus Maze (EPM):

This is a behavioral paradigm that takes advantage of the conflict behavior of rodents

between exploration of a novel area and aversion to open and elevated spaces (Hata et

al. 2001). The expression of scores as percentage of time and distance in the open

arms allows for the correction of overall changes due to exploration of the maze,

reducing the activity-induced artifacts (Hogg. 1996).

25

The device has the shape of a cross with each arm being of 5 X 35 cm. Two arms are

open and two of them have walls of 15 cm high, with the arms connected by a 5X5

cm central square. The arms are 76 cm above the floor.

Mice were tested on the plus maze in a room with low, indirect incandescent red

lighting and very low noise levels. The mouse was placed at the center of the maze

with head facing the open arm and allowed to explore for 5 minutes. The number of

entries into open and closed arms, as well as the time spent and distance travelled in

open and closed arms was recorded with the help of an automated EPM (Hamilton

Kinder, Version 3.11; Poway, CA). Animals sensitive to stressful environments avoid

the open arms of the maze. The mazes are cleaned with 70% ethanol and permitted to

dry between sessions. Automated software (Kinder Scientific Motor Monitor

Software Package, Built 08356-14) kept a record of the number of entries, distance

travelled (inches), and time spent (seconds) in the closed and open arms. Entry was

determined when all the four paws of the mouse were on the arm.

e. Y maze activity:

The Y-maze measures a mouse’s working memory status and exploits the innate

tendency to explore novel areas. The apparatus used for this test is an acrylic maze

with 3 arms at 120 degrees to each other, each arm 3.5 cm wide and 20 cm long. The

acclimatization period to the room was 1 hour. Then the animal is placed in the center

of the apparatus. The sequence of arm entries (entry defined as all four paws within

the arm), alternation sequence (entering three different arms in succession e.g. ABC

or BCA) are video recorded for 8 minutes for each sequence. The percentage of

alternation was determined by dividing the total number of alterations by the total

number of choices minus 2, multiplied by 100.

26

f. Pre-pulse Inhibition (PPI):

This is a test to evaluate psychosis-like behavior. Mice were tested in automated

startle chambers (SM100 Startle Monitor System Version 6.12; Hamilton Kinder,

Poway, CA, USA) for PPI. They were calibrated prior to the day of testing. Mice

were placed in the startle chamber and allowed to acclimate for 5 min. A background

noise of 60 dB was on in between trials. The animals were then presented with 30

trials randomly mixed of startle stimuli (loud white noise) and pre-pulse stimuli

(lower dB white noise preceded by loud). The startle stimuli consist of a 20 ms white

noise stimulus presented at 2 different intensities (85 or 100 dB). The pre pulse

stimulus consists of 20ms, 70 dB white noise pulse, presented 100ms prior to the

startle (85 or 100 dB) stimulus (70dB + 100 ms + 85 dB and 70 dB + 100 dB). The

inter trial intervals range from 9-16 s. The startle response was recorded on a pressure

plate (Sharma, Elased, and Lucot, 2012).

g. Rotarod test:

The rotarod test is used to assess motor coordination by measuring the latency to fall

off the rotating drum. The rod is 3cm in diameter and designed so that the mouse only

drops 25cm onto a plate that stops a timer. The animals were moved to the experiment

room from their home cages 30 minutes prior to testing. There were two protocols

followed, one that used a constant speed (25rpm) for 2min and a second that used

accelerating speeds for 5min (up to 30rpm). Both tests were conducted with all

subjects. Learning sessions were conducted for 3 days in which the animals were

placed on a rotarod (24rpm) for a maximum of 60s and the latency to fall off was

recorded. The animals were then returned to their home cage for 5-10 minutes and

then the session was repeated for 3 times each day of training. For the learning

sessions (3 consecutive days): Each mouse was placed on a rotating rod (24 rpm) for a

27

maximum of 60s and the latency to fall off was recorded. In test session, two trails

were conducted at a fixed speed with a maximum time of 60s, and an hour later the

animal was tested on an accelerating rod. The animal was placed on a rotating rod and

then the speed was increased every minute up to 44rpm for a maximum of 5 minutes.

The latency was recorded when the animal falls. The same test was performed again

10 minutes later. This procedure was adapted from Current Protocols in

Neuroscience, 2001 and Pallier, Drew, and Morton, 2008.

h. Forced Swim Test:

The Forced Swim Test (FST) is used to evaluate depression-like behavior. In this test,

mice were placed in a transparent glass cylinder (diameter 10 cm, height 35 cm)

which was filled up to 30 cm with water (23-25°C). The mice were tested separately

in different cylinders with an opaque white plexi glass separating them so that the

mice cannot see each other during the test. This testing is video recorded and scored

for durations of immobility. After the test the mice were dried with a towel and

returned to their home cage and placed on a thermal blanket heated to 37C. A mouse

was considered as immobile when floating motionless or making only those

movements necessary to keep its head above the water (Castagné et al., 2001; Sharma

et al., 2010).

Scoring

A new time-scoring technique was used to score several behaviors during a single

viewing session which has been shown to be both reliable and valid for detecting

antidepressant effects of both desipramine and fluoxetine in the FST (Detke et al.,

1995). The scoring had three different categories scored based on definitions,

“floating in the water without struggling and making only those movements necessary

28

to keep the head above water” which is considered immobility; “making active

swimming motions, more than necessary to merely maintain the head above water”

(i.e., moving around in the cylinder) called swimming; “making active movements

with forepaws in and out of the water, usually directed against the walls” which is

climbing; and “having entire body submerged” called diving. In these tests, diving

occurred and was not reliably altered by the compounds tested, data for diving are not

reported. All of the behavior scoring was done by a single rater, who was unaware of

treatment condition. This new procedure to score many active behaviors in the FST

was used to re-evaluate the effects of different types of anti-depressant drugs (Detke,

Rickels, and Lucki, 1995). Selective norepinephrine (NE) uptake inhibitors, such as

desipramine and maprotiline, reduced immobility and selectively increased climbing

without affecting swimming. Previously effects of serotonin (5 hydroxytryptamine; 5-

HT) uptake inhibitors in the FST were considered as false negatives in this test, but

this scoring system revealed the effects of serotonin (5 hydroxytryptamine; 5-HT)

uptake inhibitors in the FST, such as fluoxetine, sertraline, and paroxetine also

reduced immobility but increased swimming instead of climbing. Thus, an analysis of

behavioral patterns in the FST distinguished drugs that have a common therapeutic

outcome and those that act on distinct neurotransmitter systems. To avoid these false-

positives, tail suspension test (TST) can be used to test the depressive-like behavior

which has different false positives. A literature search revealed monoaminergics (e.g.,

amphetamine, paroxetime)-induced decreases in immobility time which might be

false positives in the forced swim test (Zhao et al. 2008).

i. Tail Suspension Test:

Mice were suspended by their tails for 6 minutes (50 Can, Adem 2012). The subject’s

behavior was recorded with a video camera and scored afterwards. A suspension bar

29

(1 cm. height, I cm. width, 60 cm. length) and tape (Time Med Labelling Systems,

Inc., Burr Ridge IL, 1.9 cm width were used. The mice were suspended by their tails

by attaching them to the suspension bar with tape in a way that they cannot escape or

hold on to nearby surfaces. The distance between mouse nose and floor was

approximately 20-25 cm. The mice were separated from each other using opaque

plastic to prevent interaction or observance between each other. A polycarbonate tray

was placed at the bottom of each mouse suspension to collect feces and urine.

Some strains of mice (mainly C57s) have a behavior called tail climbing and can thus

hold on to their tails during the test and negate it. We suspect that this strain of mouse

will have that response because their background strain is C57Bl/6. In order to

prevent this from happening a clear hollow cylinder approximately 4 cm long, 1.6 cm

outside diameter, 1.3 cm inside diameter, and weighing 1.5 grams made from

polycarbonate tubing was placed around the tails of the mice to prevent tail climbing

behavior of the mice.

The animals were brought to the testing room an hour before testing. The climb

stoppers were placed around the tail of the mouse and the tape was applied at the very

end of the tail leaving 2-3 mm outside the tape. The mouse was suspended by placing

the free end of the tape on the suspension bar. At the end of 6 minutes the animals

were returned to their cages and the tape was removed by gently pulling it off.

4.5 Brain dissection

Animals were sacrificed by decapitation. The brains were removed and the frontal

cortex was hand dissected. The remainder of the brain was placed in a brain matrix

(Ted Pella, Inc) and sliced into 1mm or 2 mm slabs rostral to the intraneural line,

(Caudate : 5-4, Amygdala/Hypothalamus: 3-2, Hippocampus: 2-1, Brain stem1: 1-0,

30

Brain stem2: 0- -1) which were then stored at -80C on plastic squares until further

dissection. Amygdala and the caudate nucleus were dissected using landmarks in The

Mouse Brain Stereotaxic Coordinates (Paxinos and Franklin, 2008) as a guide.

Accumbens was taken using a tissue punch (-20̊ C ice blocks) of 1mm thickness.

Other sections were dissected using landmarks in The Mouse Brain Stereotaxic

Coordinates (2008) as a guide.

Figure 7 - Brain blocker – used to dissect the brain into 1 mm slabs.

31

Figure 8 - Sagittal section of the mouse brain. The vertical line rostral to the

intraural line indicates the slots where razor blades were placed for dissection.

4.6 Neurochemical studies

a. Preparation of tissue samples

All the brain tissues were homogenized in 0.2N HCl using a homogenizer followed by

centrifugation. The supernatants were collected into 2 aliquots, which were stored at -

80ºC until they were analyzed by HPLC.

32

b. Determination of catecholamines by High Performance Liquid

Chromatography (HPLC)

HPLC is high performance liquid chromatography which is used to separate, identify

and quantify the components in a mixture. Its function relies on a high-pressure pump

which passes a pressurized liquid solvent containing the sample through a short,

narrow column which is filled with a solid adsorbent material. Each component of the

sample interacts differently with the adsorbent material causing different retention

times of each component which helps in separation as they flow through the column.

The electrochemical detector attached to the machine generates a signal proportional

to the amount of the sample component eluting from the column. Here we used BAS

LC-4B and BAS LC-4C detector with a potential of 0.75V and maintained across a

glossy carbon working electrode with a positive radial flow. This electrochemical

(amperometric) detector is attached to the system to identify the separated

components of the sample as they get eluted from the column. In this amperometric

cell the column effluent passes electrodes to which a voltage is applied. As the

molecules in the column effluent pass the electrodes they become oxidized when the

potential is sufficiently large. The amount of current drawn to drive the

electrochemical reaction is quantified and concentration determined by reference to an

external standard.

The supernatant (sample) in the aliquot is injected directly into the chromatographic

system. Chromatographic separation was conducted using a BAS 100 X 3 mm C18

column (BAS model MF-8954), 80 X 4.6mm C18 column (ESA model 68-0100)

optimized to 0.75V. The mobile phase consisted of soap (1-octanesulfonic acid),

acetonitrile and dimethyl acetamide. The flow rate was kept at 0.3ml/min.

33

c. Brain monoamine analysis

To identify monoamines, a series of standard solutions containing known amounts of

monoamines includingnorepinephrine (NE), 3,4 - dihydroxyphenylacetic acid

(DOPAC), 5-Hydroxy Indole acetic acid (5-HIAA), Homovanillic acid (HVA),

Dopamine (DA) and serotonin (5-HT) were injected into the chromatography system

before and after each sample analysis. The concentrations of mono amines in the

samples were determined by comparing the peak heights from the samples relative to

that of the standards. The concentrations of NE, DA, HVA, DOPAC, 5-HIAA and 5-

HT were expressed as ng/mg of wet tissue. The ratio between the metabolite and the

parent compounds (e.g. HVA/DA, DOPAC/DA, 5-HIAA/5-HT) was used as a

surrogate index of neurotransmitter usage.

4.7 Spot Urine Collection

Urine was collected from each animal by gently scruffing the animal and placed it on

clean surface until it urinated. The urine was collected using a pipette and placed in a

micro tube which was placed in ice until storage at -80 C until analysis. Subjects that

did not urinate after 30s were placed in a clean empty cage until they did.

4.8 Urine catecholamine extraction

Catecholamines in spotted urine were extracted over neutral alumina, activity grade 1,

from Sigma Chemical Co. product A-9003 supplemented with an internal standard

consisting of 2,3-dihydroxybenzoic acid (DHBA).This preliminary extraction over

alumina is necessary for down-stream electrochemical detection of urine

catecholamines. This procedure removes electroactive species in the urine that cannot

be separated by HPLC (Smith et al. 2013). Extraction was done as follows: alumina

was placed in 1.5mL tubes and supplemented with HCl containing sodium

34

metabisulfite. To assay samples spot urine, DHBA internal standard and tris buffer

were added. The pH was then adjusted to 8.5±0.05 by adding Tris buffer. Then the

tube was vortexed, centrifuged and then the supernatants were removed and diluted

with deionized water. This was done for 2 times. Then the supernatants were removed

and further diluted with HClO4 containing sodium metabisulfite. The tube was then

vortexed; centrifuged and the supernatants were aliquoted into different 0.5 mL tubes

and stored at -80 ̊C until analysis (Smith et al. 2013).

4.9 Determination of catecholamines by High Performance Liquid

Chromatography (HPLC)

To determine catecholamines in these processed samples, 20µL of them were directly

injected into the chromatographic system. Chromatographic separation was conducted

using 80 X 4.6mm C18 column (ESA model 68-0100) optimized to 0.55V. The

mobile phase consisted of soap (1-octanesulfonic acid), methanol and acetonitrile.

The flow rate was kept at 0.3ml/min.

4.10 Adrenal isolation and preparation of the tissue sample

Animals were sacrificed by decapitation and then the adrenal glands were dissected,

weighed onto empty 1.5ml tubes and labelled with the animal identification number.

A single weighed adrenal gland was submerged in PBS buffer and DHBA stock (as an

internal standard) and homogenized in the buffer using a homogenizer keeping the

samples on ice in between homogenizing cycles. The homogenates were added to a

tube containing HCLO4 to be filtered and centrifuged at. Supernatants were then

collected into 3 aliquots and stored at -80C until HPLC analysis.

35

5. Data Analysis

A DataQ device attached to the detector records the waveforms and using Windaq

software.

The data were analyzed by using STATISTICA data analysis software (Stat soft,

Tulsa, OK). The results are presented as group means±S.E.M. and analyzed using

STATISTICA data analysis software. Data were analyzed by 1-way ANOVA test. It

was used to evaluate the overall significance among groups. Tukey-HSD post hoc test

was used to identify individual group differences. We also used Kruskal Wallis one

way analysis of variance in the FOB and rotarod test.

6. Results

6.1 Behavioral tests

a. Functional Observational Battery

The NKCC2A-HO had significant differences in its walking behavior which is gait.

The HO mice group had higher scores compared to the HE and WT mice. As shown

in Fig.9, there were significant differences in the gait category (Fig 9) with NKCC2A-

HO having higher gait score compared to NKCC2A-HE and WT [factor ‘genotype’ F

(2, 28) = 3.524, p < 0.05 vs WT and HE].

b. Forced swim test

As shown in Fig.10, NKCC2A-HO mice were immobile for significantly less time

compared to WT and HE mice during the 6 minute forced swim test [factor

‘genotype’ F (2, 28) = 31.044, p < 0.001 vs WT and HE].

36

c. Tail suspension test

As shown in Fig.11 there were no significant differences between mice of the three

genotypes during the 6 minute lasting tail suspension test [factor ‘genotype’, F (2, 22)

= 0.59, p >0.05 vs WT and HE].

d. Peripheral Rest time

As shown in Fig.12, during the 10 min of the test session in the open field NKCC2A-

HEmice spent significantly more time in the periphery than in the central zone [factor

‘genotype’ F (2, 28) = 5.7466, p < 0.05 vs WT and HO].

e. Rearing

Fig.13 shows a significant differences between NKCC2A-WT, NKCC2A-HE and

NKCC2A-HO mice in the rearing phenomena in the open field test [factor ‘genotype’

F (2, 28) = 4.03, p < 0.05].

6.2 Neurochemical Analysis

a. The caudate of NKCC-HO mice exhibit low 5-HIAA/5-HT ratios

The 5-HIAA/5-HT ratio in the caudate of NKCC2A-HO was significantly low (Fig.

14a, p<0.05) when compared to NKCC2A WT (Fig. 14a) [factor ‘genotype’ F (2, 28)

= 4.514, p < 0.05 vs WT].

b. The caudate of NKCC2A-HO mice exhibit low DOPAC/DA ratios

The DOPAC/DA ratio in the caudate of NKCC2A-HO was significantly low (Fig.

14b, p<0.05) when compared to NKCC2A-HE and WT (Fig. 14b) [factor ‘genotype’

F (2, 28) = 5.04, p < 0.05 vs WT and HE].

37

c. The septum of NKCC2A-HE mice exhibit high DOPAC/DA ratios

The DOPAC/DA ratio in the septum of NKCC2A-HE mice was significantly higher

than that of NKCC2A-HO and WT mice (Fig. 14c, p<0.05). However, there were no

significant differences in individual neurotransmitter levels [factor ‘genotype’ F (2,

28) = 4.07, p < 0.05 vs WT and HO].

6.3 Urine

As shown in Fig 15a, 15b, 15c, there were no significant differences in urine NE, EPI

and DA levels between the mice of three genotypes.

6.4 Adrenals

There was a significant decrease in the NE (Fig 16a) levels in NKCC2A-HO mice

when compared to NKCC2A-HE and WT [factor ‘genotype’ F (2, 23) = 4.12, p <

0.05 vs WT].

There was a significant decrease in the EPI (Fig. 16b) levels in NKCC2A-HO mice

when compared to NKCC2A-HE and WT [factor ‘genotype’ F (2, 23) = 4.744, p <

0.05 vs WT].

38

7. Results

7.1 Behavior Results

Figure 9 - Functional Observational Battery

Sum of the arousal category and the gait categories in WT, HE and HO. The HO

group had significantly higher gait scores [factor ‘genotype’ F (2, 28) = 3.524, p <

0.05 vs WT and HE].

Figure 10 - Forced swim test

Immobility time. Immobility of the HO group was lower compared to the WT and HE

[factor ‘genotype’ F (2, 28) = 31.044, p < 0.001 vs WT and HE].

39

Figure 11 - Tail suspension test

Immobility time. No significant changes in the Immobility of the HO group compared

to the WT and HE [factor ‘genotype’, F (2, 22) = 0.59, p >0.05 vs WT and HE].

Figure 12 - Open field test – Peripheral resting time

Time (sec) spent resting in the periphery during a 10 min test. HE mice were

different from WT and HO mice. No other differences were significant [factor

‘genotype’ F (2, 28) = 5.7466, p < 0.05 vs WT and HO].

40

Figure 13 - Open field test - Rearing

Total number of rearing events in the ten min test. The WT were lower than either

HE or HO [factor ‘genotype’ F (2, 28) = 4.03, p < 0.05].

7.2 Neurochemistry Results

Figure 14a - 5-HIAA/5-HT ratios in caudate

5-HIAA/5-HT ratios observed in the caudate area of WT, HE and HO mice [factor

‘genotype’ F (2, 28) = 4.514, p < 0.05 – significant difference from WT].

41

Figure 14b - DOPAC/DA ratios in caudate

DOPAC/DA ratios in caudate area of WT, HE and HO mice [factor ‘genotype’ F (2,

28) = 5.04, p < 0.05 – significant difference from WT and HE].

Figure 14c - DOPAC/DA ratios in septum

DA usage levels in Septum area of WT, HE and HO mice [factor ‘genotype’ F (2, 28)

= 4.07, p < 0.05 – significant difference from WT and HO].

42

7.3 Spot Urine Results

Figure 15a - Norepinephrine levels in spot urine

Figure 15b - Epinephrine levels in spot urine

43

Figure 15c - Dopamine levels in spot urine

7.4 Adrenal Results

Figure 16a - Norepinephrine levels in adrenals

Norepinephrine levels observed in adrenals of WT, HE and HO mice [factor

‘genotype’ F (2, 23) = 4.12, p < 0.05 – significant difference from WT].

44

Figure 16b - Epinephrine levels in adrenals

Epinephrine levels observed in adrenals of WT, HE and HO mice [factor ‘genotype’ F

(2, 23) = 4.744, p < 0.05 – significant difference from WT].

45

Behavioral test

Result

Functional operational battery (FOB) Increased gait in NKCC2-HO compared to the WT and HE

Open field test (OFT) Higher rearing in NKCC2A-HO mice compared to the WT and

HE

Elevated plus maze (EPM) No differences in anxiety across the three groups

Y maze test No significant spontaneous alterations across the 3 groups

Prepulse inhibition (PPI) No significant disruption in PPI

Rotarod No differences in motor coordination and balance among the 3

groups

Forced swim test (FST) Increased immobility time of the NKCC2A-HO mice compared to

the WT and HE

Tail suspension test (TST) No significant change in immobility time of the NKCC2A-HO

mice compared to the WT and HE

Table 2 - Summary of Behavioral results

Accumb

ens

Amygdala Brain stem Caudate Hypothalam

us

Septum

NE indeterminate Higher in HE

Higher in

HE Higher in HE

DOPAC Lower

in HO Higher in HE

Lower in

HO Higher in HE

Lower in

HO

5-HIAA Higher in HE

Lower in

HO

Higher in

HE

HVA Lower

in HO

Higher in HVA

and HO

Higher in

HE

DA indeterm

inate

5-HT Lower in HO

Higher in

HE Higher in HE

46

DOPAC/DA

Lower in

HO

Higher in

HE

HVA/DA

5-HIAA/5-HT

Lower in

HO

Table 3 - Summary of Neurochemistry results

47

8. Discussion

We performed a preliminary characterization of the behavioral and neurochemical

phenotypes of mice lacking a single (HE) or both (HO) alleles of NKCC2A and

compared the results against WT. The HE tissues exhibits intermediate level of

NKCC2A expression, whereas HO does not show any expression with high

expression levels in the WT indicating all the three groups have different levels of

NKCC2A expression and its effect might be different across the groups. Based on

anecdotal behavior observations noted in NKCC2A-HO mice, we evaluated them by

using a functional observational battery followed by specific behavioral tests

including open field test, y maze activity, elevated plus maze, pre-pulse inhibition,

rotarod test, forced swim test, and tail suspension test.

There were few visually observed deficits in ambulation of the NKCC2A- HO mice.

A functional observational battery developed from a published industry version

(Irwin, 1968) was used with this group of animals which measured the categories of

general arousal, gait, reflexes, and motor behavior. The pattern of movement of the

limbs during locomotion is gait and abnormal gait is observed in several neurological

and musculoskeletal disorders. There were significant differences in the gait category

with the NKCC2A-HO group having higher impairment but no significant differences

were observed in rotarod test which suggest that NKCC2A might be involved in areas

affecting gait rather than balance. Abnormality in gait generally indicate some gross

functional deficits in the CNS which were later assessed using specific behavioral

tests. Therefore, this suggests that the NKCC2 might affect the CNS in areas involved

in locomotor activity like walking.

48

In FST, which is used to detect the anti-depressant like effect, the NKCC2A-HO mice

displayed significantly lower immobility time compared to the HE and the WT mice

(Fig 2) which was not reproduced by TST. Motor stimulants (Betin et al. 1982, De

Pablo et al. 1989) generally produce false positives in the FST (Panconi et al. 1993).

Nicotine (1mg/kg) has a weak but significant anti-depressant like effect consistent

with the excitatory substances which constitute false positives for the FST (Porsolt et

al. 1978) (Panconi et al. 1993). However, in this study, there were no significant

differences in the motor activity (basic movement and fine movement) of NKCC2A-

HO mice. Thus, the decrease in immobility time in FST in NKCC2A-HO was

independent of changes in locomotor activity suggesting that NKCC2A might have an

effect related to the swimming challenge in the FST rather than behavioral despair

challenge in the TST.

There might be the effect of thermogenesis in the FST that is producing decreased

immobility in NKCC2A-HO mice. Sympathetic nervous system and thyroid hormone

play a major role in generation and regulation of heat (Silva. 2006). Interestingly,

preliminary results from our laboratory indicate that NKCC2 mRNAs are expressed in

the thyroid at very low levels. Therefore, NKCC2A-HO mice might experience

hypothermia in FST condition leading to decreased immobile behavior which cannot

be observed in temperature-insensitive TST.

There were lower 5-HT and intraneural DA usage in the caudate nucleus which is

mainly associated with motor activity, suggesting its role in the FST effect. Caudate

nucleus receives projections from the A9 group of dopaminergic cell bodies which are

located in the substantia nigra (Feldman. 1997). The significant neurochemical change

in the caudate dopaminergic system was not supported by the results of the locomotor

activity in the open field test indicating that NKCC2A is not effecting the overall

49

motor activity. But, there is a study showing significant elevation in prodynorphin

(pDyn) mRNA levels following FST in the caudate putamen but not in the nucleus

accumbens, hypothalamus, amygdala, frontal cortex or hippocampus indicating that

caudate might be solely involved in this process (Reed et al. 2012). No changes in the

DA and 5-HT usage in frontal cortex, amygdala, nucleus accumbens and

hypothalamus which are common areas involved in both FST and TST explain that

these areas might not be responsible for the immobile behavior in the tests. We

suggest that NKCC2A might have an effect on DA system in the caudate nucleus but

not in the other areas involved in motor activity.

In the open field test, there were no significant differences in locomotor activity

which measures the fine movements (usually grooming), distance traveled in total or

in zones or total time spent in either zone. The NKCC2A-HO exhibited a higher level

of rearing compared to the WT mice in the open field test which may relate to more

exploratory behavior in these mice. There was also a difference in the time spent

resting in the periphery exhibiting thigmotactic behavior. However, this has uncertain

significance in the absence of any difference in total time in the periphery, distance

traveled in the periphery or any measurement in the central zone.

The elevated plus maze revealed no significant differences among groups in the time

spent either in the open arm or in the closed arm. This is usually interpreted as a lack

of difference in their ability to tolerate a stressful environment. This test is strongly

responsive to benzodiazepine type anti-anxiety treatments and not to other classes of

anxiolytics. No significant differences among the groups suggest that the GABA and

benzodiazepine receptors are functionally normal which generally effect anxiety and

performance in the elevated plus maze.

50

Y maze testing revealed no differences among mice of the three genotypes. Frontal

cortex is the anatomical site for working memory and dopamine neurotransmission in

the frontal cortex plays a major role in y-maze testing. The working memory was not

different across the three groups. This observation is consistent with absence of

significant changes in the DA usage in frontal cortex.

We used prepulse inhibition (PPI) of startle to evaluate genotype dependent effect on

psychosis-like symptoms in NKCC2A-HO mice. There were no significant

differences among groups in PPI of startle which measures the inhibitory response to

a loud white noise stimulus (85 or 100 dB) when it is immediately preceded by a

softer (70 dB) white noise. This is a basic circuit present in all normal species but

deficient in schizophrenics. It is used as a screen for antipsychotic drugs by measuring

their ability to reverse impairment of the PPI produced by either a dopamine

antagonist or dopaminergic neurotransmission in frontal cortex (Zavitsanou et al.

1999) or phencyclidine (Yee et al. 2004; Bakshi and Geyer. 1997). There was no

disruption in the PPI behavior which indicates absence of psychosis-like symptoms in

the NKCC2A-HO mice and also indicating that NKCC2A-HO did not affect the

hearing or sound perception. Interestingly, NKCC2 is expressed in the endolymphatic

sac (Nishimura et al. 2009). Together, these results suggest the possibility that either

NKCC2B or NKCC2F are expressed in ES or that NKCC2A has a minimal if any role

in audition. Dopaminergic neurotransmission in the frontal cortex plays a major role

in the modulation of startle reflex measured by PPI (Zavitsanou et al. 1999; Yee et al.

2004; Bakshi and Geyer. 1997; Mehler-Wex et al. 2006). Absence of changes in PPI

correlates with the insignificant changes in the frontal cortex and amygdala dopamine

usage suggesting that NKCC2A is not affecting the dopamine projections innervating

these areas.

51

There was a significant decrease in the adrenal NE and EPI levels in the NKCC2A-

HO mice compared to the wild type and heterozygous mice (Fig 16A, 16B).

SLC12A1 gene was found to be expressed in supraoptic and paraventricular neurons

of the hypothalamo-neurohypophyseal system during salt load condition. The

interactions among the hypothalamus, pituitary and adrenal gland forms the major

part of the neuroendocrine system that controls stress related reactions. Expression of

NKCC2 in the hypothalamo-neurohypopheseal system, a specialized brain region

involved in the elaboration of peptide hormones that directly control the kidney

function explains both the central and peripheral role of NKCC2 in the crucial

mechanisms to regulate salt and water balance. In the kidney NKCC2 is regulated by

arginine vasopressin (AVP). AVP released from an endocrine source or

paracrine/autocrine source might provoke NKCC2 synthesis and activity (Konopacka

et al. 2015). Thus we speculate that the NKCC2A-HO mice which had low NE and

EPI adrenal levels but not significantly lower urinary catecholamine levels,

compromise their ability to respond to the sympathetic stimulation.

Further studies

There is preliminary evidence of the presence of NKCC2A in some sections of the

brain such as the frontal cortex, somatosensory cortex and in the cerebral cortex. Its

expression was much higher in the WT mice compared to the HE mice and with no

expression in the HO mice (unpublished observation). We did not perform tests to

evaluate learning and cognitive behavior, there can be further investigations focused

on the attention and cognitive abnormalities of the NKCC2A-HO mice as well as

studies related to these brain areas showing NKCC2A expression, to determine the

effect of its presence in these areas and to pursue the FST and TST discrepancy which

is an interesting finding. Because of greater variability in urinary catecholamine

52

levels, we could not conclude there was a significant effect of NKCC2A on

sympathetic activity. High salt-loaded rats were observed to have elevated NKCC2

expression (Haque et al. 2011), therefore there is a need to look at the urinary

catecholamine levels in stress (salt-load) induced mice. Further studies can be focused

on stress induced urinary catecholamine levels to see the effect of NKCC2A and also

with a greater ‘N’ to increase the statistical significance of the study.

53

9. Acknowledgements

I am earnestly thankful to my advisor Dr James Lucot and also Dr Mauricio Di

Fulvio and Dr Nadja Grobe who advised and supported throughout my thesis with

their comprehensive knowledge. I am very grateful to Teresa Garrett for her

valuable support, guidance, time and help throughout my thesis and teach me working

on HPLC. I would specially like to thank Marie Heis and Katherine for their

constant support and help. I am heartily thankful to Dr. Mauricio for his valuable

suggestions and also Shams Kuran to teach me working on PCR. Thanks to all

graduate, undergraduate students and lab-staff for their assistance to conduct

experiments.

We are grateful to the Research Incentive Funds awarded to Dr. Lucot by Wright

State University, American Diabetes Association, and the Boonshoft School of

Medicine (MDiF) which partially supported the costs of animals and behavioral

studies, the outstanding resources of the Departments of Pharmacology and

Toxicology and Neuroscience, Cell Biology and Physiology and the Animal Facility

at Wright State University, Boonshoft School of Medicine.

Note: The results of this Master’s Thesis and their interpretations have been presented

in part or in whole in Scientific Meetings or have been submitted for publication. The

data presented are the result of a collaborative arrangement between the laboratories

of Dr Lucot and Dr Di Fulvio, which were responsible for the generation of data,

analysis and interpretation, and the maintenance, genotyping and characterization of

the NKCC2A colony of mice, respectively.

Finally I would like to thank my parents, my siblings, and my friends for being with

me and for their moral support.

54

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