Hydrogen Sulfides Actions in the Paraventricular Nucleus of the Hypothalamus

HYDROGEN SULFIDES ACTIONS IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS

Charline Sahara Khademullah

A thesis submitted to the Neuroscience Graduate Program

In conformity with the requirements for

The degree of Masters of Science

Queen’s University

Kingston, Ontario, Canada

(September, 2013)

Hydrogen sulfide (H2S) is a novel neurotransmitter that has been shown to influence cardiovascular function as well as other autonomic and endocrine functions by targeting a wide range of ion channels. Using whole-cell electrophysiology, I have investigated the potential role of H2S in the regulation of neuronal excitability in the paraventricular nucleus of the hypothalamus (PVN), which is a central relay centre for autonomic and endocrine function.

In current-clamp recordings, sodium hydrosulfide hydrate (NaHS), when perfused onto PVN slices at various concentrations (0.1, 1, 10, and 50 mM), elicited a concentration-dependent response relationship from the majority of recorded neurons, with almost exclusively depolarizing effects. Input resistance differences from baseline, and during the NaHS-induced depolarization, uncovered a biphasic response, implicating both a potassium (K+) and non-selective cation conductance.

+ In order to further investigate H2Ss effects on K conductances, we used both voltage- and current-clamp techniques to examine the effects of NaHS at either 1 or 10 mM on both the transient and sustained voltage-activated K+ currents in these neurons.

+ We applied TEA (10 mM) to isolate the transient/rapidly inactivating current (IA) and 4-

AP (5 mM) to isolate the sustained/delayed rectifier current (IK), and were able to show that both of these conductances were significantly reduced by H2S. Finally, we were able to demonstrate, using current-clamp, that when 4-AP and TEA+ were applied together with NaHS, they were able to completely eliminate the previously observed NaHS- induced depolarization, and the effects on membrane potential reversed to show a small hyperpolarization.

These data highlight the potential role of H2S as a critical modulator of the voltage-gated repolarizing conductances, IA and IK, which in turn regulate neuronal excitability within the PVN. This can have a large impact on the way neurotransmitters and hormones such as vasopressin, oxytocin, corticotrophin-releasing hormone, and thyrotrophin-releasing hormone are released from the PVN, which influence a wide range of neuroendocrine and autonomic functions such as cardiovascular function, fluid balance, and food intake.

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Acknowledgements

I would like to thank Dr. Alastair Ferguson for all of his guidance and the opportunities that he has provided me with. Thank you to Andrea Mimmee, Pauline

Smith, Rishi Malik, Markus Kuksis, Heather Hodgson and Stefanie Killen for all of your advice and assistance over the past two years. Finally, I would like to thank my parents and Devrim for all of their support and guidance.

Table of Contents

Abstract ...... ii

Acknowledgments ...... iv

Table of Contents ...... v

List of Figures ...... vii

List of Abbreviations ...... viii

Chapter 1: Introduction ...... 1

1.1 Hydrogen Sulfide ...... 1 Discovery and endogenous sources of hydrogen sulfide ...... 1 Concentrations of hydrogen sulfide and probes for detection in tissue ...... 4 Toxicity of hydrogen sulfide...... 4 Classification as a gasotransmitter ...... 5 1.2 Hydrogen sulfides potential mechanism for action ...... 7 Potassium channels ...... 7 Other Channels ...... 9 Physiological actions of hydrogen sulfide ...... 10 Hydrogen sulfides role in the brain ...... 11 1.3 The Paraventricular Nucleus of the Hypothalamus ...... 13 Anatomy ...... 13 Magnocellular neurons ...... 17 Parvocellular preautonomic neurons ...... 17 Parvocellular neuroendocrine neurons ...... 18 1.4 Research Aims ...... 19

Chapter 2: Depolarizing actions of hydrogen sulfide on hypothalamic paraventricular nucleus neurons ...... 20

2.1 Abstract ...... 21 2.2 Introduction ...... 22 2.3 Methods ...... 24 2.4 Results ...... 26 2.5 Discussion ...... 34

Chapter 3: Modulation of voltage-sensitive potassium channels by hydrogen sulfide alters neuronal excitability in the paraventricular nucleus of the hypothalamus ...... 38

3.1 Abstract ...... 39 3.2 Introduction ...... 40 3.3 Methods ...... 41 3.4 Results ...... 44 3.5 Discussion ...... 51

Chapter 4: Discussion ...... 55

4.1 Hydrogen sulfides actions in the PVN ...... 55

4.2 Consequence of H2S actions in the subpopulation of PVN neurons ...... 56 Vasopressin ...... 56 Oxytocin ...... 57 Corticotrophin-releasing hormone ...... 58 Thyrotrophin-releasing hormone ...... 59

4.3 H2S modulation of ion channels ...... 59 4.4 Summary and Conclusions ...... 61

References ...... 62

List of Figures

Figure 1. Enzymatic pathways for H2S production ...... 3

Figure 2. Anatomy of PVN and cell types ...... 15

Figure 3. Anatomy of PVN in relation to projection sites ...... 16

Figure 4. Hydrogen sulfide depolarizes PVN neurons ...... 28

Figure 5. Hydrogen sulfide has reproducible responses and effects on the various cell types in a similar manner ...... 30

Figure 6. Hydrogen sulfide has a concentration-dependent relationship ...... 31

Figure 7. Hydrogen sulfide-induced depolarization is biphasic...... 33

Figure 8. Current-voltage relationship in PVN neurons in response to NaHS application45

Figure 9. Hydrogen sulfide reduces the transient and sustained whole cell currents ...... 48

Figure 10. Hydrogen sulfide’s depolarizing actions in PVN neurons are abolished by 4- AP and TEA+ ...... 50

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List of Abbreviations

3-MST ...... 3-mercaptopyruvate transferase aCSF ...... artificial cerebral spinal fluid

ACTH ...... adrenocorticotropic hormone

AMP ...... adenosine monophosphate

ATP ...... adenosine-5'-triphosphate

BKCa ...... calcium-activated potassium channel

Ca2+ ...... calcium

CAT ...... cystine aminotransferase

CBS ...... cystathionine beta-synthase

Cl- ...... chloride

CNS ...... central nervous system

CO ...... carbon monoxide

CORT ...... corticotrophin

CRH ...... corticotrophin releasing hormone

CSE ...... cystathionine gamma-lyase

DA ...... dopamine

GnRH ...... gonadotrophin-releasing hormone

H2S ...... hydrogen sulfide

HPA ...... hypothalamic-adrenal-stress-axis

IA ...... rapidly inactivating potassium current

IK ...... delayed rectifier potassium current

K+ ...... potassium

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KATP ...... ATP-sensitive potassium channel

KCl...... potassium chloride

KV ...... voltage-gated potassium channel

MNC ...... magnocellular

Na+ ...... sodium

NaHS...... sodium hydrogen sulfide

NE ...... neuroendocrine

NMDA ...... n-methyl-d-aspartate

NO ...... nitric Oxide

NSAID ...... nonsteroidal anti-inflammatory drugs

PA ...... preautonomic

PVN ...... paraventricular nucleus of the hypothalamus

RVLM ...... rostral ventrolateral medulla

SAMe ...... s-adenosyl methionine

SON ...... supraoptic nucleus

TRH ...... thyrotrophin-releasing hormone

TRP ...... transient receptor potential channel

TRPA1 ...... transient receptor potential cation channel subfamily A member 1

TRPV1 ...... transient receptor potential cation channel subfamily V member 1

TSH ...... thyroid stimulating hormone

Chapter 1: Introduction

1.1 Hydrogen Sulfide

Discovery and endogenous sources of hydrogen sulfide

Hydrogen sulfide (H2S), a gas best known for its foul odor similar to that of rotten eggs, has long had a reputation for being toxic. This has stemmed mainly from its involvement in the world’s largest mass extinction, the Permian-Triassic Extinction [1]. Many researchers believe that this extinction is what led to the mammalian ability to endogenously produce and use the gas in the absence of oxygen for cellular respiration through mechanisms in the mitochondria [2].

The endogenous production of H2S is a result of the desulfhydration of the amino acids L- cysteine and/or homocysteine [3]. These amino acids are then catalyzed by one of three pyridoxal-5’-phophate-dependent enzymes: cystathionine gamma-lyase (CSE), cystathionine beta-synthase (CBS), and 3-mercaptopyruvate transferase (3-MST) (Fig. 1) [4–7]. CSE has been shown to be primarily expressed in cardiovascular tissues, including the aorta, pulmonary, mesenteric and tail arteries, as well as the portal vein [8–12]. CSE-knockout mouse models have provided evidence to suggest that a decrease in the production of H2S in various tissues [8,11].

The same study also found evidence of an increased risk of hypertension in these knockout animals, implying the importance of this enzyme in the maintenance of cardiovascular function

[8,11]. Kimura and colleagues [13], have also identified CSE as a regulator of cerebral blood flow through its activation of ATP-sensitive potassium (KATP) channels in smooth muscle.

Unlike CSE, CBS and 3-MST have been localized primarily to the central nervous system (CNS)

[14]. While both CSE and CBS have been found to be expressed predominantly in the cytosol, 3-

MST is the only H2S-catalyzing enzyme that has been found to be expressed in the mitochondria of neurons [15,16]. It is well-known that the three H2S catalyzing enzymes are responsible for the

1 production of H2S within different tissues in the body; however, what is less defined is how much endogenous H2S is produced.

Figure 1. Enzymatic pathways for H2S production.

This schematic illustrates the three enzymatic (CBS, cystathionine beta-synthase, CSE, cystathionine gamma;lyase, and the CAT, cysteine aminotransferase/ 3-MST, 3- mercaptopyruvate transferase) pathways from cystine that catalyze H2S.

Concentrations of hydrogen sulfide and probes for detection in tissue

Several studies have reported a wide range of endogenous basal H2S concentrations in mammalian tissue, with the general consensus being between 50-160 µM [17]. The variation regarding the range of physiologically acceptable concentrations found in tissue is the result of the lack of an accurate assay to measure the gas. Since H2S is a gas, the main difficulty in its measurement has come from the inability to contain it within the tissue while maintaining the tissues integrity [18]. Historically, H2S has been measured using a variety of probes, including colorimetric assays using methylene blue, sulfide-specific ion-selective electrodes, flame photometric analyses, and even high performance liquid chromatography, all of which require tissue homogenization (reviewed in [18]). It wasn’t until recently that a method was developed that didn’t damage the tissue in question [18–22]. This method used an azamacrocyclic copper fluorescent complex to selectively detect H2S within live cells and intact tissue samples, and therefore offers a strategy to observe the endogenous production of H2S and its concentrations while maintaining tissue integrity [18–22]. In order for H2S to be maintained at non-toxic concentrations in the body, it controls the activity of all three of its catalyzing enzymes through a negative feedback mechanism [9,23]. Once metabolised to sulfate, it is commonly excreted in the form of a free or conjugated form of sulfate through the body’s primary filtration organ, the kidney [24,25]. Failure to maintain physiological concentrations of H2S, or excessive exogenous exposure to the gas, can result in a variety of negative outcomes [24,25].

Toxicity of hydrogen sulfide

H2S exerts it toxic effects primarily through its actions within the mitochondria [26].

Evidence suggests that it forms a complex bond with iron in the mitochondria which then results

4 in the inhibition of cytochrome c oxidase [27]. This in turn halts adenosine triphosphate (ATP) production, thus preventing cellular respiration [27,28]. Symptoms of H2S toxicity in humans can range from eye irritation to olfactory nerve paralysis, to respiratory paralysis and even death at higher concentrations [28,29]. A study observing the effects of long-term H2S exposure in neonatal rats found that there were increased levels of both serotonin and norepinephrine in regions of the brain, specifically, the cerebellum and the frontal cortex [9,30]. Prolonged exposure to toxic levels of H2S has also been implicated in the malfunction of respiratory nuclei within the brainstem, which in turn result in respiratory paralysis (reviewed in [31]).

Classification as a gasotransmitter

More recently, H2S has been observed as having non-toxic effects and even in modulating the release of neurotransmitters at physiologically relevant concentrations [32]. This has led to its classification as a gasotransmitter, along with nitric oxide (NO) and carbon monoxide (CO) [9].

Wang, [9], has outlined five criteria that is required of a gasotransmitter and argues that H2S meets all the criteria [9].

The criteria are as follows:

 Does it have a gaseous composition?

 Can it easily and readily permeate the cellular membrane?

 Can it be produced both endogenously and enzymatically?

 Does it have specific functions in specific tissues at physiologically relevant

concentrations?

 And finally, does it have cellular and molecular targets to act on?

While H2S can be considered as the third gasotransmitter amongst NO and CO, its production and effects in mammalian tissues are unique [13]. As mentioned, H2S is derived from

L-cysteine, while NO is a product of citrulline and/or arginine which is then synthesized by NO synthase [33–35]. CO, on the other hand, is produced as a result of the catalyzation of heme to billverdin from heme oxygenase [36,37]. While not much is known regarding CO production as a result of neuronal activation, H2S production has been shown to be increased through calcium

(Ca2+)- and calmodulin-mediated pathways in response to neuronal excitation [38]. NO release, on the other hand, has been shown to be enhanced following the activation of N-methyl-D- aspartate (NMDA) receptors by L-glutamate as a result of neuronal excitation [39,40]. All three gasotransmitters have been implicated in learning and memory processes through the modulation of long-term potentiation in the hippocampus. However, it appears as though it is only H2S that depends on NMDA receptors to do so [39–43]. Another process that all three gasotransmitters have been implicated in is that of vasorelaxation and constriction, although they do so through different pathways [12,42–44]. Studies have even suggested the co-modulation of two or more of the gasotransmitter in some processes [12]. A study by Hosoki and colleagues, [12], found that

H2S enhanced the vasorelaxant effect of sodium nitroprusside, a NO donor. In the hypothalamus, H2S has been shown to stimulate KCl-dependent corticotrophin-releasing hormone (CRH) secretion, which can lead to many adverse effects, most notably, the abnormal regulation of the stress axis [45,46]. CO, on the other hand, has been implicated in the inhibition of K+-stimulated oxytocin and vasopressin release, which can lead to changes in feeding and drinking behaviours, as well as cardiovascular function [45–47]. To better understand how H2S modulates the release of neurotransmitters and produces its unique effects throughout the brain

6 and body, attention needs to be paid on how it is specifically affecting ions such as Ca2+ and K+, which in many cases directly control neurotransmitter release.

1.2 Hydrogen sulfides potential mechanism for action

It has been well-documented that H2S produces and regulates many of its physiological functions through various signaling pathways and produces changes in membrane excitability.

One suggested mechanism for H2S effects is its actions on a group of target proteins, more specifically, ion channels [8,14,48]. In the central nervous system, H2S has been implicated in altering K+ and Ca2+ levels, which are known to be involved in the regulation of cellular apoptosis, caspase activation, mitochondrial activity, and neuronal excitation in association with glia [49–51]. Other groups have linked H2Ss responses to a variety of nonselective cation channels, showing that H2S can produce its effects through numerous transient receptor potential

(TRP) channels to produce anti-inflammatory responses throughout the body [52]. H2S has also

2+ + + - been observed to have effects on Ca -activated K (BKCa), Na , and chloride (Cl ) channels

[53,54]. In order to better asses H2Ss therapeutic potential, the mechanisms by which it is modulating these ion channels must be better understood.

Potassium channels

One of the first studies to observe neuronal responses to H2S using whole-cell patch clamp techniques found that H2S was able to produce a concentration-dependent hyperpolarization in the membrane potential of dorsal raphe neurons [55]. The authors attributed this hyperpolarizing response to K+ channels, since in the presence of barium and/or cesium, K+ channel blockers, the

+ response was occluded [55]. Since then, the modulation of K channels, specifically, KATP

7 channels, has been a primary focus of H2Ss mechanism of action. Many studies since have found that its activation by H2S is what leads to vasodilation [53,56]. H2Ss activation of these ion channels has also been suggested to contribute to myocardial protection, protection against glutamate toxicity, and the regulation of insulin secretion and inflammation [11,14,53,56–59].

H2S, both exogenous and endogenous, produces vasodilation by activating KATP channels, which in turn causes a hyperpolarization of smooth muscle cells [60]. H2S activation of KATP channels in the rostral ventro-lateral medulla (RVLM) has also been shown to inhibit sympathetic vasomotor tone [61]. A study by Jiang and colleagues, [62], investigated how these KATP channels were modulated by H2S. KATP channels are generally made up of four inward-rectifier subunits (KIR) and four sulfonylurea receptor sites (SUR). In the absence of the SUR receptor sites, KIR subunits were unaffected by H2S, suggesting that it is the SUR sites that H2S specifically acts upon [63]. Furthermore, H2S has been shown to influence these SUR sites by sulfhydration, which was found to produce changes in gastric motility [64].

+ Another well-studied K channel with respect to H2S has been the BKCa channel. Telezhkin and colleagues [65], demonstrated that H2S produced inhibitory effects on BKCa channels in both

HEK293 cell lines and the rat carotid body. Another study supporting this finding provided evidence, in mice, of carotid body excitation in response to H2S, suggesting BKCa inhibition [54].

Hypoxia has been shown to cause an inhibition of BKCa channels, while CSE inhibitors have been found to occlude this effect and increase activation of these channels [54]. This provides evidence to suggest that H2S may play a role in the activation of BKCa channels in hypoxic conditions. Although the inactivation of these channels has been well documented, Jackson-

Weaver and colleagues, [66], found that H2S was actually responsible for the activation of BKCa

8 channels in the small mesenteric arteries, which was inhibited by iberiotoxin, a BKCa channel inhibitor. While it remains unknown as to how H2S produces opposite effects on the same ion channel, possible explanations may include differences in applied concentrations, differences in animal models and/or cell lines, and/or differences in regions tested. Evidence of this has been observed in the mesenteric arteries where H2S has been shown to produce vasodilation at high concentrations (1 mM), while producing vasoconstriction at low concentrations (10 µM) [67].

+ Voltage-sensitive K channels (KV) have also been implicated in H2S’s actions. Feng and colleagues [68], showed that NaHS reduced not only the expression, but also inhibited the KV channel, the delayed rectifier current (IK) in trigeminal ganglion neurons. A similar effect was observed in a study using cardiomyocytes, where H2S inhibited both the slow and rapid outward rectifier K+ currents [69].

Other Channels

+ While H2S has been shown to modulate a number of different K channels, as outlined above, numerous other ion channels have been shown to be responsive to, and modulated by, H2S. H2S has been implicated in the activation of TRP channels, specifically TRPV1 in the airway, and

- TRPA1 in the bladder [52,70]. Cl channels have also been shown to be activated by H2S, which has been linked to downstream protective effects that can save the cell from oxidative stress [53].

In both cerebellar granule neurons and SH-SY5Y cell lines, Ca2+ levels have been shown to be

2+ increased in response to H2S application [71,72], suggesting the activation of Ca channels,

2+ while in cardiac myocytes, Sun and collogues [73], found that H2S inhibited L-type Ca channels.

Physiological actions of hydrogen sulfide

Since the identification of H2S as a gasotransmitter and its effects on ion channels, considerable research has been focused on attempts to identify specific physiological roles for this signalling molecule. Following Abe and Kimura’s [42], identification of H2S as a neuromodulator, it has been shown to influence a wide range of other physiological processes, including blood vessel relaxation, arterial contraction, neurotransmission, inflammation, cardioprotection, neuroprotection, and regulation of the hypothalamic stress axis

[28,56,60,61,63,74,75]. Although it is known that H2S acts on a variety of ion channels to produce these physiological effects, the mechanisms of such actions remain largely unknown.

Perhaps the best understood effect of H2S to date has been its effects on vasoconstriction and relaxation. A recent study showed that the application of a variety of sodium hydrogen sulfide

(NaHS - H2S donor) doses (10 µM to 1 mM) to the superior mesenteric artery produced vasoconstricting effects at low concentrations, while at high concentrations; NaHS caused vasorelaxation [76]. Another study conducted by Zhao and colleagues [56], showed a decrease in blood pressure in rats (12-30 mmHg) when H2S was given through an intravenous bolus injection. Enzymes associated with H2S production have also provided evidence suggesting that

H2S plays a significant role in the mammalian cardiovascular system. Yang and colleagues [11], showed an increase in blood pressure in CSE knockout mice, indicating a role for endogenous

H2S in blood pressure regulation. Combining H2S with non-steroidal anti-inflammatory drugs

(NSAIDs) has also been an interesting focus of H2S research. Recent studies conducted by

Wallace and colleagues [77,78], demonstrated that, in the presence of H2S, the deleterious effects of NSAIDs, which include gastric ulcerations and bleeding, were supressed. This finding has led

10 to the combination of NSAIDs and H2S as a potential therapeutic measure for individuals that are required to take pain medication on a long term basis [77–79].

Hydrogen sulfides role in the brain

While there has been a lot of emphasis on the role that H2S plays in the cardiovascular system, there has also been a great expansion in research in recent years regarding its role and function in the CNS. While it is well-known that CBS is the main enzyme responsible for the production of H2S in the brain, a recent study by Shibuya and colleagues [80], reported H2S production in the brain of the CBS knockout mouse, suggesting that there was in fact another enzyme responsible for the production of H2S within the CNS. This enzyme was identified as 3-

MST. One of the first studies looking at H2S function in the brain was carried out by Abe and

Kimura [42], in which they observed that NaHS (10 - 160 µM) facilitated the induction of long- term potentiation in rat hippocampal slices by enhancing NMDA-induced inward currents. This finding led to further investigations that suggested that NMDA receptors were possibly activated by the activation of a cyclic adenosine monophosphate (AMP)-dependent kinase pathway [42].

With that in mind, another study found that NaHS (1 - 100 µM) increased cyclic AMP production in primary cultured rat cerebral and cerebellar neurons in rat neuronal and glial cell lines [9,81]. This resulted in an increased sensitivity to NMDA of NMDA receptors [81]. In short, researchers have been able to show that NaHS enhances the response of NMDA receptors to the neurotransmitter glutamate in neurons. This in turn leads to the increase of neuronal excitation and enhances learning and memory functions [42]. Other studies of H2Ss role in the brain have also found that the CBS gene was underrepresented in children with high IQ’s, further suggesting a possible effect in cognitive function [13,82–84]. There has also been evidence to

11 suggest that the H2S and NMDA relationship may not be the only mechanism to enhance neuronal excitation. It has been observed that H2S-induced increased neuronal excitation may also be as a result of its ability to increase Ca2+ influx in astrocytes that surround and act to regulate synapses to modulate synaptic activity [50].

While effects of H2S on neuronal excitability have been extensively explored within the hippocampus, there are other areas within the brain that H2S has been found to influence neuronal function. Early research in the dorsal raphe showed that NaHS hyperpolarized and reduced the input resistance of single neurons in a concentration-dependent manner [55]. A recent study looking at rat hypothalamic explants showed that while NaHS was without effect on basal CRH release [45,46], it was shown to inhibit KCl-stimulated CRH release in a concentration-dependent manner [45,46]. This study also showed that S-adenosylmethionine

(SAMe - an indirect H2S precursor) application to hypothalamic explants also caused an inhibition to the KCl-stimulated CRH release [45,46]. Furthermore, SAMe given twice daily in vivo caused a reduction in circulating corticotrophin (CORT) in rats [45,46]. H2S has also been implicated in protecting neurons from oxidative stress by normalizing glutathione levels that were previously decreased by glutamate and hydrogen peroxide [28,85]. H2S has also been linked to CNS diseases such as Parkinson’s disease, Huntington’s, and even Down syndrome

[86,87]. The CBS gene was found to be encoded on chromosome 21q22.3 [86–88], a region associated with Down syndrome, leading to the hypothesis that H2S may be involved in the cognitive dysfunction aspect associated with Down syndrome. This study, along with others, provided evidence to suggest that while physiologically relevant concentrations of H2S cause

12 neuronal excitation, concentrations outside of this range may act to supress neuronal activity

[86,87].

In addition to the work described above on CRH, recent work showed that microinjections of

H2S into the PVN produced cardiovascular changes, suggesting that the PVN may act as a site of action for H2S [89]. They observed that the application of NaHS (1 - 4 nM) in the PVN produced an increase in renal sympathetic nerve activity, mean arterial blood pressure, heart rate and cardiac sympathetic afferent reflex [89]. Another group using Wistar Kyoto rats found evidence of the presence of the H2S-catalyzing enzymes in the PVN, suggesting that it may be a site of action. However, the administration of H2S (0.1 – 1 nM) in the PVN was unable to produce any effect on any measured cardiac function, including lumbar sympathetic nerve activity, heart rate and blood pressure [90]. The authors suggested that the absence of an effect may have been a result of low exogenous H2S concentrations applied [90]. Despite the inconsistences, these studies do suggest that H2S may play a critical role in the modulation and regulation of endocrine and autonomic function through its actions in the PVN.

1.3 Paraventricular nucleus of the hypothalamus

Anatomy

The PVN is a small bilateral nucleus that occupies approximately 1/3 mm3 on either side of the third ventricle in the rat and contains approximately 10,000 neurons [91] (Fig. 2). It can be divided into 8 populations of cells. Of these 8 populations, 3 can be classified as magnocellular

(MNC), and include anterior, medial and posterior regions, while the other 5 populations can be considered parvocellular neurons, which can be further divided into periventricular, anterior,

13 medial, dorsal, and lateral [92–94] (Fig. 3). The medial and lateral regions of the parvocellular neurons are referred to as parvocellular neuroendocrine (NE) neurons while the periventricular, anterior, and dorsal regions are referred to as parvocellular preautonomic (PA) neurons [91] (Fig.

3). Evidence has been provided of the PVN in the regulation of both autonomic and endocrine control in the brain [95,96]. It has been implicated in the regulation of cardiovascular function

(including blood pressure control), energy homeostasis, the stress axis, immune response, feeding and fluid balance, body temperature, as well as sexual and maternal behaviours

[91,97,98]. All three cell types, the MNC, PA, and NE neurons, have distinct projection sites in the brain and therefore have unique roles [91–94]. Immunohistochemical studies have demonstrated two main distinguishing features of the PVN in comparison to other regions in the hypothalamus [91]. The first feature is that it contains almost all of the oxytocin and vasopressin secretory neurons, with the rest of these neurons contained within the supraoptic nuclei (SON)

[91]. The second distinguishing feature is that it produces many neuroactive substances: more than 30 transmitters and/or neuroactive peptides have been identified within the PVN [91]. This serves as evidence for the critical role that the PVN plays as a central relay centre for autonomic and endocrine functions.

Figure 2. Anatomy of PVN in relation to projection sites.

This illustration of the brain outlines the projection pathways from the PVN to the brainstem, spinal cord, median eminence, and posterior pituitary. The insets list some of the physiological effects that result from the secretion of oxytocin (OXY), vasopressin (VP), corticotrophin- releasing hormone (CRH), and thyrotrophin-releasing hormone (TRH) at these projection sites.

Figure 3. Anatomy of PVN and cell types.

This illustration shows a slice of the brain in which PVN lies on either side of the third ventricle. The inset shows a close-up of the PVN and its subpopulations. The table in the inset illustrates the cell-types electrophysiological phenotype in response to a hyperpolarizing pulse and the region of the brain that that population of cells project to.

Magnocellular neurons

The magnocellular region of the PVN contains cells that are generally larger

(approximately 25 microns) than other cells in the PVN and these cells have been found to predominantly secrete either vasopressin or oxytocin [91]. The region is made up of primarily bipolar and multipolar neurons with 2 to 3 simple dendrites that project medially and ventralaterally to the posterior pituitary [91] (Fig. 2). Here, oxytocin has been implicated in uterine contractions, lactation regulation, reduction in heart rate and mean arterial pressure, and acts to regulate the hypothalamic-pituitary-adrenal (HPA) stress axis [94,96,99]. Vasopressin has been implicated in vasoconstriction and in the regulation of fluid homeostasis, and as a result, energy homeostasis (reviewed in [100,101]). Another distinguishing feature of magnocellular neurons is that they contain many soma-to-soma connections through gap junctions [102]. It has been suggested that some stimuli use these soma-soma interactions to induce the release of oxytocin and vasopressin [91,102]. These interactions explain how pulsatile release of oxytocin and phasic bursting patterns are achieved in vasopressin secretory neurons [91]. These neurons can also be distinguished by their electrophysiological profiles/phenotype [103–105]. They are characterized by a large transient outward rectification followed by a fast inactivating A-type current [103,105,106]. In current-clamp, this manifests as a delay to first spike following a hyperpolarizing pulse [103] (Fig. 3).

Parvocellular preautonomic neurons

While preautonomic neurons are not quite as large in size as magnocellular neurons, they share many attributes. Similar to MNC neurons, PA neurons secrete both vasopressin and oxytocin, along with CRH and thyrotrophin-releasing hormone (TRH) [91,105]. They also have

17 fairly simple dendritic branches, much like the MNC neurons [91,105]. These dendritic branches usually branch out once or twice [91]. Bipolar cells here give rise to short, vertically orientated dendrites, while long primary dendrites tend to be horizontal in orientation [91]. The axons of theses neurons project caudally into the brain stem and spinal cord and synapse on preganglionic cells to affect both the sympathetic and parasympathetic autonomic systems (Fig. 2), thus regulating cardiovascular function [107]. Local axons from this region have also been shown to project to other parvocellular and MNC regions, which have been suggested to generate some of the outputs [91]. The electrophysiological phenotype of these neurons includes a robust low- threshold Ca2+ transient which can, in some instances, generate a burst of action potentials; these neurons also show a strong inward rectification [103,106]. In current clamp, these neurons display a spike immediately following a hyperpolarizing pulse [103] (Fig. 3).

Parvocellular neuroendocrine neurons

The NE neurons have been localized to the medial and lateral regions of the PVN [91]. They primarily project to the median eminence directly through to the external lamina [91,108,109]

(Fig. 2). These neurons secrete CRH, TRH, and vasopressin to the hypophyseal portal blood system [91,108,109]. CRH, along with vasopressin, acts to regulate adrenocorticotropic hormone

(ACTH) production, while, TRH regulates both prolactin and thyroid stimulating hormone

(TSH) [91,107,110]. These hypophysiotropic hormones then regulate the downstream production of cortisol, glucocorticoids, dopamine (DA), somatostatin and gonadotropin-releasing hormone

(GnRH) [111–115]. Electrophysiologically, these neurons display a low-threshold Ca2+ potential that tends to be relatively small, which usually does not lead to spike generation [103,116,117].

In current clamp, these neurons can be separated from the MNC and PA neurons due to the lack

18 of any specific features, such as a delay to baseline or an immediate spike in response to a hyperpolarizing pulse [103] (Fig. 3).

1.4 Research Aims

While it is known that H2S affects cardiovascular function by acting in the PVN, how it does so is relatively unknown. It is clear with regards to the different cell types within the PVN that if

H2S acts to modulate these neurons, it would have a large impact on many autonomic and endocrine behaviours. Therefore, it is important to define H2Ss action within the PVN and uncover the mechanism by which it acts through to produce its effects. In light of the recent literature which has provided some evidence of H2Ss actions in PVN, the current study was undertaken to address the following research questions:

 Does H2S affect the membrane excitability of PVN neurons?

 Does H2S have different actions among the specific cell types within the PVN?

 What is H2S’s role in ion channel regulation in the PVN?

We will also discuss what physiological consequences may result from these findings with regards to H2S’s actions in the PVN.

Chapter 2: Depolarizing actions of hydrogen sulfide on hypothalamic paraventricular nucleus neurons C. Sahara Khademullah

Department of Biomedical and Molecular Science, Queen’s University, Kingston, Ontario,

Canada

Alastair V. Ferguson

Department of Biomedical and Molecular Science, Queen’s University, Kingston, Ontario,

Canada

I acknowledge that this work has been published in the journal of PLoS ONE 8(5): e64495. doi:10. 1371/journal.pone.0064495, 2013

2.1 Abstract

Hydrogen sulfide (H2S) is a novel neurotransmitter that has been shown to influence cardiovascular function as well and corticotrophin hormone (CRH) secretion. Since the paraventricular nucleus of the hypothalamus (PVN) is a central relay center for autonomic and endocrine functions, we sought to investigate the effects of H2S on the neuronal population of the

PVN. Whole cell current clamp recordings were acquired from PVN neurons and sodium hydrosulfide hydrate (NaHS) was bath applied at various concentrations (0.1, 1, 10, and 50 mM).

NaHS (1, 10, and 50 mM) elicited a concentration-response relationship from the majority of recorded neurons, with almost exclusively depolarizing effects following administration. Cells responded and recovered from NaHS administration quickly and the effects were repeatable.

Input differences from baseline and during the NaHS-induced depolarization uncovered a biphasic response, implicating both a potassium and non-selective cation conductance. The results from the neuronal population of the PVN shed light on the possible physiological role that

H2S has in autonomic and endocrine function.

2.2 Introduction

Hydrogen sulfide (H2S) is produced endogenously in mammals in a variety of different cells types from the brain, blood, skin, liver, and kidney [9]. It is considered to be the third gasotransmitter, along with carbon monoxide (CO) and nitric oxide (NO) [9,45]. Since it was first observed to have non-toxic physiological effects in the brain by Abe and Kimura [42], the search to uncover alternative therapeutic roles for H2S has taken off. It has been implicated in the induction of long-term potentiation in the hippocampus, blood pressure regulation, inflammation, protection against oxidative stress, and the neuroendocrine stress axis [24,25].

H2S is produced by the action of one of three enzymes found in the mammalian body, cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), and 3-mercaptopyruvate sulfur-transferase (3-MST) [118], all of which which biosynthesize L-cysteine into H2S [65].

While not all aspects of endogenous H2S production are well understood, physiological sources of H2S include its production by enzymatic action, as well as release in response to physiological stimuli from stored pools in the cytosol and/or mitochondria [119]. These three enzymes have been suggested to be differentially distributed throughout the body, with CBS being highly expressed in brain tissue [42,120], CSE in the liver, kidney, thoracic aorta, ileum, gastrointestinal tract, portal vein and uterus [12], and 3-MST localized to the mitochondria [80]. A source of non- enzymatically produced H2S appears to be inorganic polysulfides in red blood cells [121]. This ability of red blood cells to convert polysulfides into H2S allows for it to be delivered through dietary means [122]. While the physiological sources of H2S remain poorly understood, there are a few mechanisms suggested in the literature.

Although difficult to measure endogenously, H2S concentrations in the brain have been estimated to be approximately 8 µM [123], while plasma concentrations have been suggested to be as high as 160 µM [9]. At these physiologically relevant concentrations, H2S has been found to play a role in vasoconstriction, as well as vasodilation. d’Emmanuele di Villa Bianca and colleagues [67], showed that the application of a variety of NaHS doses (10 µM – 1 mM) to the superior mesenteric artery produced vasoconstricting effects at low concentrations, while at high concentrations, NaHS caused vasodilation. Exogenous H2S has been shown to reduce blood pressure in various rat models [124], while Yang and colleagues [11], showed increases in blood pressure in CSE knockout mice. Studies observing H2S effects in the hippocampus showed that

H2S enhanced NMDA receptor currents and facilitated hippocampal long-term potentiation

(LTP) [81]. It was also found that NaHS abolished 6-hydroxdopamine (6-OHDA)–induced cell death and, as a result, rescued SH-SY5Y cells [125]. In the substantia nigra compacta, administration of NaHS eliminated 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP)– induced dopaminergic neuronal loss [126]. In hypothalamic tissue, NaHS induces a concentration dependent decrease in the release of KCl stimulated corticotrophin-releasing hormone (CRH)

[45]. Various ion channels have been implicated in these physiological responses to H2S,

+ including ATP-sensitive potassium channels (K ATP), large conductance calcium activated potassium channels (BKCa), both T-type and L-type calcium channels, as well as chloride, transient receptor potential (TRP), and sodium channels (Nav 1.5) [53,121,127–129].

The paraventricular nucleus of the hypothalamus (PVN), located against the third ventricle within the hypothalamus, is a critical autonomic control center of the hypothalamus

[95,96]. More specifically, it has been implicated in central cardiovascular and volume control, including blood pressure regulation [97]. The PVN is also known for its role in the activation of

23 the hypothalamo-pituitary-adrenocortical (HPA) axis through the secretion of the corticotrophin- releasing hormone (CRH) [130]. The PVN consists of three different cell types: magnocellular neurons (MNC), parvocellular preautonomic neurons (PA), and parvocellular neuroendocrine neurons (NE) [103,105]. The magnocellular neurons project to the posterior pituitary gland and secret oxytocin (OT) or vasopressin (VP). The parvocellular preautonomic neurons project to the brain stem and the spinal cord and secret OT, VP, CRH, or thyrotrophin-releasing hormone

(TRH). Lastly, the parvocellular neuroendocrine neurons project to the median eminence and secret CRH and TRH [131]. Recent evidence suggests that the PVN may act as a site of action for H2S. Microarray analysis has demonstrated that the three enzymes necessary to catalyze H2S are all present in the PVN and more recently, Gan and colleagues [89], found that microinjections of H2S into the PVN produce cardiovascular changes, although the physiological mechanisms through which H2S concentrations in the PVN might be regulated remain to be established. The purpose of the present study was to assess the role of H2S in the PVN and the downstream physiological effects it may have using patch-clamp recordings.

2.3 Methods

Slice preparation

Male Sprague-Dawley rats (Charles River, Quebec, Canada) aged 21-28 days were decapitated according to the regulations established by the Canadian Council on Animal Care and approved by the Queen’s University Animal Care Committee. The brains were removed and placed in ice-cold solution composed of (in mM): 87 NaCl, 2.5 KCl, 25 NaHCO3, 0.5 CaCl2,

7 MgCl2, 1.25 NaH2PO4, 25 glucose, 75 sucrose bubbled with 95% O2/5% CO2. The region of the hypothalamus containing the PVN was isolated, and 300 µm coronal sections were cut using

24 a vibratome (Leica, Nussloch, Germany). Slices were then incubated at 32ºC for a minimum 1 hour prior to recording in carbogenated artificial cerebrospinal fluid (aCSF) containing (in mM):

126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 10 glucose saturated.

Electrophysiology

Slices were mounted onto the stage of the recording chamber, which was continuously perfused at a flow rate of 2-3 ml/min with carbogenated aCSF at approximately 32ºC. An upright differential interference contrast microscope (Scientifica, Sussex, United Kingdom) at

40x magnification was used to visualize the neurons. Using a Sutter Instruments P97 flaming micropipette puller, borosilicate glass electrodes (World Precision Instruments, Sarasota,

Florida, USA) were pulled and filled with an intracellular solution composed of (in mM): 125 potassium gluconate, 10 KCl, 2 MgCl2, 0.1 CaCl2, 5.5 EGTA, 10 HEPES, 2 NaATP (pH 7.2 with KOH). Electrodes were optimized to have a resistance of 3-5 MΩ. Whole cell recordings were made with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, California, USA) and acquired at 10 kHz, and filtered at 2.4 kHz using a Micro 1401 interface. Once a high resistance seal was obtained, whole cell configuration was accomplished by breaking through the cell membrane to gain access to the internal contents of the cell through the application of a brief period of negative pressure. Only neurons with a minimum spike amplitude of 60mV (range 60-

100mV), input resistance of >400MΩ (range 400-1100MΩ), and series resistance of < 20MΩ

(range 8-20MΩ) were included in our subsequent analysis. The data was recorded in Spike2 software for offline analysis (Cambridge Electronic Devices, Cambridge, UK). PVN neurons were characterized as MNC (delayed return to baseline in response to a hyperpolarizing pulse),

PA (calcium spike in response to a hyperpolarizing pulse), or NE (neither of the above) using a

25 standard current pulse protocol prior to the application of NaHS [103]. Following a minimum

100s baseline recording period, specific known concentrations of sodium hydrosulfide hydrate

(NaHS) were applied to slices through a bath perfusion system. The response to NaHS was determined by comparing the mean membrane potential of the neuron before and after application and averaged across 100 s time periods. A response was considered significant if the change in membrane potential after NaHS application was at least twice the amplitude of the standard deviation of the baseline membrane potential during 100 seconds prior to application.

Descriptive statistics were also used to describe mean group data as well as the standard error of the means in all such groups. The recorded membrane potential was adjusted to correct for the calculated junction potential (+15 mV).

Chemicals and drugs

All salts used to prepare the slicing solution, aCSF, and the internal recording solutions, as well as NaHS, were obtained from Sigma-Aldrich Pharmaceuticals (Oakville, Ontario,

Canada).

2.4 Results

Hydrogen sulfide influences the excitability of PVN neurons

Hydrogen sulfide was bath applied to a total of 65 PVN neurons during current-clamp recordings. The majority (n=52, 80%) of neurons were responsive, with (96%) of responsive cells depolarizing, which was characterized by a rapid rise in membrane potential immediately after NaHS entered the bath, followed by maintenance of the effects for the duration of donor application, and a rapid recovery to baseline membrane potential associated of replacement of

26 bath solution with aCSF (Fig.4). The depolarizations were usually accompanied by an increase in firing frequency during the initial rise in membrane potential (Fig.6B, C, and D). The effects of

NaHS were reproducible, in that when the same concentration was applied for a second time while recording from the same neuron a similar second response was observed as illustrated in

Figure 5A. The remaining 2 (4%) cells influenced by NaHS·XH2O responded with a rapid, long lasting hyperpolarization followed by a return to baseline.

Hydrogen sulfide exerts similar effects on MNC, PA, and NE PVN neurons

We also examined the effects of hydrogen sulfide on PVN neurons differentially classified according to their electrophysiological fingerprints as MNC, PA or NE. NaHS influenced the membrane potential of PA neurons (73% depolarize, 27% no response n = 33),

MN neurons (80% depolarize, 13% hyperpolarize, 7% no response, n = 15) and NE neurons

(79% depolarize, 21% no response, n = 14) (Fig.5B). After categorizing the neurons according to these criteria, it appears that NaHS does not influence the distinct cell types in a different manner and therefore all cell types were grouped together for analysis.

Figure 4. Hydrogen sulfide depolarizes PVN neurons. Traces illustrate the depolarizing effects of NaHS application on PVN neurons. A) Current clamp recording trace illustrating a depolarizing response to 1 mM NaHS. Trace B) shows a current clamp recording illustrating a depolarizing response to 10 mM NaHS. Bar graph C) illustrates the various responses to NaHS (0.1 – 50mM) of PVN neurons (80%, n = 52/65 depolarized, 20%, n = 13/65 showed no response, and 3%, n = 2/65 hyperpolarized). Bar graph D) shows the percentage of PA (73%, n = 24/33), MNC (80%, n = 12/15), and NE (79%, n = 11/14) neurons that depolarized.

Hydrogen sulfide affects PVN neurons in a concentration-dependent manner

The depolarizing effects of NaHS were also found to be concentration dependent as illustrated in Figure 6E. Bath application of 50 mM NaHS elicited a depolarizing (mean 13.46 ±

1.62 mV) response in 100% (n=25) of the recorded neurons, followed by a return to baseline membrane potential which usually occurred within 60s as illustrated in Figure 6D. We also examined the effects of lower concentrations of NaHS, with 10 mM influencing 79% (n=11/14) of neurons tested with 10 of 11 showing depolarizations (mean 10.38 ± 1.34mV) and similar recovery times, as illustrated in Figure 6C and E, while the 1 cell which hyperpolarized (-8.53 mV) showed a much longer recovery time of nearly 10 minutes. A similar pattern of responsiveness was seen following 1 mM NaHS, with 83% (n=15/18) of neurons responding, the majority (n=14) of neurons showing much smaller depolarizing effects (3.00 ± 0.28mV), and similar short recovery times which can be seen in Figure 6B. Again, 1 cell hyperpolarized (-8 mV) again with a longer recovery time of 3 minutes. Bath application of 0.1 mM NaHS failed to elicit a response in 7 of the 8 cells tested as illustrated in Figure 6A, although 1 neuron responded with a small depolarization (1.7 mV). Overall, the vast majority of the PVN neurons tested responded with depolarization and the concentration dependent nature of these effects are summarized in Figure 6E.

Figure 5. Hydrogen sulfide has reproducible responses and effects the various cell types in a similar manner. A) Current clamp recording trace illustrating a PVN neuron’s rapid and repeatable response and recovery to 1mM NaHS. Trace B) shows various concentrations (10mM, 1mM, and 0.1mM) applied in the same neuron.

Figure 6. Hydrogen sulfide has a concentration dependent relationship. Traces illustrate the magnitude of the depolarizations in response to the various NaHS concentrations (0.1, 1, 10, and 50 mM). As the concentrations increase so does the response. A) Current clamp recording trace illustrating no response to 0.1 mM NaHS. B) Current clamp recording trace illustrating a depolarizing response to 1 mM NaHS. C) Current clamp recording trace illustrating a depolarizing response to 10 mM NaHS. D) Current clamp recording trace illustrating a depolarizing response to 50 mM NaHS, with an increase in firing frequency during the initial phase of the depolarization. E) Scatter plot showing the response of all recorded neurons to the various NaHS concentrations (0.1, 1, 10, and 50 mM) with the mean response and standard deviations indicated by the black square and bars

Hydrogen sulfide-induced depolarization is mediated by multiple ion channels

In order to identify potential ion channels responsible for the NaHS-induced depolarization, we applied hyperpolarizing current step protocols to the neurons to examine the changes in input resistance. This hyperpolarizing pulse protocol was applied prior to the application of NaHS, immediately following the application (initial phase), and as the membrane potential was recovering back to baseline from the effect (recovery phase). Voltage/current (V/I) plots were generated for all five points and demonstrated as illustrated in Figure 7 that NaHS resulted in an initial large increase in input resistance (control vs initial phase) during the initial phase of the depolarization, as seen in Figure 7B, followed by a large decrease (control vs recovery phase) during the recovery phase, which often lasted beyond a return to baseline membrane potential (Fig.7C). The point of intersection of these voltage current plots also provided information about the reversal potential for ion channels potentially modulated by H2S.

This point was calculated from the slope of the control line versus the slope of either the line from the initial phase or from the recovery phase. The intersecting points were then averaged for each group (initial phase vs. recovery phase). This analysis suggested the initial stage of the

NaHS-induced depolarization may result from the inhibition of potassium channels as the reversal potential was -103.6 ± 8.4mV (n=5) (Fig.7B). Conversely, the point of intersection taken from voltage current plots during the recovery phase (-53.6 ± 9.8 mV, n=5) indicated the involvement of a non-selective cation current as illustrated in Figure 7C.

Figure 7. Hydrogen sulfide-induced depolarization is biphasic. A) Current clamp recording trace illustrating the change in input resistance over the duration of the hydrogen sulfide-induced depolarization (50 mM). B) V/I plot showing the change in input resistance (point of intercept, 114mV), during the initial phase of the depolarization. C) V/I plot showing the change in input resistance (point of intercept, 62mV), during the recovery phase of the depolarization. Input resistance calculated from the slope of the line.

2.5 Discussion In this study we have used in vitro whole-cell patch clamp techniques to show that the

H2S donor, NaHS increases the excitability of the majority of neurons within the PVN , suggesting important roles for H2S in the regulation of diverse neuroendocrine and autonomic function [95]. We observed that the H2S donor caused rapid onset and reversible depolarizations in the vast majority of PVN neurons tested, these effects were similar to those previously reported in dorsal raphe serotonergic neurons and oxygen sensing cells in trout gill chemoreceptors neurons [55,132]. Nagai and colleges [50] reported that H2S produced an increase in intracellular calcium (Ca2+) concentrations and Ca2+ waves in cultured astrocytes and hippocampal slices, suggesting potentially important interactions between glia and neurons, interactions which could contribute to depolarizing responses [133]. The depolarizing effects appeared to be concentration dependent with only 1 neuron responding by depolarization to

0.1mM NaHS, but depolarizations of increasing magnitude following 1mM, 10mM, and 50mM, which was the highest concentration tested in our studies (Fig.6). Similarly, Umemura and

Kimura [51] reported that H2S has dose-dependent responses on the reduced metabolic activity in rat neuronal cultures and neuroblastomas. A concentration dependent response was also seen in hypoglossal rootlets of medullary brain slices, in which there was increased activity as the H2S concentration was increased [134].

According to the observations by Abe and Kimura [42], the lower concentration we used

(0.1mM) falls within the physiologically relevant range, which is similar to the endogenous H2S concentrations reported in brain homogenates (~160µm). It is important to note that since H2S is a fairly volatile and an unstable gas, the concentrations that were stated and desired may not have

34 been the final concentrations to reach the cells [18]. According to Olson [18], approximately 13% of dissolved H2S is lost with every minute of application, and the loss continues to increase when the solution is oxygenated. Therefore, higher concentrations of the donor may be necessary to elicit a response [18]. When dissolved in aCSF at C, H2S has a pH of 7.6 [135]. While we measured the lower concentrations (0.1mM, 1mM, and 10mM) to be within close range of what can be considered physiologically relevant by definition, it should also be noted that both the pH and the osmolality measures across all of the concentrations except for the highest (50mM) were within physiologically acceptable ranges [136,137].

Also, of importance is the ability of NaHS to produce such rapid responses and recoveries of the membrane potential in the PVN neurons, as illustrated in Figure 5A. The rapid neuronal response produced by H2S can translate into rapid onset and inhibition of physiological effects. These results are comparable to the results obtained in the carotid body in response to

H2S administration [138]. With that said, while the neurons were able to rapidly recover back to baseline, the integrity of the cell may not have fully recovered. It appeared that for an extended period of time after the neuron had recovered and achieved the pre-NaHS administration resting membrane potential it showed a decrease in input resistance, implying that several ion channels remained open (Fig.7A).

The present work is the first to suggest that H2S acts to modulate two different ion channels in the male juvenile rat as summarized in Figure 7. We observed different reversal potentials for effects on input resistance immediately after H2S application when compared to the later period during recovery to the control membrane potential. Our reversal potential data

35 indicates that NaHS in the PVN is modulating a potassium current immediately following administration. However, as the neuron’s membrane potential recovers toward baseline, a separate conductance with a reversal potential in the range of a nonselective cation conductance is apparently activated (Fig.7C). While our data implicates both potassium and non-selective cation channels in the depolarizations produced by NaHS, future voltage clamp studies will be necessary, both to confirm such actions on these specific conductances, as well as to more clearly define the membrane events underlying such interactions. In accordance with our observations, potassium channels have been implicated as one of the primary targets through which H2S exerts its vascular effects to produce changes in blood pressure and other cardiovascular variables [56]. In the central nervous system, H2S has also been implicated in altering intracellular potassium levels which are known to regulate cellular apoptosis, through effects on osmolality, caspase activation, and mitochondrial activity [49]. A recent study observing the effects of H2S in the brain, have shown that it rescues the brain from cerebral hypoxia in a concentration dependent manner, likely through a potassium channel [139]. A number of other groups have associated H2S elicited responses with numerous nonselective ion channels, showing that H2S can modulate its effects through various TRP channels to produce anti-inflammatory responses [52].

Our data also shows that the administration of NaHS produces consistent responses among the MNC, NE, and PA neurons within the PVN, as illustrated in Figure 5B. These cell types send projections to different brain regions including the brain stem, anterior pituitary, and the median eminence to mediate physiological autonomic and endocrine functions [95,103]. This finding provides some evidence that H2S may have implications on several core autonomic and

36 endocrine functions, such as, circadian rhythm, cardiovascular function, the stress axis and fluid balance, among others, as a result of the consistent depolarizations observed across all PVN cell types by the administration of NaHS. Some evidence for this was observed by another group which demonstrated an H2S concentration dependent decrease in potassium chloride stimulated

CRH release from rat hypothalamic explants, which downstream inhibited stress-related glucocorticoid release [45]. Most recently, Gan and colleagues [89], observed increases in renal sympathetic nerve activity, mean arterial pressure and heart rate in response to NaHS microinjections into the PVN, further indicating that H2S has an effect on the physiological functions produce by the PVN.

Our electrophysiological studies of the effects of NaHS on neurons in the PVN are the first to show that this neuromodulator exerts direct actions on the excitability of this neuronal population. Our results were also able to shed light on the possible ion channels involved in

NaHS modulation in the PVN, and provide insight into the possible role of NaHS in autonomic and endocrine function.

Chapter 3: Modulation of voltage-sensitive potassium channels by hydrogen sulfide alters neuronal excitability in the paraventricular nucleus of the hypothalamus

C. Sahara Khademullah

Department of Biomedical and Molecular Science, Queen’s University, Kingston, Ontario,

Canada

Alastair V. Ferguson

Department of Biomedical and Molecular Science, Queen’s University, Kingston, Ontario, Canada

3.1 Abstract

Hydrogen sulfide (H2S) is a gaseous signalling molecule that has been shown to target a wide range of ion channels, including those in the cardiovascular system. Recent work has reported that not only is there cardiovascular changes associated with H2S actions in the hypothalamic paraventricular nucleus (PVN), but H2S also has directs effects on the excitability of single neurons in this critical autonomic control center of the hypothalamus. In the current study, we investigated the effects of H2S on specific ion channels in PVN neurons. We used whole-cell voltage and current clamp techniques to examine the effects of H2S (NaHS) at either

1 or 10 mM on both the transient and sustained voltage-activated potassium currents in these

+ neurons. We applied TEA to isolate the transient/rapidly inactivating current (IA) and 4-AP to isolate the sustained/delayed rectifier current (IK), and were able to show that both of these conductances were significantly reduced by H2S. Finally, we were able to demonstrate through the use of current-clamp recording techniques that when 4-AP and TEA+ were applied together with NaHS, they were able to completely eliminate the previously observed NaHS-induced depolarization, and the effects on membrane potential in fact reversed to show a small hyperpolarization. These data highlight the potential roles of H2S as a critical modulator of the voltage-gated repolarizing conductances IA and IK and thus suggest broad based roles for this interaction in the underlying the cellular mechanisms through which H2S modulates cellular excitability involved in cardiovascular, autonomic and endocrine regulation.

3.2 Introduction

While hydrogen sulfide (H2S) is usually thought of in terms of its toxicity, it has recently been classified as a gasotransmitter, alongside nitric oxide (NO) and carbon monoxide (CO), albeit with a different mode of action [9,42]. H2S is endogenously synthesized by many types of mammalian tissues [48]. In the brain, H2S synthesis is catalyzed primarily by cystathionine-beta synthase (CBS), although cystathionine-gamma lyase (CSE), which has been found primarily in the liver, kidneys, heart, and gastrointestinal tract and 3-mercatopyruvate-tranferase (3-MST), which has been localized to the mitochondria [12,42,80,118,120], have also been implicated in its synthesis. While H2S concentrations in various human tissue samples range from 8 - 160 µM, concentrations in brain homogenates are generally closer to 8 µM [9,123]. The demonstration of physiological roles for H2S in cytoprotection, vasoconstriction, vasodilation, neurotransmission, inflammatory processes, nociception, brain-gut axis regulation and even neuronal plasticity has led to an upsurge of research focussing both on understanding integrated physiological roles for this messenger, as well as work to elucidate the cellular mechanisms underlying such actions

[42,45,53,81,124,140,141].

In the brain, Abe and Kimura [42,81], were among the first to show H2S had non-toxic effects. They found that H2S was able to augment NMDA receptor function to promote long term potentiation (LTP) in the hippocampus. H2S has also been shown to act in the hypothalamus to inhibit the release of KCl-stimulated corticotrophin-releasing hormone (CRH) in hypothalamic tissue [45], and in the paraventricular nucleus of the hypothalamus (PVN) to influence cardiovascular function [89]. The PVN is critical to the regulation of autonomic control [95,96].

It consists of three cells types, magnocellular neurons (MNC – projecting to the posterior pituitary), parvocellular preautonomic neurons (PA – projecting caudally to autonomic control

40 centers in the medulla and spinal cord), and parvocellular neuroendocrine neurons (NE – projecting to the median eminence) [103,105]. We have recently reported that H2S depolarizes the majority of PVN neurons, effects which were suggested to be the result of the modulation of potassium (K+) channels and/or non-selective cationic conductance [142].

While considerable work has demonstrated the ability of H2S to modulate ATP-sensitive potassium channels (KATP) [48,60,143], many physiological effects of H2S have also been attributed to the modulation of other ion channels [56]. Among these are transient receptor potential (TRP) channels, large conductance calcium-activated potassium channels (BKCa), voltage-gated calcium channels, chloride channels, sodium channels (Nav 1.5), and other K+

+ channels, including the delayed-rectifier voltage-gated K channel, KV2.1

[50,65,127,127,128,144] . Recently, Feng and colleagues [68], were able to show that the application of NaHS reduced not only the expression, but the function of a voltage-gated potassium (KV) channel, the delayed rectifier current (IK). While it is known that a variety of ion channels are targets for H2S modulation and regulation, the mechanisms by which this occurs remains poorly understood. The aim of the present study was to further investigate the possible ion channel targets and mechanisms of action of H2S in the PVN using patch-clamp recordings.

3.3 Methods

Slice preparation

Male Sprague-Dawley rats (Charles River, Quebec, Canada) aged 21-28 days were rapidly decapitated and the brains were removed and placed into an ice-cold solution containing

(in mM): 87 NaCl, 2.5 KCl, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 25 glucose, 75 sucrose bubbled with 95% O2/5% CO2. The region of the hypothalamus containing the PVN was

41 isolated, the section was then mounted onto a stage and 300 µm coronal sections were cut using a vibratome (Leica, Nussloch, Germany). Slices were then incubated at 32ºC for a minimum 1 hour prior to recording in carbogenated artificial cerebrospinal fluid (aCSF) containing (in mM):

126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 10 glucose saturated. All procedures were done in accordance with the regulations established by the Canadian Council on

Animal Care and approved by the Queen’s University Animal Care Committee

Electrophysiology

Slices were mounted onto the stage of the recording chamber, which was continuously perfused at a flow rate of 2-3 ml/min with carbogenated aCSF at approximately 32ºC. Neurons were visualized using an upright differential interference contrast microscope (Scientifica,

Sussex, United Kingdom) at 40x magnification. Using a Sutter Instruments P97 flaming micropipette puller, borosilicate glass electrodes (World Precision Instruments, Sarasota,

Florida, USA) were pulled and optimized to have a resistance of 3-5 MΩ when filled with an intracellular solution composed of (in mM): 125 potassium gluconate, 10 KCl, 2 MgCl2, 0.1

CaCl2, 5.5 EGTA, 10 HEPES, 2 NaATP (pH 7.2 with KOH). Whole cell recordings were made with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, California, USA) and acquired at 10 kHz, and filtered at 2.4 kHz using a Micro 1401 interface. Once a high resistance seal was obtained, whole cell configuration was accomplished by breaking through the cell membrane to gain access to the internal contents of the cell through the application of a brief period of negative pressure. Only neurons with a minimum spike amplitude of 60 mV (range 60-

100 mV), input resistance of >400 MΩ (range 400-1100 MΩ), and series resistance of <20 MΩ

(range 8-20 MΩ) were included in our subsequent analysis. Following a minimum 100s baseline

42 recording period, either 1 or 10 mM sodium hydrosulfide hydrate (NaHS) was applied to slices through a bath perfusion system. The current clamp data was recorded in Spike2 software for offline analysis (Cambridge Electronic Devices, Cambridge, UK), while the voltage clamp data was recorded in and analyzed with Signal 4 (Cambridge Electronic Devices, Cambridge, UK).

Response Criteria and Statistical Analysis

Membrane potential changes in response to NaHS was determined by comparing the mean membrane potential of the neuron before and after application and averaged across 100 s time periods. A response was considered significant if the change in membrane potential after

NaHS application was at least twice the amplitude of the standard deviation of the baseline membrane potential during 100 s prior to application. Descriptive statistics were also used to describe mean group data as well as the standard error of the means in all such groups. In voltage-clamp, the amplitudes of the peak/rapid and steady state/persistent K+ conductances were compared prior to and after the administration of NaHS using a paired Student’s t-test.

Responses were determined as significant if p < 0.05 and very significant if p < 0.01. All recorded membrane potentials were adjusted to correct for the calculated junction potential (+15 mV).

Chemicals and drugs

NaHS solution was prepared fresh prior to bath application. Tetrodotoxin (TTX; 1 µM) was added to the aCSF to block the sodium (Na+) conductance, as confirmed by voltage-clamp with the absence of the large rapid voltage-gated inward current. While 5 mM 4-aminopyridine

(4-AP) was used to abolish the transient K+ current and 10 mM tetraethylammonium (TEA+),

43 obtained from ACROS Organics (Geel, Belgium) was used to block the sustained K+ current. All salts and chemicals, unless otherwise stated, used to prepare solutions were obtained from

Sigma-Aldrich Pharmaceuticals (Oakville, Ontario, Canada). All drugs were dissolved in aCSF and applied directly through the bath perfusion system.

3.4 Results

NaHS-induced currents in PVN neurons.

Although multiple ion channels have been reported to be modulated by H2S [145] our previous work demonstrated that NaHS produces depolarizing effects in the vast majority of

PVN neurons [142]. The reversal potential from these effects suggested the modulation of a K+ conductance. Therefore, we used voltage-clamp techniques to further examine the effect of

NaHS on currents elicited during a slow (12 mV/sec) voltage ramp from -80 to 30 mV before and during NaHS’s administration, as illustrated in Figure 8A. Using this protocol, we were able to evaluate the H2S-induced current by subtracting the current measured during 10 mM NaHS from that obtained in control conditions (aCSF), with the resultant difference current (Fig. 8B) representing the H2S-induced current. These recordings revealed an effect of H2S to reduce the mean outward current measured at the 30 mV point from 4.51 ± 0.59 nA to 1.98 ± 0.41 nA (n =

5, p < 0.0025 using a paired Students t test), as illustrated in Figure 8C. The summary data through these voltage ramps presented in Figure 8D support the conclusion that H2S decreases outward currents evoked by slow voltage ramps in the depolarized range, which appear to activate at approximately -40 mV in accordance with the hypothesis that H2S is inhibiting the sustained voltage activated K+ conductance.

Figure 8. Current-voltage relationship in PVN neurons in response to NaHS application. A) Whole-cell current recording from a PVN neuron in response to a voltage ramp (-80 to 30 mV, 12 mV/sec) showing the mean currents observed during perfusion with aCSF (black) and aCSF containing 10 mM NaHS (grey) showing a decrease in current in the depolarized range. B) The bar graph illustrates the difference in current between the control (black bar) (4.51 ± 0.59 nA) and NaHS (grey bar) (1.98 ± 0.41 nA) (n = 5, p< 0.0025) measured at 30 mV. C) This I/V curve shows the difference (NaHS-induced) current obtained by subtracting the mean NaHS ramps from the control ramps. D) I/V curve represents the mean (n=5) NaHS induced current (grey lines represent standard error), and the activation profile of this current (initial activation at -40 mV) suggests the potential modulation of IK.

NaHS causes a reduction in both the transient and sustained whole cell currents in

PVN neurons.

We used standard voltage clamp protocols in which a series of 10 mV, 200 ms voltage steps (-95 mV to -5 mV) from a holding potential of -75 mV were applied to PVN neurons before and during administration of 10 mM NaHS. Using this protocol, we were able to isolate

+ both the transient current, which is indicative of the fast-inactivating K current (IA), and the sustained current, which is indicative of the IK current. We assessed the effects of H2S by averaging the peak current from the first 15 ms of each step, and on the sustained current by taking the peak current during the last 15 ms of the step. H2S produced a decrease in both the transient current observed at -25 mV (0.71 ± 0.15 nA to 0.51 ± 0.1 nA, p < 0.048), and the sustained current, observed at -5 mV (0.75 ± 0.19 nA to 0.48 ± 0.12 nA, p < 0.037), suggesting

+ effects of H2S on both of these voltage-activated K conductances. We next examined the effects of H2S on these specific conductances in isolation, using the pharmacological tools 4-AP (5 mM

+ - 5 mins) to block IA and thus isolate IK (n=5), or TEA (10 mM - 5 mins) to block IK and therefore isolate IA (n=4). We then examined the effects of 10 mM NaHS on the currents measured in response to the same voltage step protocols described above and as illustrated in

Figure 9. We observed clear inhibitory effects of H2S on both of these conductances with a recovery toward control currents following a return to aCSF. The recording presented in Figure

9A illustrates the effects of H2S on the IK current in one PVN neuron, while mean data for all neurons tested are illustrated in Figure 9B. Comparison of currents measured at the -5 mV step show that sustained current decreased from 2.65 ± 0.47 nA to 1.2 ± 0.43 nA (p < 0.021).

Similarly, the traces (Fig. 9C), show the presence of an even larger decrease in the IA current in

46 response to H2S (mean data shown in Fig. 9D). Comparison of currents measured at the -5 mV step show that transient current decreased from 3.65 ± 0.61 nA to 1.25 ± 0.39 nA (p < 0.008).

Figure 9. Hydrogen sulfide reduces the transient and sustained whole cell currents. Whole-cell current recording from a PVN neuron in response to voltage steps (-95 mV to -5 mV, 200 ms) from a holding potential of -75 mV show the difference in peak currents observed during the perfusion of aCSF and 4-AP (5 mM) and the peak currents observed during the perfusion of aCSF, 4-AP and 10 mM NaHS. A) The whole-cell currents show a decrease in the sustained current (measured during the last 15 ms of each step). B) This I-V curve shows the mean current measured across all cells tested (n=5) at each voltage step in control conditions (aCSF & 4-AP- black boxes) and following bath application of 10 mM NaHS (NaHS & 4-AP - Grey boxes). C) Whole-cell current recording from a PVN neuron in response to voltage steps (- 95 mV to -5 mV, 200 ms) from a holding potential of -75 mV showing the peak currents observed during the perfusion of aCSF and TEA+ (10 mM) and aCSF containing TEA+ and 10 mM NaHS. The whole-cell currents show a decrease in the transient current (measured during the first 15 ms of each step). D) This I-V curve shows the mean current measured across all cells tested (n=4) at each voltage step in control conditions (aCSF & TEA+ - black boxes) and following bath application of 10 mM NaHS (NaHS & TEA+ - grey boxes). Significant differences in current are indicated by asterisks (*p<0.05, **p<0.005, paired t-test).

+ The depolarizing actions of H2S in PVN neurons are abolished by 4-AP and TEA .

We examined whether the depolarizing actions of NaHS, as previously observed [142], was involved in the modulation of voltage-activated K+ currents. Using current clamp techniques we tested the effects of H2S when simultaneously applied with the pharmacological blockers 4-

AP (5 mM) and TEA+ (10 mM). We examined 4 neurons, in which we were able to first confirm depolarizing effects of H2S (1 mM NaHS) in aCSF alone (3.82 ± 1.31 mV), prior to testing in the

+ presence of K channel blockers (Fig. 10). However, when H2S (1 mM NaHS) was applied again in the presence of both K+ channel blockers, which also caused depolarizing effects (6.8 ± 4.67 mV), a hyperpolarization with recovery to baseline (-17.11 ± 8.55 mV) was elicited in strict contrast to the depolarization under control conditions (Fig. 10B).

Figure 10. Hydrogen sulfides depolarizing actions in PVN neurons are abolished by 4-AP and TEA+. Trace A) shows a whole-cell current-clamp recording illustrating a depolarizing response to the perfusion of aCSF and 1 mM NaHS. Trace B) shows a whole-cell current-clamp recording from the same neuron perfused with aCSF, 5 mM 4-AP and 10 mM TEA+ illustrating a hyperpolarizing response (-17.11 ± 8.55 mV) to the bath application of 1 mM NaHS. C) The bar graph illustrates the mean membrane potential for all cells (n=4) that were perfused first with 1mM NaHS and aCSF (black bar) and responded by depolarization, then perfused with 1 mM NaHS in the presence of TEA+ and 4-AP (grey bar) and responded by hyperpolarization. Significant differences in membrane potential are indicated by asterisks (**p<0.005, paired t- test).

3.5 Discussion

We previously proposed that H2S (NaHS – 1 and 10 mM) administration resulted in the depolarization of the majority of PVN neurons. The effects displayed a reversal potential of

103.6 ± 8.4 mV, suggesting a role for K+ channels [142], observations which are consistent with studies reporting that H2S induces membrane depolarization through the inhibition of TASK channels [146]. Our initial ramp studies, however, suggested a role for voltage-activated K+

+ + currents, specifically the sustained K current, IK (Fig. 8). These voltage-gated K channels have been shown to be involved in ﬁring patterns by influencing spike interval and action potential repolarization and therefore, may play a key role in cardiovascular, autonomic and neuroendocrine regulation in the PVN [147,148]. Using whole-cell voltage and current clamp techniques, we have been able to demonstrate that KV channels are modulated by H2S in the

PVN, suggesting that these ions channels may play an important role in H2Ss regulation of cardiovascular, neuroendocrine and autonomic function [75]. Specifically, we have found that

+ H2S reduces whole cell K currents, and when pharmacologically isolated, it reduces both the transient (IA) and sustained (IK) KV currents in PVN neurons. These observations on IK consistent with recent work form Wei et al. [69], showing that H2S (NaHS – 100 to 300 µM) similarly reduced sustained K+ currents in cardiac myocytes, although they did not examine transient K+ currents in these cells. Similarly, Feng et al. [68], has also identified H2S actions on KV channels in trigeminal ganglia, showing that CBS was co-localized with KV1.1 and KV1.4 and that the inhibition of these KV channels by NaHS resulted in increased excitability of trigeminal neurons.

By contrast, we believe our study in PVN neurons is the first to suggest that H2S is targeting IA in addition to IK.

Our data suggests that H2S causes an approximately 50% reduction in both IA and IK currents, which are again consistent with recent reports on sustained K+ currents [69] (Fig. 9). A reduction of such a magnitude may have effects on cell volume regulation, hormone and neurotransmitter secretion, and even cellular proliferation [149]. These results also suggest that both currents independently play an important role in the H2S-induced modulation of PVN neurons, in which voltage-gated K+ currents have long been known to play important roles in the regulation of cell excitability, and have even been used as an electrophysiological fingerprint to identify specific neuronal subpopulations in the PVN [106,148,150]. It has been suggested that changes associated with KV channels would have implications on bursting firing patterns, which are responsible for the secretion of multiple neurohormones, including that of oxytocin, vasopressin, CRH, and thyrotrophin-releasing hormone (TRH). These changes would also affect preautonomic neurons in the PVN, which play critical roles in communicating with caudal medullary and spinal autonomic control centers [116]. This in turn would play a critical role in cardiovascular, neuroendocrine and autonomic outputs from PVN neurons [116]. Our data show that while H2S administration in control conditions depolarizes the membrane potential of PVN neurons as previously reported [142]. In addition we found (Fig. 10A) subsequent H2S

+ administration to the same neuron during combined pharmacological block of IK (TEA ) and IA

(4-AP) abolished the H2S-induced depolarization to reveal additional hyperpolarizing actions of this gasotransmitter (Fig. 10C). We have not yet investigated the specific ionic mechanisms responsible for this hyperpolarization, although a number of potential targets have previously been shown to be modulated by H2S. Clearly, additional current- and voltage-clamp studies will be necessary to confirm what specific channels are underlying this H2S-induced hyperpolarizing response in PVN neurons. A variety of potential targets have been identified by studies

52 suggesting that H2S-induced relaxation of vas deferens smooth muscle cells may be a consequence of the modulation of TRP, KATP or BKCa channels

[11,53,55,56,58,60,65,70,128,151]. Another study has implicated H2S in the hyperpolarization of the intact and endothelium-denuded mesenteric arteries through the mediation of both the

intermediate and small-conductance BKCa channels [55,152]. Both exogenous and endogenous

H2S produces vasodilation by activating KATP channels, which in turn causes a hyperpolarization of smooth muscle cells [60]. However, Telezhkin et al. [153] demonstrated that H2S produced inhibitory effects on BKCa channels in both HEK293 cell lines and the rat carotid body to produce a depolarization and subsequent hypoxia. Taken together, these studies suggest that H2S modulates more than one ion channel to produce different and in some instances, paradoxical responses. While it is not well understood what causes these difference, there may be many contributing factors, such as differences between concentrations used, difference among models used (cell lines vs. animals), as well as differences in strain of animal used. Some evidence for this is provided in a study by Kombian et al. [55,152], that reported lower concentrations of

NaHS produced depolarizing responses, while higher concentrations produced hyperpolarizations.

The data presented here implicates both transient and sustained KV channels in the depolarization of the membrane produced by NaHS in the male juvenile rat. As suggested previously, this can have a large impact on the way neurotransmitters and hormones are released from the PVN and in turn impact a wide range of neuroendocrine and autonomic functions.

Other groups have already shown that the application of H2S in the PVN results in physiological responses, such as changes in blood pressure regulation [89]. Since it is know that the PVN contains neurons that project to the brain stem to regulate cardiovascular function among other

53 functions, our study suggests that in addition to KATP, Kv channels may also contribute to the cardiovascular effects [as proposed by the literature] [15, 18, 19].

Chapter 4: Discussion

4.1 Hydrogen sulfides actions in the PVN

The data presented in this thesis demonstrates that H2S has direct actions on the membrane properties of PVN neurons. Similar to what was observed by Andresson et al. [154], using both current- and voltage-clamp techniques in dorsal root ganglion neurons, NaHS was shown to produce depolarizations through the activation of TRPA1 channels. With the incorporation of whole-cell electrophysiology, we have shown that the majority of PVN neurons depolarize in a dose-dependent manner to H2S administration, with a strong, positive correlation.

Umemura and Kimura [51], showed that H2S produced a concentration-dependent response in rat neuronal cultures and neuroblastomas, which resulted in the reduction of metabolic activity. This concentration-dependent effect was also consistently seen among different cell types in the PVN.

The reversal potential from these experiments suggested that there were in fact two or more ion channels being modulated to produce the responses seen. Specifically, it appeared as though a type of K+ channel was primarily responsible for the initial depolarizing response, while a non- selective cation channel may be responsible for the latter portion of the response. Using voltage- clamp measurements, we were able to show that the depolarizing actions of H2S were as a result of the modulation of KV channels. Both IA and IK channels were shown to be inhibited by H2S.

When both KV channels were blocked and H2S was applied in current clamp, the depolarization was occluded and uncovered a hyperpolarization. This was an indication that these KV channels were responsible for the depolarizations produced by H2S in the PVN.

Abe and Kimura [42], were the first to observe the neuromodulatory effects of H2S in the brain. In the hypothalamus, researchers have shown that H2S can act to affect hormone secretion

55 and cardiovascular function, as well as neuroendocrine function [25,42,45,124]. This discussion will focus on the physiological consequences of H2Ss actions in the PVN.

4.2 Consequence of H2S actions in the subpopulation of PVN neurons

In Chapter 2, we provided data to show that H2S produces depolarizing effects on the cellular membranes of MNC, PA, and NE neurons; therefore, it is likely that this would result in the increased secretion of vasopressin, oxytocin, CRH, and TRH, to the posterior pituitary gland, median eminence, brain stem, and spinal cord regions [91]. Physiologically, an H2S-induced increase in the secretion of these neuromodulators may have downstream effects on electrolyte balance, blood pressure, memory formation, social behaviours, and the regulation of the HPA axis, among other functions [96,99,155,156].

Vasopressin

The consequence of our findings on vasopressin-secreting PVN neurons is possibly far- reaching, since these neurons project directly to the posterior pituitary, brain stem, and spinal cord, and if stimulated, can affect many critical physiological functions [91]. An increase of vasopressin can result in water imbalance, changes in thermoregulation, enhanced memory formation, increases in blood pressure, and the regulation of both vasoconstricton and relaxation

[157–168].

An H2S-induced escalation in vasopressin secretion from the PVN would have dramatic effects on many autonomic and endocrine functions. In light of our findings, H2S may also be used as a potential therapeutic agent to regulate vasopressin release from the PVN. H2S has also previously been observed to play a role in some of the physiological functions that are regulated by vasopressin. A hallmark study in H2S function found that while there has been no direct link

56 provided between H2S and memory function, H2S can enhance NMDA receptor-mediated responses and facilitate long-term potentiation [42]. This finding suggests that H2S, too, may play a factor in learning and memory processes, as with vasopressin [42]. Another study by Streeter et al. [166], observed that in the Spargue-Dawley rat, H2S produced concentration-dependent vasorelaxation in the middle cerebral arteries. This effect was also concluded to be a result of the

+ modulation of more than one K channel, including KV subunits. In Chapter 2, our results also show that H2S produces a concentration-dependent depolarization of the membrane potential of these vasopressin-secreting neurons, which we later show in Chapter 3 is regulated by a KV channel.

Oxytocin

Oxytocin-secreting neurons in the PVN project primarily to the posterior pituitary, although a smaller number of these neurons have been traced into the brain stem and spinal cord

[91,169]. As our results in Chapter 2 have alluded to, H2S increases the membrane excitability of the oxytocin-secreting neurons in the PVN, which may result in increased secretion from the posterior pituitary. Increased oxytocin release has been linked to pro-social behaviours, neuroendocrine responses to stress, increased lactation and uterine contractions, increased penile erections, and reduced gastric motility [170–174].

We speculate that the H2S-induced depolarization observed would be linked to many of these effects. Some studies observing the effects of H2S on neuroendocrine and autonomic function have provided further evidence for this. In lactating rats, CSE was found to be up- regulated for a 2 week period from the onset of lactation [175]. The up-regulation of oxytocin during lactation has also been observed [176]. This may suggest that since both H2S and oxytocin

57 are being up-regulated during lactation, that it may be possible that they are having effects on one another, which may be occurring at the level of the PVN. Western blot and immuncytochemical techniques have also revealed that CBS and CSE are both expressed in mouse gastric tissue and that the administration of NaHS (100 µM) in this tissue enhanced both the amplitude and frequency of spontaneous gastric contractions, as does oxytocin [177,178].

This may suggest that an H2S antagonist could be implemented as a therapeutic to target increased levels of oxytocin that ultimately results in abnormalities in gastric motility.

Corticotrophin-releasing hormone

The results in Chapter 2 show that H2S has depolarizing actions on CRH-secreting neurons in the PVN, which would also result in a wide range of physiological outcomes. These neurons project to the median eminence through to the hypophyseal portal system down to the anterior pituitary to control many endocrine functions. They also project to the brain stem and spinal cord to regulate a variety of autonomic functions. It is well known that CRH is a central regulator of the hormonal stress axis, and in turn, results in the release of corticotrophin and glucocorticoids

[179–182]. While CRH release appears to be unaffected by NaHS directly, a recent study found it inhibited KCl-stimulated CRH release in a concentration-dependent manner [45]. This may reduce the production of corticotrophin and glucocorticoids. In light of our findings, further investigation of the action of H2S on CRH release in the PVN may uncover an effect on increased secretion of corticotrophin and/or glucocorticoids from the median eminence, along with changes in stress response in relation to the brainstem and/or spinal cord, if not CRH directly.

Thyrotrophin-releasing hormone

NE TRH-secreting neurons in the PVN project primarily to the median eminence and its release regulates TSH and prolactin production [91]. Lesions of the hypothalamus usually cause an inhibition of TSH secretion and an increase in prolactin [183]. This suggests that serum levels of TRH secreted from the PVN act to regulate downstream prolactin secretion [183]. On the other hand, TRH neurons that project to the brainstem and/or spinal cord region have been implicated in food and water intake [184,185]. This suggests that the H2S-induced depolarizations in NE neurons observed in our study may influence both food and water intake.

While there is not a lot of other evidence to propose any effects of H2S on TRH, TSH, or prolactin secretion, there is some evidence to advocate there is a link between KV channels and prolactin secretion [186,187]. Our data presented in Chapter 3 observed the modulation of KV channels within the PVN by means of H2S, suggesting that H2S may be able to modulate the secretion of TRH, TSH, and prolactin through such channels.

4.3 H2S modulation of ion channels

In Chapter 3, we report that H2S produces its depolarizing actions in the PVN through the inhibition of KV channels, specifically, the IA and IK currents. A recent study by Sooner and

Stern [188], observed the presence of IA currents in neurons projecting from the RVLM to the

PVN. They reported that these currents were an integral component to how PVN neurons modulate their action potential waveforms and firing activity [188]. This is important as action potential frequency mediates neuronal excitability and therefore neuromodulator release [188].

Whole cell patch-clamp analysis has also revealed that these IA currents in the PVN specifically contribute to cardiac function [189]. Sooner et al. [189], observed that neurons, in the

59 hypertensive rat, that projected from the RVLM to the PVN, showed evidence of a diminished IA current. This resulted in the production of action potentials that were broader than under control conditions and decayed more slowly [189]. As our data indicates, H2S appears to be inhibiting these currents to produce amplified cellular excitability and enhanced secretion of peptides and transmitters.

+ The sustained slow transient K current, IK, has been identified amongst PVN neurons

[116]. More specifically, it has been observed that magnocelluar neurons, and to some extent, neuroendocrine neurons, express these currents, which affect the cell’s ability to be excited at hyperpolarized membrane potentials [116]. This effect on excitability may produce a decline in the release of both oxytocin and vasopressin from the PVN [116].

Our data demonstrates that H2S inhibits this IK current, which may therefore lead to enhanced neuronal excitability and in turn, an upsurge in secretion of vasopressin and oxytocin

[121,190]. While we’ve reported that H2S can modulate many different ion channels to produce its physiological consequences in Chapter 1, other endeavours still exist. One such possibility to explore is that of H2S’s actions on ion channel function through means of mitochondrial function. H2S is an inhibitor of cytochrome c oxidase, which is an enzyme at the end of the respiratory electron transport chain [191,192]. This inhibition of cytochrome c oxidase has been linked to the activation of KATP channels [60,193]. Although there hasn’t been much evidence to suggest that KV channels are involved in mitochondrial cellular respiration and/or the regulation of cytochrome c oxidase, it is possible that these ion channels may still be affected by the inhibition of cytochrome c oxidase via H2S’s actions on the KATP channels.

4.4 Summary and Conclusions

Research in the field of gasotransmitters has taken off over the past few years, particularly with respect to the non-toxic and possible therapeutic effects of H2S. There still remains much to be uncovered with regards to how H2S produces its physiological effects. The data presented in this thesis has provided evidence to suggest that H2S works to increase neuronal excitability in the PVN through KV channels. Although our data can only account for the initial depolarizing actions of H2S in the PVN, this alone can possibly account for many of

H2S’s physiological consequences, including blood pressure regulation, influences on body temperature, and effects on vasoconstriction and dilation [28,56,75,89,194]. Many groups have already started the quest to implement H2S as a therapeutic, specifically with regards to its anti- inflammatory effects [195]. Future investigations of H2S actions in the PVN will hopefully build upon the understanding of the mechanism by which H2S affects KV channels and how it produces the sustained depolarization observed in our data. These findings will lead to its possible therapeutic potentials for many endocrine and autonomic disorders.

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