EFFECT OF BOOPHONE DISTICHA ON THE BEHAVIOUR AND HIPPOCAMPAL NEUROANATOMY IN A BALB/c MOUSE MODEL Name: Nkosiphendule Khuthazelani Xhakaza Student number: 1509905 A research thesis School of Anatomical Sciences, University of Witwatersrand, As fulfilment for the PhD Degree in Anatomy Supervisor: Prof EF Mbajiorgu (PhD) Co- supervisor: Dr P Nkomozepi (PhD) Johannesburg, 2022 i DECLARATION I Nkosiphendule Khuthazelani Xhakaza declare that this Thesis is my own, unaided work. It is being submitted for the Degree of Doctor of philosophy at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other University. (Signature of candidate) 9 day of June 2023 in Pretoria . ii DEDICATION I dedicate this thesis to my parents, Mr Phillemon and Mrs Nelisiwe Xhakaza, who taught me and raised me in accordance with the principles of the Kingdom of God. While not educated, they sacrificed a lot to ensure that I get the education. They understood the value of an inheritance of education. They always stood in prayer and support for me. To my lovely wife Makhosi, my one and only son Nkazimulo, my two daughters, Ndumiso and Wandile, thank you so much for your prayers, love, support and enduring my unavailability during my long journey of the PhD studies. You have been my pillar of strength and joy in the midst of what has been a tough journey. You played a critical role in this achievement. To my entire family for always encouraging and believing in and praying for me. iii CONFERENCE PRESENTATIONS ARISING FROM THIS THESIS 1. South African Association for Laboratory Animal Science (SAALAS 2022) conference, North west University, March 2022: Boophone disticha attenuates effects of swimming stress on behaviour and neuroblast differentiation in Balb/c mouse hippocampus. NK XHAKAZA,1, 3 P NKOMOZEPI, 2 EF MBARGIORGU,3 1 Department of Anatomy and Histology, Sefako Makgatho Health Sciences University, Ga- rankuwa, Pretoria, South Africa, 2 Department of Human Anatomy and Physiology University of Johannesburg, Doornfontein, South Africa, 3 School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. nkosi.xhakaza@smu.ac.za 2. 49th Congress of the Anatomical Society of Southern Africa (ASSA Conference: from the 19th to the 21st of April 2022). Boophone disticha attenuates effects of swimming stress on behaviour and neuroblast differentiation in Balb/c mouse hippocampus. NK XHAKAZA,1, 3 P NKOMOZEPI, 2 EF MBARGIORGU3 1 Department of Anatomy and Histology, Sefako Makgatho Health Sciences University, Ga- rankuwa, Pretoria, South Africa, 2 Department of Human Anatomy and Physiology University of Johannesburg, Doornfontein, South Africa, 3 School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. nkosi.xhakaza@smu.ac.za mailto:nkosi.xhakaza@smu.ac.za mailto:nkosi.xhakaza@smu.ac.za iv JOURNAL PUBLICATION ARISING FROM THIS THESIS Xhakaza, N.K., Nkomozepi, P. and Mbajiorgu, E.F., 2022. Boophone disticha attenuates five day repeated forced swim-induced stress and adult hippocampal neurogenesis impairment in male Balb/c mice. Anatomy & Cell Biology. Anat Cell Biol . 2022 Oct 21. https://doi.org/10.5115/acb.22.120 (See Appendix 6). https://doi.org/10.5115/acb.22.120 v ABSTRACT Depression is one of the most common neuropsychiatric disorders and is associated with dysfunction of the neuroendocrine system and alterations in specific brain proteins. Boophone disticha (BD) is an indigenous psychoactive bulb that belongs to the Amaryllidacae family, which is widely used in Southern Africa to treat depression, with scientific evidence of potent antidepressant-like effects. The present study examined the antidepressant effects of BD and its mechanisms of action by measuring some behavioural parameters in the elevated plus maze, light dark box, open field forced swimming, brain content of corticosterone, brain derived neurotropic factor (BDNF), and neuroblast differentiation in the hippocampus of Balb/c mice exposed to the five-day repeated forced swim stress (5dRFSS) and 28 days chronic restraint stress. Male Balb/c mice were subjected to the 5dRFSS and 28 days chronic restraint protocols to induce depressive-like behaviour (decreased swimming, increased floating, decreased open arm entry, decreased time spent in the open arms and decreased head dips in the elevated plus maze test, increased time in dark box in the light dark box test, reduced frequency of rearing and increased time on the sides of the open field in the open field test), and treated with distilled water, fluoxetine and BD. Three weeks Boophone disticha treatment (10mg/kg/p.o) significantly attenuated both the 5dRFSS and chronic restraint-induced behavioural abnormalities and the elevated brain tissue corticosterone levels observed in stressed mice. Additionally, 5dRFSS exposure significantly decreased the number of neuroblasts in the hippocampus and BDNF levels in the brain of Balb/c mice, while fluoxetine and BD treatment attenuated these changes. In the chronic restraint stressed mice, similar effects of BD treatment were observed after 21 days of treatment, however, the levels of corticosterone were not different in control and stressed animals, probably due to habituation to stress. In both 5dRFSS and chronic restraint stress, the antidepressant effects of BD were comparable to those of fluoxetine, but unlike fluoxetine, BD vi did not show any anxiogenic effects, suggesting better pharmacological functions. It is important to note that in chronic restraint stress mice, it appeared that animals seemed to have habituated to stressful conditions, demonstrated in part by brain tissue levels of corticosterone that were not elevated in stressed animals treated with distilled water. However, BDNF levels remained significantly low in stressed animals treated with distilled water, suggesting that the effect of chronic stress in this parameter were not reversed when animals habituated. In conclusion, our study shows that BD exerted antidepressant-like effects in both 5dRFSS and chronic restraint stress mice, mediated in part by normalizing brain corticosterone and BDNF levels. Due to some degree of habituation in chronic stress model, caution should be exercised when evaluation effects of treatment in different parameters to evaluate antistress effects of tested agents, particularly levels of corticosterone. Furthermore, the persistent low levels of BDNF suggest that habituation of animals to chronic stress is due to normalising levels of corticosterone but not BDNF. The above occurrence could suggest that recovery from chronic stress without antidepressant treatment could alleviate other behavioural symptoms but not cognitive impairment which is influenced in part by BDNF levels. Key words: Depression, Antidepressants, Hippocampus, Stress, Behaviour vii ACKNOWLEDGEMENTS • My supervisor Professor Felix Ejikeme Mbajiorgu for the support, guidance and encouragement in the midst of multiple challenges encountered during the entire process. Your endurance is highly appreciated. • My Co-supervisor and friend Dr Pilani Nkomozepi. Your excellent scientific insight taught me a lot. Thank you for suggesting this PhD topic which not only produced this PhD but also four honors and Three masters graduates. • Ms Hasina Ali for her assistance with the ELISA’s lab work with such enthusiasm and a smile. • To the personnel at Wits Animal Research Facility (WRAF). Without you, this work would not have been possible. Thank you for standing and supporting me in the midst of all challenges experienced during the animal treatments. • Mr Lucky Japhta, thank you very much for all the sacrifice, camping with me during the behavioral tests until midnight and early morning hours. The role you played was tremendous in the success of this project. • My postgraduate students, Lammeeze, Mogale, Excellent, Luyanda and Gcinokuhle, thank you very much for assisting either for animal treatments and collection of organs during terminal procedures. The role you played was of great importance. • The colleagues in the departments of anatomy University of Johannesburg and Sefako Magatho Health Sciences University. Thank you very much for all the encouragement and support. • The funding bodies NRF via its Thuthuka instrument (TTK190215418278), and Sefako Makgatho Health Scinces University vial its University Staff Development Grant (UCDG). Without your financial support, this project would not have been a success. • Last but not least, God almighty for giving me the wisdom and strength to endure. viii TABLE OF CONTENTS PAGE DECLARATION ........................................................................................................................ i DEDICATION ........................................................................................................................... ii CONFERENCE PRESENTATIONS ARISING FROM THIS THESIS ................................ iii JOURNAL PUBLICATION ARISING FROM THIS THESIS ............................................... iv ABSTRACT ............................................................................................................................... v ACKNOWLEDGEMENTS ..................................................................................................... vii TABLE OF CONTENTS ....................................................................................................... viii LIST OF ABBREVIATIONS ................................................................................................ xiii LIST OF SYMBOLS ............................................................................................................. xvii LIST OF FIGURES ............................................................................................................. xviii CHAPTER ONE ........................................................................................................................ 1 1.0 INTRODUCTION ....................................................................................................... 1 CHAPTER TWO ....................................................................................................................... 5 LITERATURE REVIEW ....................................................................................................... 5 2.1 Overview of depression .................................................................................................... 5 2.2 Prevalence of depression .................................................................................................. 5 2.4 Neurotransmitter systems in depression ........................................................................... 9 2.4.1 Inflammatory process in depression ........................................................................ 12 2.5 Neuroanatomy of depression .......................................................................................... 14 ix 2.5.1 The role of the prefrontal cortex in depression .................................................. 14 2.5.2 The role of amygdala in depression ......................................................................... 15 2.5.3 Role of the hippocampus in depression ................................................................... 16 2.6 Stress effects on plasticity and molecular structure ....................................................... 24 2.6.1 Changes in dendritic spines ..................................................................................... 24 2.6.2 Stress and apoptosis ................................................................................................. 25 2.7 Treatment of depression ................................................................................................. 26 2.8 Antidepressants and their mode of action ...................................................................... 26 2.9 Medicinal plants with antidepressant properties ............................................................ 27 2.10.1 Alkaloids of Boophone Disticha............................................................................ 30 2.10.2 Pharmacological Activity of Boophone Disticha .................................................. 31 2.11 Rodent models of depression ....................................................................................... 32 2.12 Aims and objectives of the study .............................................................................. 33 2.12.1 Aim of the study................................................................................................ 33 2.12.2 Objectives ......................................................................................................... 33 2.12.3 Hypothesis ............................................................................................................. 33 CHAPTER THREE ................................................................................................................. 34 METHODOLOGY ............................................................................................................... 34 3.1 Experimental animals ................................................................................................ 34 3.3 Identification of phytochemicals using LC-MS ............................................................. 35 3.4 Five day repeated forced swimming stress model (5d RFSS) ....................................... 36 x 3.5 Repetitive restraint stress ............................................................................................... 37 3.6 Behavioural tests ............................................................................................................ 39 3.6.2 Open field test .......................................................................................................... 40 3.7 Terminal procedures ....................................................................................................... 43 3.7.1 Sectioning of the brain tissues ................................................................................. 44 3.7.3 Immunohistochemistry ............................................................................................ 45 3.7.4 Volumes of the hippocampus and its subfields ....................................................... 46 3.7.5 Doublecortin quantification ..................................................................................... 47 CHAPTER FOUR .................................................................................................................... 48 RESULTS............................................................................................................................. 48 4.1.1 Identification of phytochemicals in the hydro-ethanolic extracts of boophone disticha using LC-MS .................................................................................................................... 48 4.1.2 Phytochemical profile of BD based on LC-MS data ............................................... 49 4.1.3 Animal weights ........................................................................................................ 49 4.1.4 Five days repetitive forced swimming stress ........................................................... 50 4.1.5 Elevated plus maze .................................................................................................. 53 4.1.7 DCX Expression ...................................................................................................... 57 4.1.8 Brain derived neurotropic factor (BDNF) ............................................................... 59 4.1.9 Corticosterone .......................................................................................................... 59 4.2 CHRONIC RESTRAINT .......................................................................................... 60 4.2.1Chronic stress induction phase ................................................................................. 60 4.2.2 Animal weight changes after the treatment period .................................................. 62 xi 4.2.3 Elevated plus maze .................................................................................................. 62 4.2.4 Light dark box ....................................................................................................... 68 4.2.5 Open field test .......................................................................................................... 72 4.2.6 Volume of the hippocampus .................................................................................... 78 4.2.7 BDNF and Corticosterone ....................................................................................... 82 4.2.8 DCX expression ....................................................................................................... 84 CHAPTER FIVE .............................................................................................................. 85 DISCUSSION ...................................................................................................................... 85 5.1 INTRODUCTION .......................................................................................................... 85 5.2 FORCED SWIMMING TEST .................................................................................. 86 5.2.1 Changes in immobility time .................................................................................... 86 5.2.2 Elevated plus maze .................................................................................................. 88 5.2.3 Volume of the hippocampus .................................................................................... 88 5.2.4 Doublecortin (DCX) expression .............................................................................. 89 5.2.5 Corticosterone and BDNF levels ............................................................................. 90 5.3 Chronic restraint stress ................................................................................................... 92 5.3.1 Weight and food consumption changes ................................................................... 92 5.3.2 Anxiolytic effects of BD using elevated plus maze ................................................ 93 5.3.3 Anxiolytic effects of BD using light dark box ........................................................ 95 5.3.5 Volume of the hippocampus .................................................................................... 97 5.3.6 BDNF and corticosterone ........................................................................................ 97 xii REFERENCES ...................................................................................................................... 101 APPENDICES: ...................................................................................................................... 149 Appendix 1: Workflow .......................................................................................................... 149 Appendix 2: Ethics certificate 1 (Original) ............................................................................ 150 Appendix 3: Ethics certificate 2 (Repeat experiment) ........................................................... 151 Appendix 4: Plagiarism Declaration ...................................................................................... 152 Appendix 5: Turn it in report ................................................................................................. 153 APPENDIX 6: ........................................................................................................................ 154 xiii LIST OF ABBREVIATIONS ACC- Anterior cingulate cortex ACTH- Adrenocorticotropic hormone Aβ-Amyloid beta ATP- Adenosine triphosphate BDNF- Brain derived neurotropic factor BD- Boophone disticha CA1- Cornu Ammonis area 1 CA2- Cornu Ammonis area 2 CA3- Cornu Ammonis area 3 CORT- Corticosterone COX-1- Cyclo-oxygenase 1 CRH- Corticotrophin-releasing hormone CRH1- Corticotropin-releasing hormone receptor one CRH2- Corticotropin-releasing hormone receptor two CRP- C-reactive protein DA- Dopamine dACC- Dorsal anterior cingulate cortex DAT- Dopamine Transporter DCX-Doublecortin xiv D2- Dopamine receptor subtype 2 DG- Dentate gyrus DIP- Direct intrahippocampal pathway DLPFC-Dorsolateral prefrontal cortex ELS-Early life stress GC- Glucocorticoids GFAP- Glial brillary acidic protein GR- Glucocorticoid receptors HPA- hypothalamic-pituitary-adrenal axis 5-HIAA- Serotonin primary metabolite 5-HT- Serotonin 5-HT1A to 5-HT7- Serotonin receptor subtypes 5-HTTLPR- Serotonin transporter gene IL-1-Interleukin one IL-1β-Interleukin beta IL-6-Interleukin 6 IPCs- Intermediate progenitor cells LC- Layer Chromatography LPS- Lipopolysaccharide LTP- Long-term potentiation xv MR- Mineralocorticoid receptor MDD- Major depressive disorder MRIs- Magnetic resonance imaging NAT- Noradrenaline Reuptake Transporters NE- Norepinephrine NF-κB- Necrosis factor caper beta NGF -Nerve growth factor NSC- Neuronal stem cells NSF- Novelty suppressed feeding NT-3- Neurotrophin-3 OFC-Orbitofrontal cortex PFC- Prefrontal cortex PIP-The polysynaptic intrahippocampal pathway PVN- Paraventricular nucleus RGL- Radial glial cells SERT- Serotonin transporter sgACC- Subgenual anterior cingulate cortex SGZ- Subgranular zone Sox2- Sex determining region Y-box 2 SSRI- Selective serotonin reuptake inhibitor xvi SVZ- Subventricular zone TNF-Tumour necrosis factor TNF-α- Tumour necrosis factor alpha TrkB- Tyrosine kinase receptor VMPFC-Ventromedial prefrontal cortex WRAF- Wits Animal Research Facility xvii LIST OF SYMBOLS α- Alpha β-Beta xviii LIST OF FIGURES Figure 2.1: Regulation of corticosterone in HPA axis: ............................................................ 8 Figure 2.2: Schematic representation of the intrahippocampal polysynaptic pathway (Prasad et al., 2019). ............................................................................................................................. 18 Figure 2.3: Schematic representation of the direct intrahippocampal pathway (Prasad et al., 2019) ........................................................................................................................................ 19 Figure 2.4: Cell stages in neurogenesis (Adapted from Samuels et al., 2016). ...................... 23 Figure 2.5: Representing the Amaryllidaceae, bulbous plant known as Boophone Disticha (SMGrowers.co.za). ................................................................................................................. 29 Figure 2.6: Representing the structures of the Boophone Disticha Alkaloids as follows: ..... 30 (A) Buphanamine (B) Buphanisine (C) Buphanidrine (D) Acetylenerbowdine, (E) Nerbowdine, (F) Udantaline, (G) Crimanidine, (H) Crinine, (I) Distichamine and (J) Lycorine (Cheeseman et al. 2013). .......................................................................................................... 30 Figures 3.1: Schematic representation of the forced swimming test. (Adapted from: Creative biolab. Accessed on 21 December 2022). ................................................................................ 37 Figure 3.2: Picture demonstrating the restrain procedure during the chronic restraint stress induction. ................................................................................................................................. 38 Figure 3.3: Schematic representation of the elevated plus maze (Adapted from: Media Wiki pedia: Accessed on 21 December 2022). ................................................................................. 40 Figure 3.4: Schematic representation of the open filed test (Adapted from: Biomed.au.dk: Accessed on 22 December 2021). ............................................................................................ 41 Figure 3.5: Schematic representation of the light dark box .................................................... 43 Figure 4.1: Spectrum view of LCMS chromatogram, acquired in the positive-ion mode, from an extract of the bulb of boophone disticha. ............................................................................ 48 xix Figure 4.2: Graphic representation of changes in swimming times after 21 days of treatment. .................................................................................................................................................. 52 Figure 4.3: Graphic representation of the elevated plus maze results. ................................... 54 Figure 4.4: Graphic representation of the volumes of the dentate gyrus and hippocampus across the groups. ..................................................................................................................... 56 Figure 4.5: Photomicrographs showing DCX immunoreactive cells in the subgranular zone (SGZ) of the dentate gyrus of the hippocampus of the normal (control) (A) and treated (B to D) groups. ................................................................................................................................ 57 Figure 4.6: Graphic representation of the expression of DCX immunoreactive cells (DCX-ir) and biochemical analyses of brain derived neurotropic factor (BDNF) and corticosterone. ... 58 Figure 4.7: Summary of changes in food consumption and weigh changes in the intervals of days 7, 14, 21, and 28 during chronic restraint Stress induction period. ................................. 61 Figure 4.8: Graphic representation of the elevated plus maze results in day seven of the treatment period. ...................................................................................................................... 63 Figure 4.9: Graphic representation of the none-significant elevated plus maze results in the 14th day. .................................................................................................................................... 64 Figure 4.10: Graphic representation of the significant differences in the elevated plus maze results in the 14th day of treatment. .......................................................................................... 65 Figure 4.11: Graphic representation of the significant differences in the elevated plus maze results in the 21st day of treatment. .......................................................................................... 66 Figure 4.12: Graphic representation of the significant differences in the elevated plus maze results in the 21st day of treatment. .......................................................................................... 68 Figure 4.13: Graphic representation of the time spent in light and dark box in day 7 of the behavioral tests......................................................................................................................... 69 xx Figure 4.13: Graphic representation of the time spent in light and dark box in day 14 of the behavioral tests......................................................................................................................... 70 Figure 14: Graphic representation of the time spent in light and dark box in day 21 of the behavioral tests......................................................................................................................... 71 Figure 4.15: Graphic representation of the time spent in the center of the open field during behavioral tests......................................................................................................................... 73 Figure 4.16: Graphic representation of the time spent on the sides of the open field during behavioral tests......................................................................................................................... 75 Figure 4.17: Graphic representation of the frequency of rearing in the open field during behavioral tests......................................................................................................................... 76 Figure 4.18: Graphic representation of the frequency of line crossing in the open field during behavioral tests......................................................................................................................... 77 Figure 4.19: Graphic representation of the volume of the hippocampus proper/CA. ............ 79 Figure 4.21: Graphic representation of the volume of the granule cell layer of the hippocampus. ........................................................................................................................... 81 Figure 4.22: Graphic representation of the brain levels of BDNF and corticosterone. .......... 83 Figure 4.23: Demonstration of nonspecific failed DCX stain: ............................................... 84 xxi LIST OF TABLES Table 4.1: Phytochemical profile of BD based on LC-MS data ............................................ 49 Table 4.2: Body masses (grams) ............................................................................................. 50 Table 4.3: Showing weight changes during the 28 days chronic restraint induction period ... 60 Table 4.4: Showing changes in food consumption during the induction of the chronic restraint stress model. ............................................................................................................................. 61 Table 4.5: Body masses (g) ..................................................................................................... 62 1 CHAPTER ONE 1.0 INTRODUCTION World Health Organization identifies depression as one of the leading causes of morbidity and mortality worldwide (Jogdand, 2014). Depression is a complex disease that can occur as a result of a multitude of different factors, including biological, emotional, and environmental influences (Zayka et al., 2015). Diagnosis of depression is mainly based on symptomatic behavioural criteria, such as depressed mood, low self-esteem, and recurrent thoughts of death and suicide. The heterogeneity of depression suggests that multiple different biological mechanisms may underlie its aetiology (Anacker, 2014). It is estimated that 40% risk of developing depression is genetic, though the specific genes involved are partially understood (Homung and Heim, 2014). The other 60% non-genetic risk remains poorly defined, with diverse theories implicating acute or chronic stress, childhood trauma, viral infections, and even random processes during brain development as possible causes (Kessler el. al, 2003). In addition to structural, learning and memory changes observed in depression, chronic stress results in dendritic remodelling i.e dendritic atrophy and spine loss in the hippocampus (Sierakowiak et al., 2015). It has also been shown that chronic stress causes a decrease in cell proliferation and concomitant increase in granule cell death in the dentate gyrus of the adult hippocampus (Saaltink and Vreugdenhil, 2014). There is however, no evidence to date to show if stress reduces the total number of neurons in the hippocampus and if so, the cellular mechanisms by which this neuronal loss occurs (Kheirbek and Hen 2013). Other reports suggested that dendritic restructuring, decreased hippocampal neurogenesis, and decreased neuronal cell survival associated with stress may provide a cellular basis for the impairments seen in the brains of patients with depression (Qiao et al., 2016). Several studies have also shown that stress and depression causes a reduction in hippocampal neurotrophin levels, including brain derived neurotropic factor (BDNF), leading to dysfunction of the 2 cholinergic system (Smith et al., 1995; Kaneko et al., 2006). The neurotropic factor family has been the focus of much of the work on stress and depression, and the most widely studied member of this family is BDNF (Duman, 2002). Brain derived neurotropic factor, nerve growth factor (NGF) and neurotrophin-3 (NT-3), influence the proliferation, differentiation, and growth of neurons during development, but are also expressed in the adult brain and play a critical role in the survival and function of mature neurons (McAllister, 2002). High levels of BDNF are expressed in limbic brain structures implicated in mood disorders, including the hippocampus, prefrontal cortex, and amygdala, and act through a transmembrane tyrosine kinase receptor referred to as TrkB (Tanis et al., 2007). Exposure to immobilization stress results in a dramatic reduction in levels of BDNF in the rodent hippocampus (Smith et.al, 1995) relative to the major subfields of the CA3 pyramidal cell layer and granule cell layer of the hippocampus whereas dendritic atrophy and decreased neurogenesis are observed respectively in response to stress (Smith et.al, 1995). Although there is no evidence suggesting that depression associated anatomical and chemical changes in the hippocampus involve a change in neuronal number, antidepressants may supposedly mediate their long-term therapeutic effects by triggering cellular mechanisms that counteracts the structural impairments. Czen et al, (2001) further demonstrated that chronic, not acute, treatment with the atypical antidepressant tianeptine, reverses the stress-induced impairments such as changes in metabolite concentrations, decreases in neurogenesis and reduction in volume of the dentate gyrus in the hippocampus. A study by Pawluski et.,al, 2014 showed that minipump administration of fluoxetine in female rats increased cell proliferation in the hippocampal granular cell layer. Most of the current antidepressant drugs have undesirable side effects and are beyond the reach of many South Africans, and Africans in general. As a result, the populace mainly relies on alternative and complimentary traditional medicines for antidepressant therapeutics. These 3 factors have led to an investigation into natural products and herbal medicines with potential anxiolytic and antidepressant properties including Boophone disticha (BD) as affordable and easily accessible alternatives to conventional antidepressants. Therefore, the present study evaluated the anxiolytic/ fluoxetine-like action of BD in search for a cheap, easily available and accessible alternative to modern conventional antidepressant drugs. Boophone disticha (L) (family Amaryllidaceae; tumbleweed/sore-eye flower) is an indigenous psychoactive bulb that is widely used in Southern Africa. Boophone disticha belongs to the Amaryllidaceae family, Boophone genus, which can be found throughout Southern and Tropical Africa (Gadaga et al., 2011). It has been reported that South African traditional healers use the bulbs and leaves of Boophone disticha to treat anxiety (Sandager et al., 2005). The study done by Gadaga and colleagues (2011), described the neurotoxicological effects of the Boophone hydro-ethanolic extract ranging from mild tremors to limb paralysis and death at high doses. In the above study, the LD50 was estimated to be above 120 mg/kg, while 50 mg/kg dose showed little toxicity, and hence 50 mg/kg was recommended for therapeutic purposes. A dose of 10mg/kg has previously been shown to significantly reduce mean arterial pressure in mice (Pote et.al, 2014). In addition to the above, crude extracts of the leaves of Boophone disticha have been shown to have affinity for the selective serotonin reuptake inhibitor (SSRI) site of the serotonin transporter (Pedersen et al., 2008). Furthermore, two alkaloids isolated from Boophone disticha (buphanadrine and buphanamine), have been shown to be responsible for binding to the selective serotonin reuptake inhibitor (SSRI) site of the serotonin P transporter (Cheesman et al., 2012). These mechanisms may partially explain why Boophone disticha is used traditionally for the management of anxiety and depression. Thus, much needs to be done in current and future studies to test this hypothesis. The focus of the present study is to validate if a crude extract of Boophone disticha has any anxiolytic-like 4 and/or antidepressant-like activity and its effects on the chemical neuroanatomy using the established mouse models of depression. Available literature showed no report on the effects of Boophone disticha treatment on disorders due to stress on behaviour and brain structure in animal models of depression, and the present study is designed against this backdrop to elucidate the neuro-therapeutic effects of the hydroethanolic extract of the bulbs of Boophone disticha. This study further, attempts to bridge the knowledge gap between the toxicity and beneficial effects of Boophone disticha in in-vivo treatment of anxiety disorders in an effort to further extend the frontiers of the search for suitable, affordable and available antidepressant drugs from traditional herbs. Boophone disticha is expected to demonstrate antidepressant, anxiolytic and neuroprotective effects in the mouse models of depression. 5 CHAPTER TWO LITERATURE REVIEW 2.1 Overview of depression Major depressive disorder is a devastating mental illness that has profound effects in the quality of life of the affected individuals. Symptoms of depression that mostly affect the individuals quality of life include low-self esteem, poor concentration, sleep disorders, loss of pleasure and interest, and sadness amongst other things (Depression, W.H.O., 2017). While the aetiology underlying this disorder is not clearly understood, psychosocial stress is one of the major factors implicated in this condition (Costello et at., 2002; Yang et al., 2015). Stress is described as the external or internal events or conditions that affect the organism (Goldberger and Breznitz, 2010). These adverse events challenge the equilibrium in an organism’s physiological state, triggering a cascade of adaptive responses (Chrousos and Gold, 1992; Chrousos, 2009). It has been suggested that the ability of an individual to respond to stress is influenced by genetic factors, with the inheritability of 32 to 38% chances (Feder et al., 2009). The hypothalamic pituitary adrenal (HPA) axis is an essential component of the stress system, and its accurate functioning is crucial for the success of the adaptive response to stress (Tsigos, and Chrousos, 1994). The response to stress is meant to be temporal, to restore the physiological equilibrium and disappear (Tafet and Nemeroff, 2016). However, chronic stressful conditions result into maladaptive behaviours that lead to pathological conditions such as mood disorder including depression (Caspi et al., 2003; Heim et al.,2008; Nemeroff and Seligman, 2013). 2.2 Prevalence of depression The report by the Institute of Health Metrics and Evaluation indicated that 280 million people, including 23 million children and adolescents, were living with depression in 2019 (Institute of Health Metrics and Evaluation, 2021). The burden of depression has been shown to be high 6 in both males and females across the entire life span in many locations around the world (GBD 2019 Mental Disorders Collaborators, 2022). However, studies have shown that the prevalence of depression is higher in females (5.1%) compared to males (3.6%) (Ferrari et al., 2013). There is also evidence that despite many majors put in place to reduce the impact of depressive disorders globally, no reduction in prevalence of depression is realized since 1990 (Patel et al.,2016). The African continent is no exception to the pandemic of depression representing about 10% of the global burden of mental (WHO, 2017). A review by Gbadamosi et al. 2022 noted that the epidemiological data in Sub-Saharan African countries is poor with only a few countries, including South Africa, represented in the published data. The disease was aggravated by the outbreak of SARS-COVID-19 pandemic which caused a 25% increase in prevalence of depression worldwide (COVID-19, WHO, 2022). One study estimated that 53.2 million new depression patients were added globally after the outbreak of COVID-19 throughout the year 2020 (Santomauro et al., 2021). In areas like the African continent where poverty and mental health issues already have a negative interaction in these vulnerable population, COVID-19 had a significant negative impact (Álvarez-Iglesias et al., 2021). The increase in prevalence of depression after COVI-19 outbreak has become one of the main driving factors for plant derived antidepressant agents that can be used with easy access and minimal negative effects (Fedorova et al., 2021). 7 2.3 Endocrine process in depression Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is among the most prevalent biological findings in major depression (Baumeister et al., 2016). Altered levels of cortisol is one of the important varieties of hormonal abnormalities, indicating the existence of endocrine disturbances, more specifically the dysfunctions in the hypothalamic-pituitary- adrenal (HPA) axis (Brigitta, 2002). The HPA axis consists of positive and negative feedback loops that involve the brain, pituitary, and adrenal glands, which regulates glucocorticoid production (Keller et al., 2017). When environmental stress is experienced, monoamines, serotonin, norepinephrine and dopamine are released from the amygdala, hippocampus, and other brain regions (Manke, 2019), which triggers the paraventricular nucleus (PVN) of the hypothalamus to synthesize corticotrophin-releasing hormone (CRH). This hormone binds to corticotropin- releasing hormone receptor one (CRH1) and two (CRH2) in the anterior pituitary. The binding of the CRH causes secretion of the adrenocorticotropic hormone (ACTH) into the circulation, which activates the production and release of glucocorticoids (GCs) in the adrenal glands (cortisol in humans, corticosterone in rodents). Homeostasis is restored by the negative feedback mechanisms initiated by binding of GCs to glucocorticoid receptors (GR) of the hippocampus, hypothalamic paraventricular nucleus (PNV) and the anterior pituitary gland, inhibiting further release of CRH (Figure 2.1) (de Kloet., 2005). However, in the major depressive disorder (MDD), the sensitivity of the GR is impaired leading to a reduced negative feedback mechanism resulting in hypersecretion of CRH and an elevated production of GCs (Holsboer, 2000). These elevated levels result into HPA shifting into high set point causing prolonged elevated HPA activity in patients with MDD (Raison and Miller, 2003, Young et al., 2003). In 2020, a Human Sciences Research Council conducted a study which showed that 33% of South Africans were depressed (Nguse and Wassernaar, 2021). 8 Figure 2.1: Regulation of corticosterone in HPA axis: Exposure to stress stimulate the hypothalamus to secret corticotrophin releasing hormone (CRH). The CRH activates the adreno-corticotropic hormone (ACTH) in the pituitary gland. Aceto-corticotrophin hormone stimulates the adrenal gland to release corticosterone (CORT). Corticosterone will than bind to the glucocorticoid receptor and form GR complex which subsequently undergoes dimerization. The binding of the GR complex to CRH and ACTH makes a closed loop that results into a negative feedback (Sriram et al., 2012). Furthermore, changes in serotonin receptors are some of the effects of hypercortisolism (Angst et al., 2007, de Kloete, 1998), which are thought to be the mechanism for specific symptoms observed in severe MDD (Leonard, 2005). In humans, association between MDD and cortisol levels seems to be dependent on the stage and severity of the disease (Nandam et al., 2020). A study using dexamethasone suppression testing recorded no differences in salivary and serum cortisol levels between controls and patients who had chronic MDD for more than two years (Watson et al., 2002). On the contrary, 9 high cortisol levels were observed in remitted MDD patients compared to controls after exposure to a visual stress cue (Holsen et al., 2013). The above suggests that in remitted MDD, persistent hyperresponsiveness to dexamethasone can be used to predict relapse, while in chronic MDD, cortisol levels do not provide prognostic information (Zobel et al., 2001; Watson et al., 2002). In addition, a number of studies have shown that severity of MDD is proportional to cortisol levels, with more severe subtypes, like melancholic and psychotic, consistently displaying elevated baseline cortisol levels than atypical MDD which is a less severe subtype (Zobel et al., 2001; Posener et al., 2000; Schatzberg et al., 2014; Keller et al.,2006; Karlović et al., 2013). Early life stress (ELS) is one of the factors that are responsible for programming responses to abnormal adult corticosterone during stressful events (Hunter et al., 2011). Changes to GR and mineralocorticoid receptor (MR), glucocorticoid resistance and increased central CRH activity are the potential mechanisms via which responses to corticosterone changes are programmed (Heim and Binder, 2012, Juruena, 2014). Impairment of HPA axis in adulthood has been recorded in a number of childhood trauma associated psychiatric disorders including MDD, suggesting persistent abnormal cortisol into adulthood (Heim et al., 2001). 2.4 Neurotransmitter systems in depression Neurotransmitters are important connecting molecules in the transmission of neural signals with precision and intensity at either presynaptic or postsynaptic level (Branco and Staras, 2009). In the 1750’s, effective antidepressant drugs were introduced stimulating the interest in the brains neurochemistry and neurotransmission system (Palazidou, 2012). Deficiency of mono-amine neurotransmitters together with the abnormal function of the neurotransmitter receptors is the biological etiology of the major depressive disorder (MDD) (Liu et al., 2020). Abnormalities in expression of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) 10 monoamine neurotransmitter systems has been observed in several neuronal circuits in different regions of the brains of subjects with MDD (Castren, 2005; Hamon and Blier, 2013). The disruption of monoamine neurotransmitters has a potential to affect the function of their receptors. Decreased levels of DA in the receptors causes over excitability of the amygdala due to failure of inhibition from the frontal cortex, resulting in fear and pathological anxiety (Liu et al., 2018). The D2 receptor subtype of DA has been specifically found to have lower density in patients with anxiety disorders compared to their heathy counter parts (Shin and Liberzon, 2010). Reports by previous researchers recorded evidence that decreased expression of DA receptors in the striatal pathways, with enhanced activity of the insula and adjacent operculum, play a role in mood alterations and related behaviours including eating disorders (Stice et al., 2009; Frank et al., 2012), suggesting that the regulation of mood by the dopaminergic system is through the insula. Furthermore, it has long been hypothesized that decreased activity of 5-HT pathways is part of the mechanism in the pathophysiology of depression (Coppen A, 1967; Hogenelst et al., 2016). The administration of reserpine in humans has been shown to deplete brain stores of 5-HT and to interfere with its synaptic vesicles, resulting into depressive symptom (Shore et al., 1955). Further research noted that reserpine induced depressive symptoms could be reversed by monoamine precursors (Hirschfeld, 2000). In addition, patients with MDD have been shown to have lower 5-HT serum concentrations compared to healthy controls, suggesting 5-HT deficiency in patients with MDD (Bot et al., 2015; Phillips, 2017). A number of studies have recorded low levels of primary metabolite of 5-HT (5-HIAA) in patients with MDD, specifically those exhibiting suicidal behaviour (Kruesi et al., 1990; Asberg, 1997; Placidi et al., 2001). The low concentrations of 5-HT observed in MDD are probably due to low neuronal 5-HT synthesis and abnormal function of 5-HT receptors (Artigas, 2013). At least five of the 11 fourteen 5-HT receptor subtypes: 5-HT1A, 5-HT1B, 5-HT4, 5-HT6, and 5-HT7 presumably play a role in depression (Hamon et al., 1990; Beck et al., 1992; Riad et al., 2000; Richardson- Jones et al., 2010). The subsequent development of selective serotonin reuptake inhibitors (SSRIs), that work by increasing availability of 5-HT in the receptors ameliorating depressive symptoms in patients, supported the theory that 5-HT systems are a biochemical basis for MDD (Cowen, 1990). A preclinical study in transgenic mice discovered that altering levels of raphe 5-HT1A autoreceptors is a determining factor to response and no response to antidepressant treatment (Richardson-Jones et al., 2010). The above study found mice with lower levels of 5-HT1A autoreceptors to be more resistant to stress, with better response to SSRI treatment than mice that had elevated levels of 5-HT1A autoreceptors. Furthermore, mice exhibiting decreased levels of 5-HT1A autoreceptors have also been shown to respond to acute treatment with SSRIs in novelty suppressed feeding (NSF), a behavioural paradigm that usually responds to chronic antidepressant treatment of at least 14 days (Richardson-Jones et al., 2010; Samuel and Hen, 2011) indicates that the negative feedback of raphe 5-HT1A autoreceptors has a temporal limiting effect in behavioural response to SSRI treatment (Yohn et al., 2017). The involvement of NE in the pathophysiology of depression was proposed in 1979, whereby it was suggested that depressive symptoms are caused in part by the reduction of NE in the central nervous system (Zis and Goodwin, 1979). Further research has shown that inability to adequately regulate stress response is one of the key factors in the pathophysiological of depression (Carboni et al., 2010; Weger and Sandi, 2018). Several studies have shown that there is overstimulation of NE in MDD patients (Yehuda et al., 1998; Mausbach et al., 2005; Nutt et al., 2006). In addition, norepinephrinergic neuronal cell bodies have been recorded to have increased binding of agonist ligands at α2-adrenergic autoreceptors, indicating higher 12 activity of these NE autoreceptors, which is indicative of reduced noradrenergic neurotransmission in MDD (Hamon and Blier, 2013). 2.4.1 Inflammatory process in depression Activated inflammatory pathways and increased proinflammatory cytokines have been observed in patients with major depression (Raison et al., 2006; Miller et al., 2009, Stetler and Miller, 2011). Furthermore, cytokines reach the brain and interact with neurotransmitter metabolism, neuroendocrine function, neural circuitry, and synaptic plasticity, all of which plays a role in pathophysiology of depression (Borden and Parkinson, 1998). In the brain, cytokine signals stimulate synthesis, release, and reuptake of dopamine, norepinephrine and serotonin, which are mood-relevant neurotransmitters (Felger et al., 2007; Anisman et al., 2008; Miller, 2009). Other effects of proinflammatory cytokines are on the HPA-axis where they cause increase in levels of CRH and ACTH, as well as cortisol in patients suffering from depression (Besedovsky and del Rey, 1996; Pariante and Miller, 2001). Prolonged activation of cytokine networks in the brain systems can cause dysregulation of cognitive function and glial/neuronal circuits thereby contributing to the pathophysiology of depression (Miller et al., 2009). Increased serum and plasma concentrations of interleukin 6 (IL-6) and C-reactive protein (CRP) are frequently observed inflammatory markers in depressed patients (Howren et al., 2009). In addition, BDNF, an important requirement of antidepressant response which fosters neurogenesis has been shown to be reduced by increased levels of interleukin beta (IL-1β), tumour necrosis factor (TNF) and necrosis factor caper beta (NF-κB) in stress-induced animal models of depression (Goshen et al., 2008; Koo et al., 2010). Furthermore, inflammatory mediators have been associated with depressive symptoms, whereby both acute and chronic administration of cytokines/cytokine inducers like lipopolysaccharide (LPS) can cause behavioural symptoms similar to those seen in patients 13 with major depression (Reichenberg et al., 2001; Brydon et al., 2008). Elevated levels of hippocampal TNF-α and IL-1 and cognitive impairment have been observed after peripheral administration of LPS resulting from reduction in expression of hippocampal BDNF (Wu et al., 2007). Additionally, hippocampal dependent learning and memory, synaptic plasticity and neuronal survival are linked to BDNF and its signalling pathways, partially explaining the cognitive impairment seen with elevated levels of proinflammatory cytokines (Sairanen et al., 2005). 2.4.2 Genetic predisposition in depression Epidemiological evidence suggests that heritability of bipolar disorders is about 80% based on the studies done on twins, adoption and family, where genetic factors were found to play a significant role in the aetiology of affective sicknesses (Berrettini, 1999). Furthermore, development of the MDD has been found to have a very strong dependence on gene- environmental interactions (Kendler et al., 1997; Klengel and Binder, 2015; Binder, 2017). Gene–environment interaction is a different effect of an environmental exposure on disease risk in persons with different genotypes or a different effect of a genotype on disease risk in persons with different environmental exposures (Ottman, 1996). The pioneering study on gene-environment interaction in depression was done in 2003, and demonstrated close interaction between a functional polymorphism in the serotonin transporter gene (5-HTTLPR) and stressors that predict depression (Caspi et al., 2003). Additionally, a study done in healthy male adults who were exposed to public speaking stress task showed significant effect of the BDNF Val66Met polymorphism on HPA-axis reactivity (Alexander et al., 2010). 14 2.5 Neuroanatomy of depression The connection between the cingulate gyrus, hippocampus, the hypothalamus and the anterior thalamic nuclei, the structures collectively referred to as a limbic system, was first described by James Papez in 1937. He described the above circuit as a communication between these brain structures that plays a role in the storing of memory and also enabling the cortex to control the emotions. Further advances in technology including the introduction of neuroimaging techniques broadened the knowledge on the importance on ‘neurocircuit of emotion’ where the prefrontal cortex (PFC) was also found to play a role in the circuit (Pelazidou, 2012). This circuit has been shown to be responsible for maintaining emotional stability, and the destruction in the structures involved in it is believed to be central to the pathophysiology of depression. 2.5.1 The role of the prefrontal cortex in depression The PFC is located anterior to the premotor and the primary motor area of the frontal cortex. It is divided into three main parts: (i) the dorsolateral, (ii) the paralimbic, and (iii) the anterior cingulate cortex (ACC). The ventromedial (VMPFC) and the dorsolateral (DLPFC) connect with each other via the cingulate gyrus and the hippocampus (Pelazidou, 2012). Decreased activity of the PFC, including its connections to and from other depression linked limbic and subcortical structures, has been observed in depressed patients (Savitz and Drevets, 2009). Previous studies conducted on MRIs of MDD patients have shown large volumetric reduction in anterior cingulate cortex (ACC) and orbitofrontal cortex (OFC) (Koolschijn et al., 2009; Gray et al., 2020). In addition, MMD patients with large grey matter volume in the right dorsal anterior cingulate cortex (dACC) and right inferior frontal gyrus have better clinical outcomes and fewer symptoms of depression (Serra‐Blasco et al., 2016). Studies in rodents found the right ventromedial prefrontal cortex (vmPFC), an analog of the human subgenual 15 cingulate cortex (sgACC) is of critical importance in the regulation of HPA axis (Sullivan and Gratton, 1999). Chronic stress that lasts for a number of days to weeks is known to induce behavioural depressive symptoms and brain structural changes (Ferenczi et al., 2016; Willner, 2017). The structural changes due to chronic stress are not only limited to the amygdala and hippocampus, but they also involve the PFC that includes dendritic spine density amongst other changes (McEwen and Morrison, 2013; McEwen et al., 2016). 2.5.2 The role of amygdala in depression The amygdala is a brain structure connected to the superior aspect of the hippocampus. It plays an important role in processing emotions, affective state, conditioning of fear and social behaviours (Fox and Shackman, 2019; Hur et al., 2019; Janak and Tye, 2015; Lindquist et al., 2012; Putnam and Chang, 2021; Shackman et al., 2016). Studies indicate that the amygdala function acquires a sustained increase in the activity during acute phase of depression, which normalises after antidepressant treatment (Sheline et al., 2001; Fu et al., 2004; Siegle et al., 2007). The recorded increase in amygdala activity has been shown to be associated with severity of symptoms of depression (Drevets et al., 2002). Furthermore, depressed patients with first episode of MDD were shown to have increased volume of the amygdala while on depression treatment (Frodl et al., 2002; Lange and Irle, 2004; Weniger et al., 2006). On the other hand, patients who recovered from recurrent depression showed no structural or volumetric increase of the amygdala, suggesting that the volume normalised after antidepressant treatment (Mervaala et al., 2000; Caetano et al., 2004; Hastings et al., 2004). It has been suggested that the increase in the volume of amygdala is related to the increase in the generation of oligodendrocytes (Wennström et al., 2004). 16 2.5.3 Role of the hippocampus in depression The hippocampus is a key hub of the limbic system known to be dysfunctional in the neuropathology of the MDD (Campbell and MacQueen, 2004). The hippocampus is an elevated tissue of gray matter that lies within the parahippocampal gyrus in the floor of the temporal horn of the lateral ventricle (Fogwe et al., 2018). It consists of three distinct zones: the dentate gyrus (DG), the hippocampus proper, and the subiculum, which is a transitional zone joining the DG and the hippocampus proper (Fogwe et al., 2018). Definitions may also include the presubiculum and parasubiculum, which are the anatomically adjacent areas, and the functionally connected areas which includes entorhinal, parahippocampal cortices, and white matter areas including the fimbriae and the fornix (Amaral et al., 2007). The hippocampus proper consist of the cornu ammonis subfields 1 to 4 (CA1–CA4) (Amaral et al., 2007). CA3 and CA2 form the borders of the hilum of the dentate gyrus on either side (Fogwe et al., 2018). CA3 forms the larger subfield of the hippocampus that receives fibers from the proximal dendrites of the granule cells of the DG (Daugherty et al.,2016). Each subfield of the hippocampus is further organized into distinct layers. The main cellular layer is the pyramidal cell layer which is also referred to as stratum pyramidale which contains approximately 300,000 – 400,000 cells in rodents (Miettinen et al., 2012). The neurones of the above layer have been shown to connect to different cortical and subcortical structures with the exception of few cases where one neurone project into two different regions (Ishikawa and Nakamura, 2006). The above connections are believed to project between the ventral hippocampus to the medial nucleus of the amygdala, whereby they modulate bulbar cardiorespiratory control circuits (Ajayi et al., 2018). Other layers include a noncellular stratum oriens which lies deep to the pyramidal cell layer, and the noncellular stratum lucidum, which lies above the pyramidal cell layer in the CA3 sub- field (Ajayi, 2019). The stratum lucidum is also present immediately superficial to the 17 pyramidal cell layer in CA1 and CA2 subfields, where it contains the apical dendrites of the cells in the pyramidal cell layer. In addition, mossy fibres projecting from the DG to the CA3 subfield mainly occupy the above layer (Ajayi, 2019). Lying superficial to the stratum lucidum is the stratum radiatum which constitutes intrinsic connections within the subfield, and lastly, the superficial area of the hippocampal regions is the stratum lacunosum-moleculare, which receives terminal projections from the thalamus, entorhinal cortex, and other cortical areas (Amaral, 1995). The DG is made up of three layers namely, the superficial stratum moleculare that has scattered neuronal cells, the middle stratum granulare with densely packed oval shaped granule cells, and the stratum multiforme which consists of polymorphic neuronal cells (EI Falougy et al., 2008). The main hippocampal circuit functions within the hippocampal formation which includes subiculum inferiorly, the DG medially and the molecular layer (Schultz and Engelhardt, 2014). Autopsy and imaging studies have shown that the hippocampus, parahippocampal region, and the neocortical association are essential for memory processing (Fogwe et al., 2018). Bilateral damage to the above regions has been shown to cause impairment of short-term memory resulting into an inability to form new memories (Kizilirmak et al., 2019). Learning and memory circuit comprises two important pathways, the polysynaptic (Fig. 2.2) and direct (Fig. 2.3) intrahippocampal pathways (Anand and Dhikav, 2012). The polysynaptic intrahippocampal pathway (PIP) comprises entorhinal cortex, DG, CA and the subiculum (Prasad et al., 2019). Inputs to the PIP originate from the occipital cortex, temporal cortex, and posterior parietal cortex via the entorhinal cortex as summarised in Figure 2.2 (Prasad et al., 2019). 18 Figure 2.2: Schematic representation of the intrahippocampal polysynaptic pathway (Prasad et al., 2019). The direct intrahippocampal pathway (DIP) receives input from the inferior temporal association cortex and connect directly with CA1 through layer III of the entorhinal cortex as illustrated in Figure 3 (Prasad et al., 2019). 19 Figure 2.3: Schematic representation of the direct intrahippocampal pathway (Prasad et al., 2019) 20 The hippocampus is intricately sensitive to stress and the stress hormone class, glucocorticoids. The DG sub-region has a high density of GC receptors that respond to increased levels of circulating GCs resulting into hippocampal atrophy (De Kloet et al., 1998). Hippocampal atrophy has been linked to a number of mechanisms (Dhikav and Anand, 2011). One of the important mechanisms resulting into hippocampal atrophy is stress resultant from increase in circulating glucocorticoids (Gilbert and Brushfield, 2009). Deposition of beta amyloids, suppression of hippocampal neurogenesis, impairment of long term potentiation, oxidative and metabolic stress are five mechanisms implicated in stress induced hippocampal atrophy (Hiramatsu et al., 1992; Hamaguchi et al., 2006; Howland and Wang, 2008; Srivareerat et al., 2009; Ondrejcak et al., 2010; Rothman, and Mattson, 2010; Tran et al., 2010; Yassa and Stark, 2011). 2.5.3.1 Beta amyloid deposition and hippocampal atrophy Amyloid beta (Aβ) is a peptide that is secreted by neurones in an activity-modulated manner as observed in the pathophysiology of Alzheimer’s disease whereby it removes dendritic spines, and sites of excitatory synaptic transmission (Wei et al., 2010). The effects of Aβ accumulation are profound, including loss and deterioration of synapse, inflammation and eventually, cell death (Cline et al., 2018). Previous studies have shown that large accumulation of Aβ accompanies reduction in the volume of hippocampus (Knopman, 2013; Jack et al., 2014; Gordon et al., 2016). Similarly, chronic stress has been implicated in dendritic atrophy and loss of dendritic spines in areas CA1, CA3, and loss of hippocampal volume probably due to the same Aβ mechanism (McEwen, 1999; Sousa et al., 2000; Pawlak et al., 2005). 21 2.5.3.2 Suppression of neurogenesis and hippocampal atrophy A process of production of new neurons in the adult hippocampus, referred to as neurogenesis, is now believed to be taking place in several mammalian species (Peter et al., 1998; Lucassen et al., 2010). Neurogenesis is believed to play an important role in the pathophysiology of depression, however, there is still a lack of explanation as to how the newborn neurons play a role in mood and other symptoms of depression (Kempermann et al., 2008). A view that neurogenesis is part of the mechanism of action of the antidepressant drugs has been suggested (Santarelli et al., 2003). The above is because reduction in neurogenesis cannot produce depressive symptoms on its own even though the prolonged reduction in the formation of new granule cells in the dentate gyrus (DG) can impair the composition of the cell population in the DG and make it vulnerable to impaired functioning (Lucassen et al., 2014). It is for this reason that part of the success of antidepressant treatment should be the restoration of the normal rate of neurogenesis (Kempermann et al., 2008). Most neuronal stem cells (NSC) in the adult brain are inactive, however, NSCs in the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) slowly divide to generate new neurons, a process called neurogenesis (Kempermann and Gage, 1999; Encinas et al., 2011; Ming and Song, 2011). The belief in the existence of adult neurogenesis started in the 1960s when adult generated brain cells were identified in rodents (Altman, 1962). This philosophy of newly generated adult brain cells was met with great skepticism for three decades until confirmed in numerus mammals including humans and non- human primates (Cameron and Gould, 1994; Peter et al., 1998; Gould et al., 1999). Recent studies have also shown the persistence of neurogenesis in Balb/c mice and avians, noting that the proliferation and migration of newly formed cells decline with age (Nkomozepi et al., 2019a; Nkomozepi et al., 2019b). In animals, the newly formed cells migrate into the olfactory bulb and subsequently get integrated into the olfactory neuronal circuitry (Alvarez-Buylla and 22 Garcıa-Verdugo, 2002; Sun and Moon, 2010; Faiz et al., 2005). Five stages of developing granule cells have been described (Fig. 2.4) (von Bohlen Und Halbach, 2007). The first stage is the proliferation stage which is represented by neuronal stem cells or by type 1 radial glial cells (RGL) which can be labelled with glial brillary acidic protein (GFAP), Sex determining region Y-box 2 (SOX2) and Nestin or even other markers of stem cells. Stage 2 is marked by intermediate progenitor cells (IPCs) that arise from RGLs. The IPCs continue to divide and show expression of doublecortin (DCX). In stage 3, IPCs give rise to migrating neuroblasts which also express DCX and give rise to stage 4 mature dentate gyrus neurones. In stage 5, newly formed neurones are integrated into the hippocampal circuitry. However, research has shown that neurogenesis is upregulated by a number of factors such as pregnancy, environmental enrichment, exercise, antidepressant treatments, while other factors like aging and stress downregulates neurogenesis (Fan et al., 2017; Trinchero et al., 2019; Wan et al., 2019). The reduction in neurogenesis has been shown to cause reduction in the volume of the dentate gyrus and CA3 in the hippocampus after four weeks of suppression of neurogenesis, and reduction of whole hippocampal volume after eight weeks (Schoenfeld et al., 2017). However, suppression of neurogenesis is not a main contributor of hippocampal volume loss compared to factors such as reduction in number of inhibitory interneurons and astrocytes seen in depression throughout the hippocampus (Czéh et al., 2015; Tata et al., 2006). 23 Figure 2.4: Cell stages in neurogenesis (Adapted from Samuels et al., 2016). 2.5.3.3 Long term potentiation and hippocampal atrophy Long-term potentiation (LTP) is described as a persistent strengthening of synapses leading to long-lasting increase in signal transmission between neurons (Fu and J hamandas, 2020). Long- term potentiation can be disrupted by chronic stress and/or glucocorticoid administration which may lead to depression (Srivareerat et al., 2009). Deposition of Abeta, glucocorticoid administration, and chronic stress are some of the factors thought to be responsible for disruption of LTP in long term depression (Hamaguchi and Yamada, 2006; Howland and Wang, 2008; Adekar et al., 2010). It is believed that enhanced postsynaptic potentials following presynaptic stimulation serves as a substrate in the hippocampal neuronal circuit during learning and memory (Molnár, 2011). 2.5.3.4 Oxidative stress and hippocampal atrophy Stress and Abeta (Alpha beta) is broadly known to increase production of free radicals in the brains of experimental animals (Howland and Wang, 2008; Srivareerat et al., 2009). A number of authors have shown that the pyramidal cells of CA3 and granule cells of the dentate gyrus 24 are prone to oxidative stress, with some authors suggesting that pyramidal cells of CA1 are the ones that are more susceptible to oxidative damage (Chang et al., 2012; Huang et al., 2012, 2013; Uysal et al., 2012). 2.6 Stress effects on plasticity and molecular structure As mentioned in sections above, the most widely understood explanation of mental and brain illnesses is the imbalances in the monoaminegic neurochemical system. Modern research has shown that pathophysiology of stress involves abnormalities in the plasticity and the changes in the volume of limbic structures of the brain (Lucassen et al., 2014). The above changes include apoptosis, remodeling of dendrites, and changes in glial cells (Czéh and Lucassen, 2007; MacQueen and Frodl, 2011; Rajkowska and Stockmeier, 2013). These changes in limbic brain structures are thought to be coupled with behaviors observed in stressed individuals, even though it is uncertain whether the structural changes are reversible adaptations to stress or pathologic changes (Lucassen et al., 2014). 2.6.1 Changes in dendritic spines The importance of the dendritic spines is their involvement in storing the information and formation of synapses, all of which are impaired by prolonged chronic stress (Tasker and Herman, 2011). Exposure to chronic stress and prolonged administration of high doses corticosterone is known to cause blunting of the apical spines of the dendrites of neurons in CA3 and CA1 of the hippocampus after a couple of weeks of stressful conditions (Woolley et al., 1990). Other effects of chronic stress include changes in vesicles and mitochondria at synapses, larger surface area of postsynaptic density and reduction of synapses of mossy fibers (Sandi et al., 2003; Tata et al., 2006). Reduction in dendritic spine densities and atrophy which was reversible with training or recovery period has been recorded in stressed animals (Magariños et al., 1996; Sandi et al., 25 2003; Stewart et al., 2005). The above effects have been observed specifically in the prefrontal cortex and pyramidal cells of the hippocampus (Goldwater et al., 2009; Radley and Morrison, 2005; Radley et al., 2005). In addition, the morphology of both pre- and post-synaptic neurons is markedly altered after prolonged exposure to stress and glucocorticoids (Liston et al., 2013). The opposite of the above effects has been recorded in the nucleus accumbens and amygdala whereby exposure to chronic stress resulted in increased density and hypertrophy of dendritic spines (Mitra et al., 2005; Vyas et al., 2004). 2.6.2 Stress and apoptosis Apoptosis, also called programmed cell death is a process whereby cell growth and division ceases resulting into the dead of cells that are subsequently removed from the system by the process of phagocytosis (D’Arcy, M.S., 2019). Prolonged exposure to glucocorticoids has also been shown to causes shrinkage and volume reduction of the hippocampus and cerebral cortex, probably due to increase in neuronal cell apoptosis (Swaab et al., 2005; Lucassen et al., 2006; Sapolsky et al., 2002; McKernan et al., 2009; Shelton et al., 2011). Contrary observations have been reported in patients and animal models of depression where no neuronal loss or hippocampal shrinkage was observed (Lucassen et al., 2001; Müller et al., 2001). In a previous study where the dentate gyrus of unmedicated patients showed fewer granule cells, it was suggested that the cellular changes in the hippocampus of depressed individuals could be related to the length of time of the disease (Boldrini et al., 2013). In rodent stress models, apoptosis has been observed in acute stress (Heine et al., 2004; Lucassen et al., 2006; Yu et al., 2011), while in chronic stress, apoptosis only happens in some hippocampal structures and normalises after treatment (Lucassen et al., 2004). The above is crucial in interpretation of the structural and neuronal changes observed in the experiments of the depression animal models to properly explain both the effects of depression and antidepressant treatment (Huang et al., 2013; Lucassen et al., 2001). 26 2.7 Treatment of depression Given the known pathophysiology of depression, the medical sciences have developed antidepressant drugs that would counteract the symptoms of depression in order to maintain healthy living (Khushboo and Sharma, 2017). The principle of treatment is based on the foundation of neurotransmitter functionality of the pre and post synaptic transmission, which is highly regulated by enzymes and several mechanical steps (Bondy, 2012). The steps of neurotransmitter function include synthesis, storage, release, induction of cellular response and termination of the neurotransmitter release (Bondy, 2012) . Firstly, the synthesis step is the process where neurotransmitter amino acid precursors, go through facilitated transport from the blood to the brain. In the brain, the precursors will be converted by enzymes into a specific neurotransmitter (Bondy, 2012). Secondly, in the storage step, the synthesized neurotransmitter will get stored in synaptic vesicles which are found within the neurons in the brain (Bondy, 2012) . Thirdly, the release step is the process of regulated release of the neuro-transmitter into the synaptic cleft (Bondy, 2012). Fourthly, the induction of cellular response step is the process where the neurotransmitter molecules within the synaptic cleft induce a signal transduction cascade reaction (Bondy, 2012) . It is also important to note that the induction reaction will be triggered by binding of the neurotransmitter on the surface of the neurotransmitter receptors located on the post-synaptic membrane (Whalen, 2018). However, neurotransmitter molecules do not cross or go through the post-synaptic membrane (Whalen, 2018). Finally, the termination step is carried out by the enzymatic process (Bondy, 2012). The antidepressant drugs will act by intervening in one of the steps above as described below. 2.8 Antidepressants and their mode of action Antidepressant drugs are divided into five categories which are: tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), serotonin noradrenaline reuptake inhibitors (SNRI), monoamine oxidase inhibitors, and atypical antidepressants (Ferguson, 2001). 27 Selective Serotonin Reuptake Inhibitors are a group of commonly used anti-depressant agents, mainly because of their high efficacy in alleviating depressive episodes (Whalen, 2018). According to Fasipe (2018) the mechanism of action of SSRIs is explained by their selective inhibition of the serotonin transporter (SERT). A more precise mechanism of SSRI is however its ability delaying the inhibition of serotonergic neurotransmission in about four pathways that occur after desensitization of 5-HT1A and 5-HT1B auto-receptors (Fasipe, 2018). However, SSRIs affect a wide range of postsynaptic receptor subtypes, which result in a wide range of side effects including nausea, headaches, dry mouth and sexual dysfunction (Ferguson, 2001). Thus, though effective, the resultant side effects limits its overall usefulness in the treatment of depression and necessitates the search of a cheap and better alternative treatment drug from natural plant sources. 2.9 Medicinal plants with antidepressant properties There is a fair amount of literature documenting the use of medicinal plants to treat depression (Aderibigbe, 2018; Ahmadpoor et al., 2019; Jahani et al., 2019; Rabiei and Setorki, 2019; Ilkhanizadeh et al., 2021). The study done on the antidepressant effects of Albizia adianthifolia, one of the plants used by traditional healers in African countries to treat depression showed reduction in immobility time in both forced swimming tests and tail suspension test in Swiss mice (Aderibigbe, 2018). Adiantum capillus-veneris (Pteridaceae), a cosmopolitan plant that is widely distributed in parts of the world that have tropical climate and high humidity is one of the plants that has been shown to have a wide range of medicinal properties including analgesic, anti-inflammatory, and anti-oxidant properties (Trojan-Rodrigues et al., 2012; Rajurkar and Gaikwad, 2012; Gaikwad et al., 2013). In addition to the above medicinal properties, a study conducted in male Balb/c mice exposed to chronic stress showed that Adiantum capillus-veneris possesses antidepressant properties in elevated plus maze and forced swimming test where the frequency of entry in open arms was increase and the immobility time 28 reduced in forced swimming test and elevated plus maze respectively (Ahmadpoor et al., 2019). From the African perspective, the study of Amaryllidaceae alkaloids began in the late 1990s with the investigation of the alkaloidal content of Crinum bulbispermum (Elegorishi et al., 1999). Since ancient times, medicinal plants belonging to this family have been used to treat depression in different cultures (Elgorashi, 2019). The rationale behind the use of Amaryllidaceae in traditional medicine and their medicinal efficacy is attributed largely to the presence of a unique type of alkaloids. These alkaloids are present exclusively in this family and have been isolated from each member of Amaryllidaceae investigated so far. Few South African Amaryllidaceae species have been investigated for their affinity for the serotonin re- uptake transport protein (Nielsen et al, 2004). Extracts of the leaves and bulbs of B. disticha have exhibited strong affinity to the SSRI site, while leaf extracts of Brunsvigia grandiflora and root extracts of G. ciliaris have moderate to low affinity to SSRI site, respectively (Nielsen et al, 2004). In addition, ethanolic extracts of BD showed functional inhibition of serotonin transporter, noradrenalin transporter, and dopamine transporter upon screening in a functional inhibition assay using COS-7 cells (Pedersen et al, 2008). The efficacy of Amaryllidaceae alkaloids from Boophone disticha (BD) stimulated the screening of many Amaryllidaceae alkaloids isolated from a number of Crinum and Cyrtanthus species for their affinity to the SSRI site (Elegorishi et al., 1999). These activities were also confirmed in vivo using the tail suspension and the forced swim tests in mice and rat models (Viladomat et al, 1997, Pote et al, 2018, Gadaga et al., 2010). There is currently a paucity of information regarding the mechanism by which BD exerts its antidepressant effects on the brain. The current study used mice model to close this gap. Mice and rats possesses numerous advantages for research purposes, such as sharing 90% genome homology with humans, in addition to many genetic, physiological, and organ anatomical similarities (Tabassum et al, 2015). 29 2.10 Overview of Boophone Disticha Boophone disticha (BD) belongs to a large family of bulbous flowering plants known as Amaryllidaceae (Fig. 2.5) (Nair and Van Staden, 2014). The genus “Boophone” consists of only two species, namely: Boophone disticha and Boophone haemanthoides (Nair and Van Staden, 2014). Boophone disticha plant has been used for centuries by traditional healers within the Southern African regions (Neuwinger et al.,1997). Rock paintings and Khoi-San mummies that contained Boophone disticha scales, further substantiate the fact that this plant has been used by South African natives for centuries (Nair and Van Staden, 2014). Figure 2.5: Representing the Amaryllidaceae, bulbous plant known as Boophone Disticha (SMGrowers.co.za). The Amaryllidaceae plants have been extensively used for medicinal purposes by some of the most popular South African tribes namely: Sotho, Zulu and Xhosa (Nair and Van Staden, 2014). Different parts of the plants are used to treat different conditions. For example, dry bulbs are used for the feeling of weakness, fresh bulbs are taken to increase sexual potency, leaves 30 are taken for gastrointestinal wash and roots are burned and ground to powder in order to be applied to an area where one would experience paralysis (Gadaga et al. 2011). 2.10.1 Alkaloids of Boophone Disticha The medicinal activity can be attributed to the alkaloids of the plant (Neuwinger, Leon-Rot and Mebs, 1997). There are eleven alkaloids that have been isolated in the BD plant, which are buphanidrine, undulatine, buphanisine, buphanamine, nerbowdine, crinine, crinamidine, and lycorine, distichamine, acetylnerbowdine and buphacetine (Fig. 2.6) (Neuwinger et al., 1997) Figure 2.6: Representing the structures of the Boophone Disticha Alkaloids as follows: (A) Buphanamine (B) Buphanisine (C) Buphanidrine (D) Acetylenerbowdine, (E) Nerbowdine, (F) Udantaline, (G) Crimanidine, (H) Crinine, (I) Distichamine and (J) Lycorine (Cheeseman et al. 2013). A previous study on the inhibitory binding affinity of the alkaloids, of BD extract, on the Serotonin Reuptake Transporters (SERT) showed that Buphanadrine, Buphanamine and Distichamine have high binding affinity for the serotonin reuptake site (Neergaard et al., 2009). 31 According to Gadaga et al. (2011), buphanidrine is the most abundant alkaloid found in BD, occurring at a percentage abundance of 19.4%. The plant is well known for its powerful analgesic and neurotoxicity effects. Furthermore buphanidrine has a high hepatotoxicity at profile at high doses (Neuwinger, Leon-Rot and Mebs, 1997). Buphanidrine, has an LD50 of 8.9 mg/kg for mice administered Subcutaneously. A dose of 10 mg/kg has previously been shown to significantly reduce mean arterial pressure in mice (Pote et al., 2014). 2.10.2 Pharmacological Activity of Boophone Disticha The alkaloids of the BD give the plant a source of its pharmacological activity (Nair and Van Staden, 2014). Boophone disticha exerts its anti-inflammatory effects by inducing the production of Adenosine triphosphate (ATP) on isolated human neutrophils while simultaneously inhibiting the release of superoxide from neutrophils (Gadaga et al. 2011). The above mechanism could account for the plant’s traditional use to alleviate rheumatic pains, muscular sprains and other inflammatory conditions. Additionally, BD inhibit cyclo-oxygenase 1 (cox-1) enzyme (Gadaga et al. 2011). This enzyme is responsible for maintaining hemostasis by inducing platelet aggregation (Whalen 2018). A previous study showed that BD displaces more than 50% of the transport protein bound citalopram at various concentrations (Neergaard et al., 2009). A study on a serotonin binding assay showed inhibition of serotonin (Nair and Van Staden, 2014). A study done by Gadaga et al, (2011) showed that BD has a high affinity for binding to the receptors of the SSRIs, and inhibition of Serotonin Reuptake Transporters (SERT), Noradrenaline Reuptake Transporters (NAT) and affinity for Dopamine Transporter (DAT). The anticholinesterase effects (inhibition of cholinesterase enzyme) of BD have been demonstrated using thin Layer Chromatography (LC), explaining the use of this plant for memory enhancement and tretment Alzheimer’s disease (Gadaga et al., 2011). 32 2.11 Rodent models of depression Despite a tremendous effort dedicated to neuro-psychiatric pharmacological research, not much has been attained to counteract the onset and progression of affective disease, with most treatment strategies simply targeting the mono-aminagic neurotransmitter system (Hyman, 2014). This setback is due to the lack of detailed understanding of the molecular, cellular and neuronal circuits involved in the pathophysiology of the affective disorders, with some suggestions genetic and environmental interplay in the disease process (Caspi and Moffitt, 2006; Sullivan, 2015; Han and Nestler, 2017; Wray et al., 2018). To acquire a deeper understanding of the pathophysiology of affective disorders, rodent models have become increasingly relevant in the research of mental disorders (Italia et al., 2020). Mice are the most commonly used animals model in the research of mental disorders due to ease of handling and versatility in different behavioral tests (Hylander et al., 2022). Forced swimming test (FST) is one of the commonly used stress models in mice to demonstrate the effects of mild unpredictable stress (Becker et al., 2021). On the other hand, chronic restraint stress is useful for modeling chronic life psycho-emotional stressors which might have more longer lasting effects (Huang et al., 2015; Xu et al., 2017; Wu et al., 2018). Based on the argument that FST is a test for copying mechanism and not necessarily a stressor, it is important to combine the FST with chronic stress in drug testing as chronic stress might be more resistant to treatment than the acute stress (Molendijk and de Kloet, 2022). For example, a previous study demonstrated that neuropeptide Y is more effective in reversing the effects of acute stress and less effective in reversing the effects of chronic stress (Andriushchenko et al., 2022). 33 2.12 Aims and objectives of the study 2.12.1 Aim of the study To determine the effect of Boophone disticha on the behaviour and hippocampal neuroanatomy in male Balb/c mouse model of depression. 2.12.2 Objectives (i) To determine the anxiolytic like and antidepressant-like activity of orally gavaged hydroethanolic extract of Boophone disticha and compare with similar effects of fluoxetine using open field test, elevated plus maze and light dark box in established mouse models of depression. (ii) To compare the effects of orally gavaged hydroethanolic extract of Boophone disticha and fluoxetine on levels of brain derived neurotropic factor (BDNF) and corticosterone in the brains of mice subjected to different established mouse models of depression by ELISA technique. (iii)To compare the effect of orally gavaged hydroethanolic extract of Boophone disticha to fluoxetine on the volume of the hippocampus and its dentate gyrus of the mice subjected to different established mouse models of depression on Nissl stained sections using Volumest plugin of image J. (iv) To compare the effect of orally gavaged hydroethanolic extract of Boophone disticha and fluoxetine on the expression of doublecortin (DCX) in the dentate gyrus of the hippocampus using immunohistochemical techniques (immunostained brains sections) in mouse brain subjected to different established mouse models of depression. 2.12.3 Hypothesis Buphoone disticha attenuate the effects of stress on the behaviour and neuroanatomy of Balb/c mouse model. 34 CHAPTER THREE METHODOLOGY 3.1 Experimental animals Seventy-two adult male Balb/c mice (Postnatal day 30) were used in the current study. Animals were purchased and housed in the Wits research animal facility (WRAF) and handled with the help of professional personnel. Mice were randomly allocated to one of the following two established models of depression, 1) five days repetitive forced swimming forced swimming model (5d-RFSS), 2) 28 days repetitive restraint stress model. The procedure for induction of the stress models is described below (section 3.4 - 3.5). Animals randomly assigned to each stress model were treated for 21 days post stress induction as shown in Appendix 1. Briefly, each stress model had four groups of animals (Control, distilled water, BD and fluoxetine groups) with 9 animals in each group (n=9), adding up to 36 animals per stress model. The Control group was the normal animals that were not exposed to stress, the distilled water group was the stressed animals that were orally gavaged with 0.2 ml of distilled water, the BD group was the group that was orally gavaged with 10 mg/kg of BD dissolved in 0.2 ml distilled water, the fluoxetine group was the positive control that was orally gavaged with 10mg/kg of fluoxetine. During days 7, 14 and 21 of the treatment period, the animals underwent the following behavioural tests: open field test, elevated plus maize and light dark box as described in section 3.6.1 below. For a forced swimming stress model, the forced swimming was used for both induction of stress and a behavioural test during the treatment period. The tests were done in a random order. Following behavioural studies, animals were anesthetized using isoflurane for a short period, followed by perfusion using normal saline. After this procedure, brains were harvested as described in section 3.7 below. Male mice were chosen in the current study to eliminate the confounding hormonal factors as the female mice could be at different oestrous cycles. While it is not impossible to monitor the 35 individual for differences in the estrous cycles, the current study design did not cater for that procedure. 3.2 Plant material and extraction of phytochemicals Bulbs of BD were purchased from Random Harvesters in Muldersdrift Johannesburg, South Africa and were authenticated at the Department of Botany, University of Johannesburg, South Africa. A table knife was used to chop the fresh bulbs of BD into small pieces and sun dried for seven days. The dried plant material was ground into a fine powder using a kitchen blender. The powder was then mixed with 70% ethanol in a ratio 5:1 (70% ethanol: BD powder) and placed on an automatic shaker for 24 hours at room temperature. After 24 hours, plant debris were filtered using Whatman No. 1 filter paper. The resultant extract was concentrated using a rotary evaporator at 70ºC. The final product was a dark brown solid paste which was air dried, crushed into powder and stored at room temperature. The administered dose of the powder was weighted in grams according to each animal’s body weight and dissolved in 2ml distilled water and administered through orogastric gavage. 3.3 Identification of phytochemicals using LC-MS A Thermoscientific ultimate 3000 Ultra High-Performance Liquid Chromatography (UHPLC) in conjunction with Bruker Compact Q-TOF high resolution mass spectrophotometer (HRMS) was used to analyse 20 ul of BD extract dissolved in methanol. Gradient elution techniques was applied to elute 20 ul of the sample that was injected into the UHPLC in a period of 14 minutes. Solvent A with a concentration ranging from 95%-5% of 0.1% formic acid in water (v/v) and a solvent B with a concentration ranging from 5%-95% of 0.1% formic acid in acetonitrile (v/v) was used through Raptor ARC C18 Column (2.7 um 2.1 x100 mm). Analyses of the sample was done using the UHPLC-HRMS analyses, while Bruker Daltonics analyses was used for mass spectrum analyses. 36 3.4 Five day repeated forced swimming stress model (5d RFSS) Forced swimming stress model was first introduced by Porsolt et al, in 1977. The procedure for this model is to place the mouse for 6 minutes into a glass-polycarbonate cylinder (25 cm high×10 cm wide) filled to a depth of 10cm with water maintained at 24°C (Fig. 3.1). Immediately following the 6 minutes exposure into the water cylinder, the animals are removed from the cylinder and dried using a water absorbent pad. In the current study, we used a recently described 5 d repeated forced swim stress (5d-RFSS) protocol (Serkov et al., 2015), in which mice were forced to swim in an open cylindrical container (diameter, 10 cm; height, 25 cm) containing 10 cm deep water (25°C) for five consecutive days. The five days procedure constituted the induction phase of the stress model. This protocol had been shown to produce the depressive symptoms that last for at least four weeks, allowing for a time window to test and study therapeutic interventions (Serkov et al., 2015). After the five days induction period, animals were treated for 21 days vial oral gavage as described in section 3.1, while behavioural tests were conducted and video recorded at intervals on days 7, 14 and 21, after 5d RFSS. The period/duration of swimming and immobility during the 6 minutes test was measured from the video recordings and recorded using a timer. While the FST has received extensive criticism regarding its validity and reproducibility, a meta-analysis of 73 behavioral studies that used FST in mice demonstrated external validity and reproducibility of the FST where different types of antidepressant drugs reduced immobility in the test (Kara et al., 2018). The issue of ethics regarding the use of the FST has been raised due to its perceived suffering to animals (PETA, 2020). However, there is currently no regulation against the use of the FST. The fact sheet published by British Association for Psychopharmacology in conjunction with, the Laboratory Animal Science Association and Understanding Animal Research advocated for the continued use of FST as a valuable behavioral screening tool (LASA, 2020). A recent 37 commentary suggested the use of none-behavioral neurochemical measures like BDNF as an alternative to the FST (Sewell et al., 2021). Figures 3.1: Schematic representation of the forced swimming test. (Adapted from: Creative biolab. Accessed on 21 December 2022). 3.5 Repetitive restraint stress Clinical observations suggest that stress can act as a precipitating factor in the onset of affective illnesses, especially major depression (Bidzinska, 1984). The pathophysiology of depression and the neurobiology of stress are linked by their shared association with the hypothalamic pituitary-adrenal (HPA) axis and in particular with serotonin and norepinephrine containing neuronal systems (Breslow et.al., 1989). In animals, the behavioural deficits and the abnormalities in the nervous system and neuroendocrine system that are induced by exposure to uncontrollable and unpredictable chronic stress, such as hypo-activity in the open field, aberrations in the HPA system, and alterations in neuroplasticity and neurogenesis, can be 38 reversed by antidepressant treatments (Xu et.al., 2009). In this study, mice were restrained in acrylic cylinders (6.5 cm inner diameter, 20 cm long, with air ventilation at the sides and at the nasal end of the cylinder, Fig. 3.2) for 6 h (10:00–16:00 h) for 28 consecutive days (Makhathini et al.,2017). After the induction of chronic restraint, the 21 days treatment was conducted as described in section 3.1 with the behavioural tests in days 7, 14 and 21. Figure 3.2: Picture demonstrating the restrain procedure during the chronic restraint stress induction. (Adapted from: https://midsci.com/item/ASRATREST/Rat-Restraints/). https://midsci.com/item/ASRATREST/Rat-Restraints/ 39 3.6 Behavioural tests On the test day, mice were brought into the test room for at least one hour before the test. All testing was performed between 9:00 am and 15:00 pm by the experimenter. Behavioral tests were done randomly (Elevated plus maize, open field test, light dark box and forced swimming test). All behavioral activities were video recorded via an overhead video camera (Canon Legria, HF R806, Makro, South Africa) and later analyzed by the independent observer blinded