Categorization of Neurons in the Lateral Hypothalamus by Projection and Morphology Elin Hermann [email protected] under the direction of Prof. Konstantinos Meletis PhD. Daniela Calvigioni PhD. Janós Fuzik Department of Neuroscience Karolinska Institutet Research Academy for Young Scientists July 11, 2018 Abstract To cure brain disorders scientists must figure out how the brain functions. Thus the aim of this study is to see which neurons in the lateral hypothalamus (LHA) are connected to the Lateral habenula (LHb) and to periaqueductal gray (PAG). Additionally, the diver- sity of neurons will be investigated by projection and morphology in order to categorize neurons in the LHA. Retrograde injections were administrated in the LHb and PAG, re- constructions of the morphology of some neurons was performed. Immunohistochemistry with melanin-concentrating hormone (MCH), parvalbumin (PV), and estrogen (ER) in the LHA were conducted in different coordinates of the brain. It was shown that MCH varied in different coordinates, that PV marked neurons barely existed in the LHA and neurons with ER were common in the LHA area. Furthermore, the reconstruction of cell morphology showed two different-looking neurons projected to the LHb. One type looked similar to the neurons projecting to the PAG and the other had a simpler morphology. Future studies need to be performed in order to aquire more accurate results of both the reconstruction and the staining with immunohistochemistry to prove their reliability. Additionally, immunohistochemistry of the two different neurons projected to the LHb should be done in order to see a difference and potentially categorize them. Acknowledgements I would like to thank Prof. Konstantinos Meletis who let us to be in his lab at Karolinska Institutet. My deepest gratitude to my mentors PhD. Daniela Calvigioni and PhD. Janós Fuzik who guided, discussed with and supported me throughout this study. I also want to thank my lab partners, Gina Christoffersen, Hedieh Mohammadi and Naixuan Pei who worked in the lab. Thank you, all the xRays who gave me feedback and Rays for excellence who gave me the opportunity to do this project. Lastly I would like to thank Rays partners AstraZeneca and Kjell och Märta Beijers Stiftelse. Authors Contribution K.M., D.C. and J.F. designed the project. D.C. injected the mice, removed the brains from the mice, did CUBIC reagents for tissue clearing, imaged the 350 µm slices and J.F. biocytin-filled the neurons. J.F., D.C., G.C., H.M., N.P. and E.H. imaged the 50 µm pieces in the Zeiss LSM 880 confocal microscope. G.C., H.M., N.P. and E.H. did cell distribution analysis on IMARIS, morphology reconstructions with the ”simple neuritis tracer” function of Fiji and did the immunohistochemistry experiment. Contents List of Abbreviations 1 1 Introduction 2 1.1 Brain Coordinates . 2 1.2 Protein Descriptions . 3 1.3 Retrograde Injection, Tissue Clearing and Morphology . 4 1.4 Immunohistochemistry . 6 1.5 Brain Regions and Previous Research . 7 1.6 Aim of the Study . 8 2 Materials and Methods 8 2.1 Histology . 9 2.2 Injections and Neouronal Filling . 9 2.3 Immunohistochemistry . 9 2.4 Software Analyses . 10 3 Results 10 4 Discussion 14 References 16 A Appendix 19 List of Abbreviations α Anti. DAPI Diamidino-2-phenylindole. ER Estrogen. LHA Lateral Hypothalamus. LHb Lateral Habenula. MCH Melanin-Concentrating Hormone. NDS Normal Donkey Serum. PAG Periaqueductal Gray. PB Phosphate Buffer. PV Parvalbumin. RT Reticular nucleus of the Thalamus. 1 1 Introduction The human brain is complex and its functions are difficult to comprehend. However, it is necessary that they are understood in order to figure out how different brain disorders such as Alzheimer are developed, how they could be cured and how to prevent them. The mouse brain is closely related to the human brain as the same neuron types are found in the same brain regions in both humans and mice, thus the mapping of the mouse brain can be used as a substitute to mapping the human brain [1]. This project aims to figure out which regions are connected and how to categorize neurons in order to be able to map the brain. 1.1 Brain Coordinates Every mouse brain is unique, therefore scientists have a universal mouse brain atlas containing 132 images of the mouse brain in 100 µm thick slices with coordinates to compare their brain slices with. The coordinates of the brain are counted from bregma, which is an evident mark on the skull and brain seen in Figure 1. The parts anterior of bregma have positive numbers and the parts posterior to it have negative. Also from the bregma line in Figure 2 the length and depth are mentioned in order to know where the experiment was done in 3D. The coordinates are written, Bregma x, y, z (x= anterior/ posterior from bregma, y= right or left from bregma, z= depth). This is a necessary system in order for all scientists to know exactly where an experiment in the brain was performed [2]. 2 Figure 1: A mouse skull showing the bregma position, image from [3]. Bregma line Figure 2: A mouse brain slice showing the bregma line, image from [4]. 1.2 Protein Descriptions Interneurons are neurons that have their axons and dendrites in the same brain region as their soma (inhibitory cells). These neurons are the ones controlling activity levels in the brain [5] and are mainly found in the cortex which possesses the cognitive functions [6, 7]. Parvalbumin (PV) is proved to be an interneuronmarker, therefore it is used in order to find and mark interneurons in the brain [8]. Melanin-concentrating hormone (MCH) is a neuromodulator and neurotransmitter which regulates food and induces food intake [9, 10]. The protein is mainly produced in the LHA area [11]. Previous studies have shown that the protein is related to stress, which led to further research about MCH receptors and antidepressants [12]. 3 Estrogen (ER) is the female sex hormone and is a common protein that plays a big role in the central nervous system (brain and spinal cord) [13]. For women, a lack of ER is connected to depression, anxiety, panic disorders and irritability, and more so during menstruation. ER is expressed more in brain regions related to anxiety behaviors (e.g. amygdala and hypothalamus) [14]. 1.3 Retrograde Injection, Tissue Clearing and Morphology Every neuron has dendrites (input) and axons (output) handling the communication be- tween different neurons in the brain. Both the axons and dendrites can split into branches, see Figure 3. Thus the soma (cell body) can get information from different regions of the brain [15]. Figure 3: The morphology of three neurons in the LHA. During a retrograde injection fluorescent latex microspheres (retro-beads) are injected into a certain brain region and are then picked up by axons (output) of a soma in a different region [16]. The retro-beads are picked up by axons since of the synapse which opens whenever an ion impulse arrives [17]. Retro-beads are colored proteins that are 4 engineered to emit a certain color in the visible spectra (wavelength 300-650 nm) [18]. By performing an injection with colored retro-beads in a certain brain region, neurons that have axons connected to the injected region will pick up the colored particles. In Figure 4 the injection area was in the lateral habenula (LHb) and it is shown that neurons from the lateral hypothalamus (LHA) are colored red. Retrograde injections is a useful tool in neurology since it shows which regions are connected to each other, as shown in Figure 4. Figure 4: A mouse brain slice with a retrograde injection containing red retro-beads. The large red spot is the injection point in the LHb, the smaller red dots are neurons in the LHA connected to the LHb. Image taken by a Zeuss LSM 880. In order to get a reasonable complete morphology of neurons, 350 µm thick brain slices are necessary. Otherwise the neuron might be cut off which will give a false image of the morphology. To see the colored neurons in a microscope a tissue clearing is performed. During a tissue clearing, the lipids in the tissue are removed to make it more transparent [19]. Reconstruction of neuron morphology is interesting in order to determine different 5 cell types and to make it possible to create a model of the brain in the regions of interest. 1.4 Immunohistochemistry Antibodies can be used to mark certain proteins in neurons due to the specific protein- markers they give by their unique construction. This method is called immunohistochem- istry. By injecting proteins and antibodies into different species of animal, their body will not recognize the injected protein or antibody, thus the immune system will create a defense (antibody). Fluorescent color Secondary antibody Primary antibody Protein Neuron Figure 5: Primary antibodies bind to proteins on the neuron surface. The secondary antibodies bind to the primary antibody. A fluorescent color is connected to the secondary antibody in order to mark the protein in a choosen color. As seen in Figure 5, by injecting proteins for instance into a rabbit, the rabbit will produce antibodies against the proteins (primary antibody). The antibodies created by the rabbit are then injected into a donkey. In turn, the donkey will produce antibodies for the rabbit antibody, which a fluorescent color will be engineered to. This antibody is called donkey α (anti) rabbit (secondary antibody). By doing this, different proteins in neurons can be detected when a mouse brain slice is exposed to e.g. donkey α rabbit. Before the antibodies are added to the mouse brain, it is covered in a liquid with normal donkey serum (NDS) so that the secondary antibodies will bind to the primary antibodies 6 instead of the mouse brain [20].