Categorization of Neurons in the Lateral Hypothalamus by Projection and Morphology

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 diversity 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, reconstructions 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 between 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 immunohistochemistry. 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]. The proteins examined in this experiment were MCH, PV and ER.

1.5 Brain Regions and Previous Research

The hypothalamus has a significant purpose in the brain. The diversity of cell populations control different functions such as feeding, energy balance and reproduction [21]. Also the hypothalamus covers the neuroendocrine and metabolic responses to stress. In an article from Journal of Affective Disorders, it has been shown that a decreasing volume of the hypothalamus generates anxiety and stress but is not involved in panic disorders [22]. Other studies show a development of anxiety when exposed to stress which differs between males and females. Women have greater risk of developing anxiety disorders, which is also proven in animal models [23]. The habenula and periaqueductal gray (PAG), two other brain regions, are also related to anxiety and receive inputs from the LHA [24]. PAG’s major function is related to pain, fear and anxiety [25]. The habenula connects different brain regions and is divided into two parts, the medial habenula and the lateral habenula (LHb). Both parts have a distinct gene expression from one another. The LHb is thought to be involved in motivation and social behaviour. A recent study, in vivo, in rhesus monkeys found that the LHb have a big role in the reward system. Depression models in rodents show an increased activity in the LHb [26]. In Figure 6 shows positions of lateral hypothalamus (LHA), LHb and PAG.

7 ern eefildwt ictnadte”ipenuietae”fnto fFj,from Fiji, of function tracer” neurite ”simple the marker and nucleus biocytin the with with filled slice individual were brain recordings neurons the patch-clamp in During nuclei determine Hoechst). all to (DAPI/ membranes target diamidino-2-phenylindole cell to the and on types proteins MCH cell and different EG done PV, was the with Immunohistochemistry in neurons projected. neurons target they to the region which where to of and statistics located and were from LHA software calculations a make IMARIS, to were. location used their was where BITPLANE, and LHA the the to from PAG projected to neurons and which LHb determine to performed were retro-beads with Injections Methods and Materials 2 brain. brain human mouse the the map use to to order is in aim model term the a in long LHA as neurons The the categorize morphology. in to and is neurons projection aim which study Another see PAG. by to the LHA and order LHb on the brains to mouse connected use are to that is study this of aim The Study the of Aim 1.6 [27]. subtypes neuronal identify published to study used a were In recordings neurons. electrophysiological in 2016, viruses in rabies injecting and injections retrograde istry, periaque- (red), (green). hypothalamus habenula lateral the lateral showing and brain (magenta) mouse gray the ductal of image 3D 6: Figure ieetmtosaeue nodrt a h ri,frisac immunohistochem- instance for brain, the map to order in used are methods Different

8 the company IMAGEJ, were used in order to recreate the morphology of the ﬁlled neurons.

2.1 Histology

Three months old wild type female mice were used for histological examination. Mice were perfused in 4% paranormalaldehyde and ﬁxed brains were removed. The brain was placed on a VT1200S vibratome and sliced into 50 µm or 350 µm thick coronal sections of the LHA. 350 µm slices were placed in a liquid in order to be cleared, CUBIC reagent 1 (25 wt% (weight percent) urea, 25 wt% N,N,N’,N’-tetrakis(2-hydroxypropyl) ethylenediamine and 15 wt% polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100) was used. The pieces were in the liquid for three days. After clearing, the samples were washed in 0.1 M phosphate buﬀer (PB). CUBIC reagent 2 (50 wt% sucrose, 25 wt% urea, 10 wt% 2,20,20’-nitrilotriethanol and 0.1% v/v% Triton X-100) was used for mounting and imaging the cleared tissue in a Zeiss LSM 880 confocal microscope.

2.2 Injections and Neouronal Filling

Retrograde injections in the LHb and the PAG with 50 nL retro-beads (ﬂuorescent latex microsphere [28]) were performed in vivo in anesthetized mice using a stereotaxic injection setup. The coordinates of the injection were LHb Bregma −1.65, −0.25, 2.3 and PAG Bregma −4.0, −0.3, 1.7. Biocytin was injected in 2-5 neurons in each (350 µm) slice during previously performed patch-clamp recordings in order to see the morphology.

2.3 Immunohistochemistry

Three diﬀerent combinations of antibodies were stained on two (50 µm) slices from two, three months old, female mice wild type. Blocking solution were added (100 µL NDS, 875 µL carrier solution and 25 µL Triton X-100) to all brain slices, and left for two hours at room temperature. Slices were washed with PB three times with two hours between the washes at room temperature. Primary antibodies were added to the brain

9 slices (Appendix, table 1) and the slices were incubated for two days at 4◦C. Slices were washed in PB three times for two hours. Secondary antibodies were added (Appendix, table 2), and were incubated overnight at 4◦C. The slices were washed twice with PB, and nuclear staining DAPI/ Hoechst (6 µL PB and DAPI 1 µL) were added. The slices were then incubated for 10-15 min and washed with PB three times with two hours in between. Thereafter the stained slices were mounted and imaged in a Zeiss LSM 880 confocal microscope.

2.4 Software Analyses

The neurons labeled by retro-beads which projects to the LHb and to PAG from the LHA, in the 350 µm slices, were counted in IMARIS (software from BITPLANE an Oxford Instruments company). Thus making a calculation of the amount of neurons in the LHA projecting to the LHb and PAG. The morphology of the biocytin-ﬁlled neurons were reconstructed in the ”simple neurite tracer” function of Fiji (From the company IMAGEJ).

3 Results

The retrobead injections marked neurons projected to the LHb and PAG from the LHA. As shown in Figure 7, neurons projected to the LHb are mostly located in the anterior parts of the LHA and fewer are located in the posterior parts. Neurons projected to the PAG are mostly deﬁned to the medial and posterior parts of the LHA. However, they are more commonly accumulated in the medial part and mixed with the neurons projecting to the LHb in the anterior parts of the LHA.

Figure 7: Row B are magniﬁcations of row A (dark red areas). The projections from the LHA to the LHb (green) and to PAG (magenta) are shown at three diﬀerent coordinates of the LHA, from anterior (right) to posterior (left).

The staining with immunohistochemistry targeted neurons with MCH-, EG- and PV- proteins, and a nuclei staining were made. In the overview image of the brain slices, shown in Figure 8, it can be seen in which regions the diﬀerent neurons with the MCH and PV are located. DAPI, the nuclei staining, colored every nuclei in the brain slices blue. The MCH colored only the neurons in the LHA. PV stained cells were not in the LHA, in fact they were mostly located in the cortex, hippocampus and the reticular nucleus of the thalamus (RT). In Figure 9, the ER positive neurons are concentrated in the LHA, more so in the ventral regions of LHA rather than the medial and dorsal regions. Furthermore, the immunohistochemistry staining showed that MCH increased in number from anterior to posterior positions in the brain, see Figure 10. In the anterior region of LHA there are almost no MCH positive neurons meanwhile in the posterior region they are commonly accumulated.

11 DAPI_blue MCH_green PV_red DAPI_blue MCH_green PV_red

A B C D

Figure 8: A) Complied image of the neurons positive for MCH (green), PV (red) proteins and DAPI (blue) nuclei staining. B) Nuclei staining of all existing nucleus in the brain slice. C) MCH positive neurons located in the LHA. D) PV positive neurons. Image taken with a Zeiss LSM 880.

A B C

Figure 9: The LHA are damaged in the outer parts, however the staining worked as con- sidered. A) DAPI (blue) colored nucleus in the LHA. B) ER (magenta) positive neurons. Image taken with a Zeiss LSM 880.

PV_red MCH_green PV_red MCH_green PV_red MCH_green

Anterior Medial Posterior Figure 10: MHC (green) and PV (red) anterior, medial and posterior coordinates in the LHA. Image taken with a Zeiss LSM 880.

12 As seen in Figure 11 (A, B and C) the neurons projecting to LHb are divided in two different groups, thus revealing the neurons from LHA projecting to the LHb have different morphology. LHb type 2 have the greatest total length of the neurons and LHb type 1 have the shortest. PAG and LHb type 2 have the most primary branch number while LHb type 1 have the least. LHb type 2 have the most branches in total and LHb type 1 have the least branch numbers. Neurons projecting to LHb which are type 2 are more alike the neurons projecting to PAG than LHb type 1, even though they project to the same region. The change in morphology can be seen in Figure 12 (Appendix) where both the reconstruction of the neurons and the graphs show an obvious difference between LHb type 1 and LHb type 2. Also morphological similarities between LHb type 2 and

PAG are shown.

A B C

Figure 11: Box plots describing the total length of the neurons, the primary branch numbers and the total branch numbers. 95% of the neurons are in the boxes, the whiskers describes the maximun and minimum values 95-99% from the average. The tiny squares are the average values and the center lines represent the median values. In A the total length of the neurons are shown, B describes the primary branch numbers and C (next page) the total branch number of the diﬀerent neurons.

13 4 Discussion

The immunohistochemistry stained neurons with the proteins MCH, PV and ER on their membranes so that neurons with the proteins were colored. Neurons in the LHA projecting to the LHb did not have the same morphology which could, in fact, help future experiments to categorize neurons in the LHA. The morphology gave possible outcomes where neurons projecting to the LHb from LHA were interesting, result seen in Figure 12 (Appendix). From what this result shows, the LHA had two different cell morphology types project to the LHb. Conclusions can be made that the neurons thar are projected to the LHb are different and should therefore have different purposes in the LHA. During the immunohistochemistry, three different antibodies were used in each brain slice. This was done in order to know if one neuron contained several of the proteins that were targeted. It could tell more about connections between neurons and different cell- types. However no results of this could be seen. In theory, more antibodies could have been used in order to get other combinations of antibodies in one slice. However, the visible light-spectra do not have that much color diversities, therefore more colors would have led to unreliable results. The unreliability increases with more colors thus the combinations of two (or more) different colors will be similar to one single color mark. In Figure 10 it is seen that the MCH antibody stained neurons only in the LHA proving neurons with the MCH protein is only located in the LHA, in these coordinates. The neurons with MCH increased in number more posterior in the brain. It was already known that the MCH neurons should be found in the LHA, however what became interesting was the sectioning throughout the LHA. The interneuronmarker PV, colored mostly the cortex and the hippocampus, is seen in Figure 8. It was already known that PV were in those regions and the results confirmed its reliability. A few PV marked neurons could be seen in the LHA, which shows that there are almost no interneurons there. It is interesting because a conclusion can be made

14 that the LHA do not control a significant amount of activity in the brain compared to the cortex and the hippocampus. Furthermore, a pattern in the ER stained neurons could be seen in Figure 9 showing there was a high concentration of ER in the LHA. It is problematic to draw any reasonable conclusions from this result us too few experiments were performed with the ER protein. Many speculations can be done regarding both the morphology and the immunohistochemistry results. However more neurons in the LHA project to the LHb need to be investigated to get better statistics of the LHA neuron morphology to clarify its plausi- bility. Hopefully further experiments could confirm what was found in this study. What would be a reasonable next step is immunohistochemistry on the brain slices with the two cell morphology types projecting to the LHb, in order to see if they express different proteins on the membrane. It could in fact lead to interesting results which could be a clue for mapping the brain. The result of where the MCH marked neurons were located showed that they are something to investigate further. As a conclusion, further studies are necessary in order to determine the functional differences between LHb type 1 and LHb type 2 as well as confirming and developing the immunohistochemistry results. Both these parts of the project will be further investigated in the DMC-lab (Dinos and Marie Carlén-lab) at the Karolinska Institutet of Neuroscience.

15 References

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18 A Appendix

The following tables (1, 2) are the recipes of the primary and secondary antibodies used in immunohoistochemistry.

Table 1: Primary antibody solutions

Solution Content Dilution Amount (µL) 1 Carrier solution 971 Triton 20% 25 ER α Rabbit 1:500 2 SST α Rat 1:1000 1 PV α Guinea Pig 1:1000 1 2 Carrier solution 970 Triton 20% 25 ER α Rabbit 1:500 2 PAX6 α Mouse 1:500 2 SST α Rat 1:1000 1 3 Carrier solution 972 Triton 20% 25 PV α Guinea Pig 1:1000 1 CHAT α Mouse 1:1000 1 MCH α Rabbit 1:1000 1

Table 2: Secondary antibody (1:500) solutions with ﬂuorescence

Solution Content Wavelength (nm) Amount (µL) 1 Carrier solution 994 Donkey α Rabbit 633 (Blue) 2 Donkey α Rat 555 (Red) 2 Donkey α Guinea Pig 488 (Green) 2 DAPI (1:50000) 1 2 Carrier solution 994 Donkey α Rabbit 633 (Blue) 2 Donkey α Mouse 555 (Red) 2 Donkey α Rat 488 (Green) 2 DAPI (1:50000) 1 3 Carrier solution 994 Donkey α Guinea Pig 633 (Blue) 2 Donkey α Mouse 555 (Red) 2 Donkey α Rabbit 488 (Green) 2 DAPI (1:50000) 1

A B C

Figure 12: Row A) The morphology of three diﬀerent neurons (row 1,2 and 3) in A30S3C4 (Animal 30, Slice 3, Cell 4), A28S3C1 and A29S5C2. A30S3C4 is LHb type 1, A28S3C1 is LHb type 2 and A29S5C2 is PAG. Row C) The graphs describes the number of crossings the neuron does in a circle (row B), thus explaining how complicated neurons are due to the number of crossings.