Comparison of the effects of lithium orotate and lithium carbonate on locomotion and memory in a Drosophila melanogaster model of Alzheimer’s disease

Comparison of the effects of lithium orotate and lithium carbonate on locomotion and memory in a Drosophila melanogaster model of Alzheimer’s disease | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Comparison of the effects of lithium orotate and lithium carbonate on locomotion and memory in a Drosophila melanogaster model of Alzheimer’s disease View ORCID Profile Ishan Reddy Alla , Ryan Chung doi: https://doi.org/10.1101/2025.08.13.670149 Ishan Reddy Alla 1 Academies of Loudoun Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ishan Reddy Alla Ryan Chung 1 Academies of Loudoun Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: 924546{at}lcps.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Alzheimer’s disease (AD) is a terminal neurodegenerative disease characterized by cognitive decline and memory loss resulting from the buildup of amyloid-beta plaques and tau protein tangles. Lithium salts, particularly lithium carbonate (Li 2 CO 3 ), inhibit the enzyme Glycogen Synthase Kinase-3, which is upregulated in AD, reducing tau and amyloid-beta accumulation, inflammation, and oxidative stress. However, Li 2 CO 3 has declined in use due to long-term neurotoxicity. Lithium orotate (LiOr), an alternative lithium salt, has greater bioavailability–delivering more ions to the brain–but was discontinued in the 1970s due to toxicity concerns at equivalent doses. It was hypothesized that LiOr would be more effective than Li 2 CO 3 at lower doses (5 mM) due to higher lithium ion transfer, while Li 2 CO 3 may outperform LiOr at higher doses (10 mM) due to LiOr’s toxicity. This study compares LiOr and Li 2 CO 3 in treating locomotion and memory in a Drosophila melanogaster model produced by crossing UAS amyloid-beta 42 and Act5c GAL4 flies via the GAL4/UAS system. Once aged to 25 days, flies were assessed for locomotion using the negative geotaxis assay and for short-term memory using the aversive phototaxic suppression (APS) assay. These behavioral assays aim to quantify changes in cognitive and motor function resulting from treatment. Results suggest Li 2 CO 3 improves cognitive and motor performance in healthy flies, but LiOr was not found to be significantly more effective. This study offered a novel comparison of two lithium compounds in an Alzheimer’s model and may guide research into safer, more effective AD and lithium treatments. Introduction Alzheimer’s Disease (AD) is a terminal neurodegenerative disease, the most common form of dementia, that affects an estimated 6.7 million seniors living in the United States, a number expected to almost triple by the year 2060 (“ Alzheimer’s & Dementia”, 2023 ; Rocca et al., 2011 ). Characterized by progressive memory loss, cognitive dysfunction, and neuronal death, AD is pathologically marked by the accumulation of amyloid-beta plaques, tau protein tangles, and synapse loss ( Tue et al., 2020 ) linked to upregulated enzymes such as Glycogen Synthase Kinase-3 (GSK-3) which disrupts key cellular processes (Hooper et al., 2008). AD’s upregulation of these enzymes exacerbates neuroinflammation (immune response to damage in neurons) and mitochondrial dysfunction ( Alzheimer’s Association, n. d. ). Of the several hypotheses proposed to explain AD pathology, the amyloid-beta (Aβ) hypothesis is the best-developed, suggesting that an imbalance in the production and clearance of Aβ peptides forms Aβ plaques that disrupt brain function and trigger the remainder of AD processes ( Hardy, 2002 ). The immediate cause for AD onset remains unknown; a combination of genetic, environmental, and lifestyle factors are found to contribute to disease onset (“What causes Alzheimer’s disease”, 2024). As of now, AD has no cure, however, several treatments aim to improve the quality of life for patients by alleviating symptoms. Despite eight FDA-approved AD medications, only two target disease progression (via reducing Aβ levels) ( National Institute on Aging, 2024 ), but even those have limited success in improving cognitive function and are often accompanied by severe side effects such as brain swelling, bleeding, nausea, and dizziness ( Shen et al., 2024 ). This lack of an effective treatment underscores the need for further pharmacological research to develop safer and more effective treatments. Lithium, historically successful in mood stabilization, has shown promise for its neuroprotective potential in neurodegenerative diseases such as AD ( Brown & Tracy, 2013 ). Lithium’s inhibition of GSK-3 prevents β-catenin degradation (allowing β-catenin to accumulate and regulate gene transcription in the nucleus), which ultimately reduces Aβ deposition, enhances neuron growth, and improves cognitive function in AD ( Shen et al., 2024 ). Numerous animal studies have demonstrated that lithium treatment reduces Aβ and tau accumulation, attenuates neuronal cell death, and improves cognitive function. Shin et al. (2023) concluded that most studies have consistently reported that lithium reduces Aβ aggregation, phosphorylated tau formation, and neuronal death and improves learning and working memory in APP mouse models. Sofola-Adesakin et al. (2015) determined that, in an AD Drosophila model, lithium caused a reduction in Aβ42 protein synthesis. At all doses tested in the experiment, lithium attenuated the locomotory defects induced by Aβ42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. The most effective lithium drug in disease treatment, Li 2 CO 3, has a narrow therapeutic index (range of dosages with effective treatment) along with side effects ranging from inconvenient, such as nausea, to life-threatening, such as renal dysfunction ( Pacholko & Bekar, 2021 ). Research on Li 2 CO 3 ’s neurotoxicity after extended usage has caused a rapid decline in usage over two decades ( Pérez de Mendiola et al., 2021 ). Another lithium drug, lithium orotate (LiC 5 H 3 N 2 O 4 ; referred to as LiOr), is an aqueous lithium salt of lithium and orotic acid. Because orotic acid is a bioavailable (effective ion transmitter) mineral carrier that can more readily transport inorganic ions across biological membranes with a more consistent Li + ion transfer than Li 2 CO 3 , LiOr is predicted to cross the blood–brain barrier and enter cells more readily than Li 2 CO 3 , reducing dosage requirements and lessening toxicity concerns for LiOr ( Pacholko & Bekar, 2021 ). After LiOr was introduced, studies using rodent models determined that LiOr was more bioavailable than Li 2 CO 3 . In 1973, Hans Nieper found that LiOr yielded higher brain concentrations of lithium than Li 2 CO 3 , but possibly had higher toxicity. In 1978, a research experiment found that equivalent dosages of LiOr and Li 2 CO 3 led to LiOr having 3x the lithium transfer into the brain and a much more progressive pattern of lithium transfer compared to Li 2 CO 3 (Kling et al.). Although evidence for enhanced brain bioavailability in LiOr was initially found, research into LiOr was discontinued for decades due to renal toxicity concerns with equivalent doses of LiOr and Li 2 CO 3 ( Smith & Schou, 1979 ). These experiments failed to test for LiOr’s effectiveness at lower doses than Li 2 CO 3 due to its bioavailability, and as such, currently, there is a notable gap in the data regarding LiOr’s therapeutic index, particularly its impact on behavioral symptoms. LiOr has never been tested to see how varying doses affect any behavioral symptoms in AD. In the past decade, reviews have highlighted the wealth of shortcomings in these past comparisons between LiOr and Li 2 CO 3 ( Pacholko & Bekar, 2021 ). There is very limited data regarding the therapeutic index LiOr in comparison to Li 2 CO 3, as current lithium research has only been conducted with equivalent dosages of LiOr and Li 2 CO 3 . This experiment aimed to help fill the gap in research by systematically comparing LiOr and Li 2 CO 3 across a range of doses to determine their respective therapeutic indices in terms of memory and locomotive function in a Drosophila melanogaster AD model, leading to the research question: how do varying dosages of LiOr and Li 2 CO 3 compare in improving the locomotion and memory of a Drosophila melanogaster model of Alzheimer’s disease? Given the need to analyze the therapeutic potential of the lithium salts, Drosophila melanogaster models provide a potential platform for these needs. Drosophila melanogaster models preserve many mammalian processes, including some neurological processes. Not only do Drosophila melanogaster share 60% of human genes, specifically having fundamental similarities in the brain, but they also have a rapid generation and a short life span/cycle ( Mirzoyan et al., 2019 ). Their similarity to human brains implies that AD treatments that work on Drosophila could work on humans as well, whilst rapid generation and life cycle allow for Drosophila to be tested on in a relatively short period, allowing for more trials to be conducted. Drosophila melanogaster models are much less complex emotionally than mammalian models, which makes them an ethical model to use for research. To create a Drosophila melanogaster model for AD, the GAL4/UAS system is commonly utilized. This system allows for precise, tissue-specific control of gene expression. In order to use this system, a GAL4 Drosophila melanogaster with a specific promoter is maintained and crossed with a Drosophila melanogaster stock with a UAS target gene. The GAL4 protein is a transcription factor driven by the promoter. The GAL4 protein will bind to binding sites that are present along the UAS sequence, which means that any gene under the control of the UAS has its transcription activated when GAL4 attaches to the UAS sequence in the tissue location designated by the GAL4, leading to targeted gene expression ( Tue et al., 2020 ). For this experiment, a UAS stock with the Arctic mutation of Aβ-42 will be used ( Bloomington Drosophila Stock Center, n.d. ). The Arctic mutation was chosen because it produces more aggressive effects on both locomotion and memory compared to other genes ( Prüßing et al., 2013 ). More aggressive effects of AD will lead to a more distinct comparison of the effects of lithium treatments. An Act5C-GAL4 that expresses GAL4 ubiquitously under the Act5C promoter will be used ( Bloomington Drosophila Stock Center, n.d. ). ( Lee & Koh, 2023 ). The cross between the Arctic mutation of Aβ-42 and Act5C-GAL4 was chosen due to crosses between Aβ-42 and Act5C-GAL4 leading to locomotive and memory defects in the past ( Tan et al., 2023 ). Therefore, a cross between the Arctic mutation of Aβ-42 and Act5C-GAL4 should be effective for testing the effects of lithium on both locomotion and memory. It was hypothesized that if a D. melanogaster model of AD was subjected to both Li 2 CO 3 and LiOr treatments across various dosages, LiOr would have a superior therapeutic effect to Li 2 CO 3 on the locomotion and memory of the AD D. melanogaster at lower doses because of its higher bioavailability, allowing for reduced toxicity with lower doses while maintaining neuroprotective benefits. At higher dosages, LiOr is expected to be less effective than Li 2 CO 3 due to its toxicity at standard Li 2 CO 3 dosages ( Pacholko & Bekar, 2021 ). Using varying dosages of LiOr to test for its effect on memory and locomotion may identify advantages of LiOr in mitigating symptoms of AD at lower doses. This experiment will allow us to fill in much-needed gaps in the knowledge of LiOr’s therapeutic index, providing invaluable insight into the viability of LiOr as a potential treatment for AD. If LiOr is found to be a viable alternative to Li 2 CO 3 to treat locomotive and memory impairments in AD, treatments using LiOr can be developed in order to help improve the quality of life of AD patients. To examine the locomotive function of Drosophila , a negative geotaxis assay will be used. The negative geotaxis assay measures the natural climbing behavior of flies; deficits in this behavior can indicate motor function impairments associated with neurodegeneration. This assay was chosen due to its being very simple, as well as giving a solid understanding of the locomotive capabilities of the flies involved, which will allow for the analysis and comparison of locomotive impacts of lithium salts. To study memory in Drosophila , the aversive phototaxic suppression assay (APS) will be utilized. The APS assay measures the ability of the Drosophila to learn and remember to avoid light associated with an aversive stimulus, indicating cognitive function and memory. First, Drosophila are trained to associate the aversive smell with light, which goes against their natural phototaxic (attracted to light) behavior. Some time is spent waiting, then their behavior is tested again after some time to see if they remember the association. This assay was selected to measure memory due to the relative ease required to conduct while still providing a good measure of memory and the prevalence of APS in present Drosophila research ( Torre et al., 2023 ). Materials and Methods The independent variables were the dosages of either lithium treatment (LiOr and Li 2 CO 3 ) in the fly food and the presence of AD in the flies (Act5C-GAL4 D. melanogaster vs Alzheimer’s Disease D. melanogaster obtained via the crossing scheme). Both lithium drugs were administered at 5 mM and 10 mM to compare multiple studied doses of other lithium drugs ( Castillo-Quan, 2016 ). When converted from mM to mg/mL using the molar masses of LiOr (162.0 g/mol) and Li 2 CO 3 (73.89 g/mol), these doses correspond to 0.81 mg/mL & 1.62 mg/mL for LiOr and 0.369 mg/mL & 0.739 mg/mL in Li 2 CO 3 . Relevant dosage calculations are shown in Table 2 for LiOr and Table 3 for Li 2 CO 3 . View this table: View inline View popup Table 1 Control and Experimental Groups Download figure Open in new tab Figure 1 Groups Note. This figure presents a diagram of the groups compared. The dependent variables were locomotive function as measured using the negative geotaxis assay by the percentage of flies that successfully climb to a specified height in 10 seconds, and memory as measured using the Aversive Phototaxic Suppression (APS) assay by the percentage of flies that go toward the dark vial. Constants for this experiment included the age of flies (28 days), the time of data collection (1:50 PM - 3:30 PM EST), the sex of flies (male), the lab equipment used, and the location (Dr. Eliason’s Lab - Academies of Loudoun Room 2419). This project included six control groups (One negative control, one disease control, two Li 2 CO 3 toxicity controls and two LiOr toxicity controls for 5 and 10 mM) and four experimental groups (two LiOr treated AD and two Li 2 CO 3 treated AD for 5 and 10 mM). The negative control group will use Act5c-GAL4 D. melanogaster with no AD present and will not be treated with any lithium salts. This group provides insight into what the expected results of the assays are, allowing for the comparison of experimental groups with healthy flies. The negative control should perform well in both the climbing and memory assay due to there being no AD present. It should be noted that parent flies, flies without AD, will be used rather than wildtype, as transgenic flies may perform differently than wildtype. The disease control group will demonstrate the effects of the AD model being used. The disease control should perform worse than the negative control, because of the presence of AD. The presence of the disease control allows for the determination of whether or not the AD model works, or if either lithium salt makes any significant improvement over the base AD Drosophila . The other control groups are the toxicity control groups, which consist of D. melanogaster without AD present that are treated with Li 2 CO 3 or LiOr. These control groups provide insight on the effects of Li 2 CO 3 and LiOr on healthy flies, which is important specifically for this project due to the negative side effects associated with lithium salt treatments. These control groups are expected to perform slightly worse than the negative control group due to the aforementioned negative side effects of lithium salt treatments. Materials [AOS] indicates that the material will be sourced from the Academy of Science, rather than purchased online. Flies D. melanogaster Act5C-GAL4 [Bloomington Drosophila Stock Center - Stock #4414] D. melanogaster UAS-APP.Abeta42.E693G [Bloomington Drosophila Stock Center - Stock #33774] Fly Storage [AOS] ○ Sharpie ○ Tape ○ Clear plastic vials ○ Foam plugs (flugs) Fly Food ○ Yellow Corn Meal [AOS] ○ Distilled water [AOS] ○ Light corn syrup [AOS] ○ Soy flour [AOS] ○ Yeast [AOS] ○ Agar [AOS] ○ Li 2 CO 3 [AOS] ▪ https://www.fishersci.com/store/msds?partNumber=L119500+productDescription=LITHIUM+CARBONATE+CR+ACS+500G+vendorId=VN00033897+countryCode=US+language=en ○ Propionic acid - (10%, AOS) ▪ https://www.fishersci.com/store/msds?partNumber=A258500+countryCode=US+language=en ○ LiOr ( betterLife.com, 2019 ) ▪ https://www.chemicalbook.com/msds/Lithium-orotate.pdf ○ Cheesecloth [AOS] Lab Equipment [AOS] Paper towels Thermometer Pipette Graduated cylinders (100mL, 500mL) Beaker (500 mL, 1L) Weigh boats Scales Freezer Scoopulas Microwave (Panasonic 1250W Genius Sensor) Funnel Glass stirring rod Sorting Equipment [AOS] Weigh paper Magnifying glass Feather Cold sorter (AGP-301CPV) CO 2 plate Arc3 gasses CO 2 tank Genesee scientific Flystuff Blowgun Labomed Microscope Luxeo 6z Ice Ice Bucket Climbing Assay Equipment [AOS] Cotton balls Graduated cylinder (100mL) Marker Ruler Sharpie Tape Timer Video recording device APS Assay [AOS] > 95%, FG Quinine Hydrochloride Dihydrate ○ https://www.fishersci.com/store/msds?partNumber=AAH33474+productDescription=keyword+vendorId=VN00024248+countryCode=US+language=en Clear plastic vials Index card 0.45 mm Filter paper Fume hood LED fairy lights Opaque black tape Timer Safety Equipment [AOS] Oven Mitts Goggles Lab Coats Closed-toe shoes Fly food preparation Before experimentation, food for the treated and untreated groups was created. To prepare food, a weighboat and scoopula were used to measure out 6.75 g of yeast, 3.90 g of flour, 28.50 g of yellow cornmeal, and 2.25 g of agar and combined with 390 mL of distilled water (H 2 O) and 30 mL of light corn syrup into a 1L beaker using graduated cylinders and the scale. A glass stirring rod was used to mix the contents of the beaker until all materials were dissolved. The mixture was then placed into the microwave, where it was heated in 1-minute intervals until the mixture bubbled and rose. Following each minute, the beaker was removed from the microwave and stirred with the stirring rod. This process was repeated 3 times per batch of food made. Cheesecloth was put over the mixture and the mixture was cooled to 70°F. Once at 70°F, a micropipette was used to add 1.88 mL of propionic acid to the mixture and then mixed in using a stirring rod. Once stirred, about 10 mL of the food was placed into each empty vial, each vial was labelled, and cheesecloth was placed on the vials. Once all vials were filled, a flug was then placed on each vial and the vial tray was put into the fridge until use. Food for treated groups was produced using the same procedure mentioned above, with amounts of LiOr and Li 2 CO 3 added into the beaker and dissolved. For LiOr, 375 mg was added for 5 mM and 751 mg was added for 10 mM as calculated in Table 2 . For Li 2 CO 3 , 171 mg was added for 5 mM and 342 mg was added for 10 mM as calculated in Table 3 . View this table: View inline View popup Download powerpoint Table 2 LiOr Diet Preparation Calculations View this table: View inline View popup Download powerpoint Table 3 Li 2 CO 3 Diet Preparation Calculations Sorting flies Two different sorting methods were used, carbon dioxide (CO 2 sorting) and cold sorting. CO 2 sorting was used to sort flies that would be used for crosses, whilst cold sorting was used to sort flies that would be used for experimentation due to the damage that CO 2 can cause to the brain of flies. To sort using CO 2 , firstly the CO 2 canister and CO 2 sorter lights had to be turned on so that CO 2 was flowing. Once both were turned on, the fly vials were flipped upside down and tapped so that the flies were on the flug. After the flies were on the flug, the CO 2 gun was inserted into the side of the flug and CO 2 flowed into the vial, causing the flies to fall asleep. Consequently, once the flug was removed, the flies would fall out of the vial and onto the CO 2 sorter, which worked as a pad that would keep the flies asleep. On the pad, the flies remained asleep, allowing the usage of a microscope and feather to sort through the flies and group them based on characteristics (sex, wing shape, virginity, etc.). The flies were placed into new vials facing sideways using the feather based on the traits sorted, and the CO 2 was turned off. To sort using cold sorters, the following procedure was used. First, the cold sorter was turned on and set to 0°C. While it cooled to that temperature, flies that needed to be sorted were tapped into empty in an ice bath for 3-4 minutes, or until the flies were asleep (checked every 30 seconds). Once asleep, the flies were placed on the cold sorter plate to ensure they would not wake up. A magnifying glass was used to determine the traits of the flies and a feather was used to move the flies into their respective groups on the cold sorting plate. After grouping the flies based on traits, the flies were tapped back into vials facing sideways using the feather. To determine the sex of the flies, the visual appearance of the fly was analyzed. Female flies are lighter, larger, have more stripes on the abdomen, a pointed tip, no sex combs or claspers, whilst males flies have claspers, sex combs, and a round, dark tip, while being much smaller than the female. The microscope or magnifying glass was used in order to figure out which traits a given fly matches most closely with, allowing for the identification of the sex of the fly. To identify virgin females, a closer examination of the flies took place. Virgin flies have larger, pale bodies, with folded wings or the presence of a meconium patch, guaranteeing the virginity of the fly, whilst the presence of sex combs was used to determine sex. Fly stocks Two stocks were obtained from Bloomington Drosophila Stock Center at Indiana University: Act5C-GAL4 (stock #4414) & UAS-APPAbeta42.E693G (stock #33774). When obtained, the stocks were put into vials with fly food. To tap or transfer files to a different vial, a vial with flies in it and a vial with fresh food were gathered. With one vial in each hand, the flug was removed from the vial with fresh food. The new vial was held upside down and the vial with flies was tapped ∼2-3 times until the flies were at the bottom. The flug was removed from the vial with flies and the fresh vial was placed on it, ensuring there was no space for the flies to escape. The two vials were then flipped upside down while maintaining contact, and then tapped onto the table to drop the flies to the bottom of the fresh vial. New flugs were then placed on the fresh and old vial while keeping the old vial upside down. The new vial was then labelled. Crossing stocks In order to cross stocks to obtain progeny modeling AD, 5-6 virgin females of UAS and 4 males of GAL4 were gathered as indicated in the crossing scheme in Tables 4 & 5 and Figure 2 . Because new flies hatch in 14 days and can reproduce, parent flies were tapped out before hatching. Drosophila with curly wings (CyO marker) were removed to ensure only heterozygous AD model flies were tested as modeled in Table 4 , Table 5 , and Figure 2 . View this table: View inline View popup Download powerpoint Table 4 AD D. melanogaster model Punnett Square for Chromosome 3 View this table: View inline View popup Download powerpoint Table 5 AD D. melanogaster model Punnett Square for Chromosome 2 Download figure Open in new tab Figure 2 AD D. melanogaster model crossing scheme Note. This figure presents a design of the crossing scheme performed. Negative geotaxis (Climbing) assay The climbing assay was used to determine the locomotive capability of the flies tested. To begin, male flies were cold sorted into groups of 10 flies each. For the apparatus, a rule was used to mark 8 cm above the bottom of a graduated cylinder, which was marked using a piece of tape. Flies were tapped into the graduated cylinder, which was sealed using a cotton ball. A video recording device (e.g., a phone) was used to record each trial, ensuring the tape and flies were visible, as well as a stopwatch as well. The recording was started along with the stopwatch, and the flies were tapped to the bottom of the graduated cylinder. The flies were given 10 seconds to climb along the graduated cylinder, then disposed of. The video was then analyzed to determine the total number of flies that crossed the tape (passed), using the time seen on the stop watch when the flies are tapped as the start time, and 10 seconds after as the end time. It should be noted that each fly could only count once, once a fly crossed it counted no matter where it finished the assay, and flies had to fully cross the 8cm mark. The pass rate for each trial was then calculated using the formula: Pass rate (%) = (Number of passes/total number of flies) * 100%. Download figure Open in new tab Figure 3 Climbing/Negative Geotaxis assay diagram Note. This figure presents a diagram of the negative geotaxis (climbing) assay used. Aversive phototaxic suppression (APS) assay The APS assay was used to determine the memory capability of the flies tested. First, solid quinine hydrochloride was diluted under a fume hood to create 200μL of 0.1 M quinine hydrochloride by the Chemical and Laboratory Safety Specialist (dissolve 7.9382mg of solid quinine hydrochloride in 200μL of water; 0.1mmol/mL x 0.2 mL * 396.91 mg/mmol = 7.9382mg). Then, male flies were cold sorted into groups of 10 each. Two vials with a flug in them were made, one of which was covered in opaque black tape surrounding it, and the other had fairy lights surrounding it. In the vial with lights, 200μL of 0.1 M quinine hydrochloride was put into the flug using a micropipette. To train the flies, the group of 10 flies was tapped into the dark vial, where they were allowed to acclimate for 30 seconds. After, they were tapped into the light vial for another 30 seconds. The flies were then tapped back and forth for a total of 10 cycles. An hour after training, flies were tapped into the middle of two odorless vials (one dark & one light), and given 30 seconds to go towards either side. If the flies ended up in the dark vial, it was considered a pass, while if a fly ended up in the light vial it was considered a fail. The pass rate of the group was then calculated using the same formula as the climbing assay. Download figure Open in new tab Figure 4 Aversive phototaxic suppression (APS) assay diagram Note. This figure presents a diagram of the aversive phototaxic suppression (APS) assay used. Statistical analysis Post data collection, statistical analysis was conducted to determine if there was any significant difference between groups. Because a normal distribution could not be confirmed due to a sample size less than 30 samples, nonparametric statistical tests were used. The Mann-Whitney U test was used when comparing APS and negative geotaxis assay pass rates between healthy and AD flies & Li 2 CO 3 and LiOr at 5 mM and 10 mM. The Kruskal-Wallis test was used when comparing APS and negative geotaxis assay pass rates between healthy flies, 5 mM lithium drug healthy flies, & 10 mM lithium drug healthy flies for both LiOr and Li 2 CO 3 within both healthy flies and AD flies. Safety Throughout the experiment, certain general safety procedures took place to ensure no injuries or accidents occurred from experimentation. To begin, the experiment occurred in a clear, safe environment where closed-toed shoes were worn. Personal Protective Equipment (PPE) of safety goggles and a lab coat was worn to protect the eyes from glassware (hazard if it breaks) and skin from chemical exposure when either glassware or chemicals that required it were utilized. Hands were also washed with soap and water thoroughly before and after the experiment. Heat-resistant mitts were worn when boiling fly food to protect skin from the heat. Specific chemicals also had safety protocols associated with them as well. For propionic acid these protocols included keeping the acid away from sparks, heat, open flames, and hot surfaces as propionic acid is flammable, storage in a well-ventilated area, and the disposal of pipette tips or anything else in contact with propionic acid in special waste disposal units or bags. For Li 2 CO 3 , these protocols included storage in a dry, cool, well-ventilated area. The avoidance of inhalation of mist gas or vapors and dust collected nearby was followed, as lithium could remain on the dust particles. Disposal of anything that came in contact with the lithium in a special waste disposal unit or bag and discharge in the environment were followed. The procedures for LiOr were the same as the protocols for Li 2 CO 3 . For quinine hydrochloride, these protocols included the avoidance of inhalation or ingestion, no dust being created nearby, the avoidance of sparks, heat, open flames, and hot surfaces due to quinine being combustible, and the disposal of anything that came in contact with the quinine in waste disposal units or bags. These protocols, along with the general safety measures listed before, were followed in order to avoid dangerous events. If an event had happened, the lab supervisor would have been contacted along with any emergency personnel, the MSDS protocols would have been followed given the circumstances, and first aid would be administered accordingly to the accident. Data View this table: View inline View popup Download powerpoint Table 5 Climbing Assay Pass Rate Data View this table: View inline View popup Download powerpoint Table 6 APS Assay Pass Rate Data Results Download figure Open in new tab Figure 5 Climbing Assay Pass Rate Graph Note. This graph presents the data collected from the climbing assays by pass rate %. Error bars represent + 2 SE. Download figure Open in new tab Figure 6 APS Assay Pass Rate Graph Note. This graph presents the data collected from the climbing assays conducted by pass rate %. Error bars represent + 2 SE. The standard deviation and standard error of each group were also calculated. View this table: View inline View popup Download powerpoint Table 7 Standard deviation results from each group in each assay View this table: View inline View popup Download powerpoint Table 8 Standard error results from each group in each assay View this table: View inline View popup Download powerpoint Table 9 Nonparametric Statistical Testing Results Discussion/Analysis A difference was found between the memory of control groups of healthy flies and healthy flies treated with Li 2 CO 3 for the APS assay ( p = 0.0121), but this difference was not present for the negative geotaxis assay ( p = 0.405). The difference in the APS assay supports previous research that lithium can improve cognitive function at lower doses in animal models and humans ( Forlenza et al., 2014 ; Shin et al., 2023). The control for Li 2 CO 3 ’s effectiveness in healthy flies was similarly effective in the APS assay ( p = 0.0434), but not effective for the negative geotaxis assay ( p = 0.6121). Locomotion yielding insignificant results while memory yields significant results in both controls highlights a likely inaccuracy of data due to low sample size. Because comparisons for experimental groups failed to yield significant results with an alpha level of 0.05, it was concluded that Li 2 CO 3 or LiOr did not significantly affect the locomotion of healthy flies or the locomotion or memory of AD flies. The comparisons between other control groups highlights the lack of data within this project or procedural errors. While past studies ( Kling et al., 1978 ; Pacholko & Bekar, 2021 ) suggested that LiOr’s higher bioavailability could lead to greater neuroprotective effects at lower dosages, the findings did not support a significant advantage of LiOr over Li 2 CO 3 at the tested dosages, which may have resulted from differences in experimental design, dosage range, or the specific AD model used. Because other assays such as the lifespan and toxicity assays were not used, the optimal dosage of the lithium drugs was unclear with full certainty. Rather than using less mainstream, aggressive models like the ‘Arctic’ AD model used, a more researched GAL4-UAS model should be prioritized for more consistent controls to highlight the effectiveness of treatments. Using an Alzheimer’s Disease model without a crossing scheme necessary would significantly decrease the time needed for trials and could increase the sample size. A notable limitation was the low sample size and resulting high variability, as reflected in the error values in Tables 7 and 8 . The high standard deviation and standard error are likely due to the inconsistencies in the data that resulted from the variance associated with small samples. Higher standard deviations also leads to less significant differences as the variance is higher, providing a possible explanation for the lack of statistical significance found. A major hindrance was the weather conditions that hindered the research progress in January, along with a fire at the school causing closure. These closures meant that for a portion of the year, students were not allowed into the research labs at the school, halting progress and fly stock maintenance for a period. The fly stocks themselves faced some issues as well, for example the lithium concentrations used seemed to have more toxic effects than expected, causing a large portion of the flies (especially the groups with AD) to die. Along with that, the research was halted by the contamination of the UAS fly stocks, making a large portion of the UAS stocks difficult to cross, and may have led to some ineffective crosses. These incorrect crosses may also explain the outlier seen in the disease control of Table 6 , contributing to the high standard error of the group. Each of these factors ultimately contributed to the low sample size and high error measurements collected from this experiment, likely contributing to the insignificant difference found from testing. Some assumptions were also made regarding the experiment. For example, the lithium salt concentrations used were extrapolated from past research on Li 2 CO 3 and assumed to be safe and effective for Drosophila melanogaster (Wang et al., 2022), and as such no toxicity assay was conducted for the Alzheimer’s flies. The lack of a toxicity assay may have contributed to the issues with fly stocks mentioned above, as the lithium dosage may be too high for the AD model used. Conclusion Graphical analysis of behavioral assay performance in Figure 5 and Figure 6 suggested that AD flies treated with 5 mM LiOr performed better on average than those treated with 5 mM Li 2 CO 3 , while the reverse was true at 10 mM. These trends were consistent with the original hypothesis, but lacked statistical significance and must be interpreted with caution. However, the hypothesis that LiOr is more effective than Li 2 CO 3 at lower dosages (5 mM), but less effective at higher dosages (10 mM), was rejected because no significant differences were observed at an alpha level of 0.05 in experimental data. Factors such as limited sample size and lack of effective control groups makes the result inconclusive, indicating that future research investigating LiOr may be warranted. Future research should prioritize expanding sample sizes and including additional behavioral assays to elaborate on these preliminary findings. A clear extension of this experiment would be to complete data collection and get enough samples to draw statistically significant results, if possible, to get a better understanding of the effects of either lithium salts. 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Share Comparison of the effects of lithium orotate and lithium carbonate on locomotion and memory in a Drosophila melanogaster model of Alzheimer’s disease Ishan Reddy Alla , Ryan Chung bioRxiv 2025.08.13.670149; doi: https://doi.org/10.1101/2025.08.13.670149 Share This Article: Copy Citation Tools Comparison of the effects of lithium orotate and lithium carbonate on locomotion and memory in a Drosophila melanogaster model of Alzheimer’s disease Ishan Reddy Alla , Ryan Chung bioRxiv 2025.08.13.670149; doi: https://doi.org/10.1101/2025.08.13.670149 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41909) Biophysics (21435) Cancer Biology (18576) Cell Biology (25479) Clinical Trials (138) Developmental Biology (13366) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24301) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40359) Molecular Biology (17162) Neuroscience (88529) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4820) Physiology (7636) Plant Biology (15125) Scientific Communication and Education (2044) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)