The spine provides structure and support to the body, yet how it develops its characteristic morphology as the organism grows is little understood. This is underscored by the commonality of conditions in which the spine curves abnormally such as scoliosis, kyphosis, and lordosis. Understanding the origin of these spinal curves has been challenging in part due to the lack of appropriate animal models. Recently, zebrafish have emerged as promising tools with which to understand the origin of spinal curves. Using zebrafish, we demonstrate that the urotensin II-related peptides (URPs), Urp1 and Urp2, are essential for maintaining spine morphology. Urp1 and Urp2 are 10-amino acid cyclic peptides expressed by neurons lining the central canal of the spinal cord. Upon combined genetic loss of Urp1 and Urp2, adolescent-onset planar curves manifested in the caudal region of the spine. Highly similar curves were caused by mutation of Uts2r3, an URP receptor. Quantitative comparisons revealed that urotensin-associated curves were distinct from other zebrafish spinal curve mutants in curve position and direction. Last, we found that the Reissner fiber, a proteinaceous thread that sits in the central canal and has been implicated in the control of spine morphology, breaks down prior to curve formation in mutants with perturbed cilia motility but was unaffected by loss of Uts2r3. This suggests a Reissner fiber-independent mechanism of curvature in urotensin-deficient mutants. Overall, our results show that Urp1 and Urp2 control zebrafish spine morphology and establish new animal models of spine deformity. The backbone, or spine, is an integral part of the human body, providing support to our torsos so that we can sit, stand, bend and twist. If this structure does not form correctly, it can lead to pain, neurologic problems, and mobility issues. The spine normally has curves, but these can become deformed for many reasons, including genetic and muscular factors. There are also cases in which the cause of a spine distortion is unknown, such as in scoliosis (where the spine twists to the side), lordosis (where the lower part of the spine curves excessively), and kyphosis (where the upper part of the spine shows extreme curvature).The structure of the spine is laid out during embryonic development and maintained throughout life. Experiments in zebrafish have shown that a crucial element in preserving the shape of the spine is the flow of cerebrospinal fluid or CSF. Propelled by the movement of little ‘hairs’ at the surface of specialized cells, this liquid runs through our central nervous system along a cavity lined with neurons. These nerve cells produce Urp1 and Urp2, two short molecules (or peptides) built from the same components as proteins. In zebrafish embryos, lowering the levels of these peptides had previously been shown to cause early body deformities. But what role, if any, do Urp1 and Urp2 play in maintaining the shape of the spine in adult zebrafish?Bearce et al. set out to answer this question. First, they generated mutant zebrafish which did not carry either Urp1, Urp2 or both peptides. Contrary to previous findings, all three of these mutants developed normally as embryos. Once they were adults, zebrafish lacking Urp1 exhibited normal spines, while those lacking Urp2 had slightly deformed curves. However, zebrafish lacking both peptides had prominent curves in the tail-region of their spines, somewhat akin to lordosis in humans. This indicates that both peptides are necessary for adult spine structure, but work in a semi-redundant manner. Interestingly, the defects observed first appeared in adolescent fish and gradually worsened as they grew; many forms of human spinal abnormalities follow a similar trajectory.Bearce et al. also tested the role of the protein Uts2r3, a receptor for peptides which belong to the urotensin family (such as Urp1 and Urp2). Fish lacking this protein showed normal spine structure as embryos, but distorted spinal curves as adults, suggesting that Urp1 and Urp2 might control spine morphology by signaling via the Uts2r3 receptor.Together, Bearce et al.’s observations show that disturbing urotensin signaling leads to a lordosis-like condition in adult zebrafish, with evident deformities in the tail-region of the spine. Considering the broad similarities in structures between the zebrafish and the human spine, these results point to a possible involvement of urotensin signaling in spine distortion in humans. More studies using zebrafish will likely provide further insights into the principles that control the shape of the spine and what goes wrong when it breaks down. Understanding how the shape of organisms is acquired is a central goal of developmental biology. The chordate body axis forms during embryonic development, when it is based around the rod-like notochord . Later, A breakdown of spine morphology occurs in scoliosis, lordosis, and kyphosis. Scoliosis is medically defined as lateral curvatures of the spine greater than 10°  and can Danio rerio), have emerged as prominent animal models of spinal deformity (sspo) which sits in the CSF in brain ventricles and the central canal  gene, ∆Purp1 and ∆Purp2 because they lack the peptide coding sequence. In addition, mRNA quantitation revealed downregulation of urp1 and urp2 in their respective mutant backgrounds, indicating transcript decay  along with Cas9 into wild-type embryos at the one-cell stage, we generated mosaic mutants, called crispants, that were then assessed for body shape phenotypes . In posi mutants . Quantit mutants . By contas9 only . Using Tas9 only . We usedkgrounds . Overall∆Purp1 mutant adults at 3 months post fertilization (mpf) appeared normal whereas ∆Purp2 mutants exhibited minor body dysmorphologies and kinked tails  while ∆Purp2 mutants showed slight sagittal curves , corresponding to a standard length between 3.9±0.7 mm and 5.9±0.4 mm  when we ended this analysis   than either ∆P;urp2∆Purp1 mutants (197.5±38.9°) or b1436uts2r3 mutants (146.1±47.4°)  . Second, mutants . Third, e curves . By contviations . These rtm304;urp1∆P;urp2∆Pcfap298 triple mutants that were embryonically rescued by temperature shifts. Triple mutants exhibited significant curves, similar to tm304cfap298 single mutants  and kyphosis-like (icm02pkd2l1) models.Overall, our phenotypic data show that urp1 and urp2 expression . In sibling controls, Sspo localized into an RF throughout the central canal  is a cyclic peptide that was first identified from the teleost urophysis  and subs∆Purp1 and ∆Purp2 mutants that lacked the genetic region coding for the Urp1 and Urp2 peptides, respectively, then phenotyped skeletal morphology by µCT. This revealed that Urp1 and Urp2 function semi-redundantly to control spine morphology, with double mutants, as well as Uts2r3 receptor mutants, developing spinal curves in the caudal region during adolescent growth. By contrast, single ∆Purp1 and ∆Purp2 mutants developed more subtle curves that nevertheless worsened with age. The lack of major vertebral defects, the location and direction of the curves coupled with phenotypic differences compared with mutants that model AIS and kyphosis, suggested that urotensin-deficient mutants model a lordosis-like condition.In this study, we discovered a role for two of the URPs, Urp1 and Urp2, in zebrafish spine morphology. To do so, we generated sspo), has been implicated in controlling body axis and spine morphology  ∆Purp1 and ∆Purp2 mutants showed urp1 and urp2 transcript downregulation, respectively, in addition to deletion of the peptide coding regions; (2) urp1 and urp2 single and double crispants, in which gRNAs targeted several regions of the gene, also showed normal straightening; and (3) exogenous addition of Urp1 and Urp2 peptides, without pro-domains, rescued CTD of a cilia motility and RF mutant , b1421urp2 , b1436uts2r3 as well as previously published lines: tm304cfap298  with 30 cycles of the middle three steps. PCR product was purified  and then used for in vitro RNA synthesis using a MEGAshortscript T7 Transcription Kit . Synthesized gRNAs were purified , then 150 pg along with 320 pg/nl Cas9  were injected into one-cell stage fertilized eggs. The mosaic mutant fish resulting from these injections (F0 fish) were raised and outcrossed to AB wild-types, and DNA was extracted from the resulting F1 embryos. Mutant alleles were screened by PCR coupled with restriction enzyme digestion and/or Sanger sequencing (GeneWiz). Embryos from F1 clutches harboring mutations were raised to adulthood and outcrossed to AB wild-types to generate F2 families which were screened for mutations and raised. The nature of mutations was identified by sequencing DNA extracted from adult fin clips of F2 heterozygous fish using CRISP-ID to deconvolute . DNA was digested into 150 bp fragments, and paired-end sequencing was performed using a NovaSeq 6000 Sequencing System. Trimmomatic  was used to remove Illumina adaptor sequences from paired-end reads. Illumina short-read sequences were then aligned to the GRCz11 reference sequence of chromosome 24 using BWA-MEM (version 0.7.01). SAMtools (version 1.8) was used to sort and index reads. Aligned reads in BAM format were analyzed in IGV (version 2.13.1). Mutants were routinely genotyped by PCR amplification with oligos sspo_geno_1 and sspo_geno_2, followed by BsaI-HFv2  restriction digestion to produce 300 bp and 99 bp bands from wild-type DNA and a protected 399 bp band from mutant DNA.The nature of the cfap298, sspo, urp1, and urp2 were chosen from a look-up table , 50°C (10 min), and 72oC (10 min). Assembled oligos were purified and used as templates for in vitro RNA synthesis, as described in ‘Generation and genotyping of mutant lines’ section. For mutagenesis, 1000 pg of gRNAs along with 1600 pg/nl Cas9  were injected into one-cell-stage embryos. To assess rates of mutagenesis, DNA was extracted from 1 dpf crispants and subjected to T7 endonuclease I assays .Four gRNA oligos per gene for Zebrafish larvae at 28–30 hpf were imaged using a Leica S9i stereomicroscope with integrated 10-megapixel camera. Body angles were calculated using ImageJ  as descrrpl13 and used the ∆∆CT relative quantitation method in which fold changes are calculated as 2−∆∆CT. The efficiency of amplification was verified to be close to 100% with a standard curve of RNA dilutions.RNA was extracted using a Zymo Direct-Zol RNA Miniprep kit . cDNA was prepared from 25 ng of RNA using oligoDT primers in a 20 µl reaction using a High Capacity cDNA Reverse Transcription Kit . qRT-PCR reactions were performed in real time using 5 µl PowerUp SYBR Green Master Mix , 0.8 µl of 10 µM forward and reverse primers, 1.4 µl of nuclease-free water, and 2 µl of diluted cDNA. PCR was performed using a QuantStudio Real Time PCR System (Applied Biosystems) with cycling parameters: 50°C (2 min), 95°C (10 min) then 40 cycles of 95°C (15 s), and 60°C (1 min). Each reaction was performed in quadruplicate. Quantitation was relative to Larvae were incubated in water containing 0.2% calcein  for 10 min then rinsed two to three times in water (5 min per rinse). Larvae were immobilized with 0.005% tricaine, mounted in 0.8% low melt agarose, and imaged with a Leica THUNDER stereoscope.pkd2l1, urp1, or urp2 were prepared by heating 10 pmol of each to 95°C for 90 s then snap-cooled in the dark for 30 min. Embryos were then incubated overnight in 500 µl of amplification buffer containing 30 pmol hairpins at room temperature in the dark. Embryos were washed five times in 5× SSCT, stored at 4oC, and then mounted for confocal microscopy. Images were acquired using a Zeiss LSM880 using either a ×20 air or ×40 water objective. Acquisition settings were derived using wild type embryos and then applied to all embryos. Images were exported to IMARIS 9.5.0 (Oxford Instruments). A Gaussian filter of width 0.42 µm (×20) or 0.21 µm (×40), and a rolling ball background subtraction of 10 µm was applied.Embryos were fixed in 4% paraformaldehyde at 4°C overnight, washed with phosphate buffered saline (PBS) then serially dehydrated to 100% methanol, and stored at –20°C. Embryos were rehydrated, washed with PBS containing 0.1% Tween-20, incubated in hybridization buffer (Molecular Instruments), then incubated in 2 pmol of probes at 37°C overnight in a total volume of 500 µl of hybridization buffer. Embryos were washed in wash buffer (Molecular Instruments), washed twice in 5× SSCT (sodium chloride sodium citrate with 0.1% tween-20), and then incubated in amplification buffer (Molecular Instruments) for 1 hr. RNA hairpins designed to bind either Scans were performed using a vivaCT 80  at 18.5-µm voxel resolution (for 3 mpf and 12 mpf fish) or 10-µm voxel resolution (1 mpf fish) as previously described . DigitalQuantitation of lateral spine curvature was performed in ImageJ by orienting isolated spine images in a dorsal view with heads to the left and with the otic vesicles and first Weberian vertebrae parallel to the x-axis. Landmarks were assigned to the narrowest point of each centrum rostral to caudal; where maximum projection resulted in hidden or overlapping vertebrae, the appropriate number of points was added in closest approximation. The y-value from each landmark was subtracted from the point rostral to it, resulting in a map of local deflections where positive values indicate rightward displacement, and negative values indicate leftward displacement.Embryos (28 hpf) and larvae (12 dpf) were anesthetized in tricaine until touch response was abolished and then embedded in 0.8% low-melt agarose laced with tricaine in inverted imaging chambers . In larvae, care was taken to align the posterior body close to the coverslip to the RF within the working distance of the objective. A Nikon Ti2 inverted microscope equipped with Plan Apo ×40 and ×60 WI DIC (1.2 NA) objectives, a Yokogawa Spinning Disk and pco.edge sCMOS camera were used to capture 512×256 images in time series. Exposure time varied with age (100 ms–300 ms) as Sspo-GFP brightened in intensity over time; exposure, camera settings, and laser power were kept constant between age-matched individuals. Images were cropped, rotated, and intensity-adjusted in ImageJ . This is a beautifully executed study on the role of Urp signaling in spine morphogenesis in zebrafish. This work challenges the model that Urp1/ 2 controls the extension and straightening of the body axis of the zebrafish embryos, and thus makes a significant contribution to the literature. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  General StatementsWe were immensely pleased that the reviewers considered our conclusions “well supported” and our study “beautifully executed”. Reviewers also recognized the significance of our work. Reviewer 1 stated that “building a model that describes one of these pathways will allow us to begin to test therapies to treat or prevent scoliosis” then noted that we “help to build a larger model of normal spine morphogenesis” and that this is “important”. Reviewer 2 called our work an “exciting advance in our understanding of one of the essential signaling pathways that help regulate body axis straightening and spine morphogenesis in zebrafish” and mentioned that our work “may also help to further our understanding of the etiology and pathophysiology of multiple forms of neuromuscular scoliosis in humans”. Reviewer 3 agreed, stating that our work “adds important information on the role of urotensin signaling in spine formation” and noted that it is timely: “findings are of special significance in the light of recent reports that mutations in UTS2R3 show association with spinal curvature in patients with adolescent idiopathic scoliosis”.We thank the three reviewers for reading our research and providing feedback. In all cases, we have incorporated their suggestions, and we believe this has made our manuscript much stronger. Indeed, reviewers had only a small number of “major points”, and all have now been addressed. We have also addressed all “minor points” raised by reviewers.Major pointsReviewer 1The key conclusions are well supported, see below for my two major issues.1. Please don't call this lordosis. Lordosis or hyperlordosis effects lumbar vertebra. The curve in the lumbar region shifts body weight so that human gait is more efficient that in the great apes, or so the story goes. Zebrafish do not have lumbar vertebra equivalents or a natural curve in the caudal region. Similarly, fish do not have the equivalent vertebra to generate kyphosis, which is again a hyper flexion of a normal human spinal curve. Instead zebrafish have Weberian, precaudal and caudal vertebra. It would be so much more useful for the field if the authors used these terms and specified ranges, i.e. numbered vertebrae, that are effected so we can directly and accurately compare regions of defects between zebrafish mutants. It would help to make the point that the uts2r3 mutant has more caudally located curves than urp1/2 double mutants.We agree with the reviewer and have made changes in line with their suggestions. For example, at the end of the abstract, we have changed “new animal models of lordosis-like curves” to “new animal models of spine deformity”; at the end of the Introduction, we say “mutants model human spinal deformity” rather than “mutants model a lordosis-like condition”.We have also added a new paragraph to the Discussion about whether zebrafish can truly model scoliosis, lordosis and kyphosis which, among other points, mentions some of what the reviewer brought up about lumbar vs. caudal vertebral differences.When we do mention lordosis (less often than previously) in the text, we are careful to say “lordosis-like” and we fully explain what we mean by this, including the aspects of the zebrafish phenotypes that are not lordosis-like. Last, when describing our phenotypes, we now use the appropriate names for zebrafish vertebrae, i.e., caudal rather than lumbar.2. The observation that urp1/2 double mutants have curves only in the D/V plane and almost completely lack side-to-side curves is noteworthy. Does the urp1-/-urp2-/- mutant uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be very interesting. It is particularly important to describe how penetrant the phenotype is and how many times it was observed. See 9 minor comments. It would help the reader if the authors explicitly described the features that they see in the cfap298 mutant that constitute lateral curves and that are lacking in urp1/2 (e.g. in figure 4E).tm304cfap298 and ∆P;urp2∆Purp1 mutants and, in the new version, also b1436uts2r3 and tm304;urp1∆P;urp2∆Pcfap298 triple mutants. These new data are:To address this comment, we have supplied more data on the extent of lateral curvature in Figure 4Fi-Fii).New data showing the extent of lateral curvature in various mutant conditions  to assess mutagenesis in crispants. These assays revealed significant insertion-deletion mutations at all gRNA targeting sites as well as cases of large deletions between sites.urp1 and urp2 targeting was effective for mutagenesis :New data demonstrating that Figure 1—figure supplement 2C-D).”These data are referenced in the main text in the following way: “Using T7 endonuclease assays, we confirmed that high levels of insertion-deletion mutations were generated at gRNA sites in crispants  show normal axial straightening. These mutants completely lack the coding region for the Urp1 and Urp2 peptides.urp1 and urp2 crispants with significant indels (as assessed by T7E1 assays) also showed normal straightening. By contrast, all other positive control crispants showed expected axial phenotypes.urp1 and urp2 mutants and also reported lack of axial defects .A preprint produced independently of our work also generated Thus, we conclude that Urp1 and Urp2 are dispensable for axial straightening in zebrafish. We devote several paragraphs in our Discussion to this somewhat unexpected finding.Reviewer 31. The addition of the F0 crispant experiment to show that the pro-peptide of urp1/2 does not have a function and is responsible for the difference between the observed morpholino and the crispr phenotype was important. However, since no phenotype was observed in crispants it is important to add evidence of induced cuts for all guide RNAs used in the crispant experiment. These control experiments might have been done already. If not, they can easily be done in a short period of time by performance of T7 assays on injected fish and would not require additional reagents.urp1 and urp2 crispants .Please see response to Reviewer 2 above including Figure1-figure supplement 2. In summary, we have now performed the suggested T7 assays and they indicated that indels were indeed generated in Overall, the evidence we have that Urp1 and Urp2 peptides do not contribute to axial straightening is:∆Purp1 and ∆Purp2 single and double mutants  show normal axial straightening. These mutants completely lack the coding region for the Urp1 and Urp2 peptides.urp1 and urp2 crispants with significant indels (as assessed by T7E1 assays) also showed normal straightening. By contrast, all other positive control crispants showed expected axial phenotypes.urp1 and urp2 mutants and also reported lack of axial defects .A preprint produced independently of our work also generated 2. The authors claim that there were no structural defects observed in urp1/2 double mutants. However, the hemal arch in figure 3 E seems to be deformed. This could be normal variance or a phenotype. This can be addressed by simple reinspection of the scans.∆P;urp2∆Purp1 mutants. We now explicitly state this in the text. We also note that we do not see significant vertebral defects such as fusions or hemivertebrae and nor do we see any patterning defects at 10 dpf based on calcein staining. Additionally, we have added vertebral body shape quantitation (length and height aspect ratios):Reinspection of the scans shows no significant hemal arch defects in Figure 3F)New data showing vertebral body shape quantitation  need be explicit about what we mean with "without vertebral defects." What do we count as defects? Vertebrae can be fused, bent, shortened or the growing edges can be slanted. In Figure 3E, and movie7, it is clear that the highlighted mutant vertebrae are shorter than WT. The growing ends of normal vertebra are perpendicular to the long axis of the vertebra. In the mutants the ends are slanted. Please define in the text what you consider a relevant vertebral defect, because these vertebrae have defects. Or are you only considering the calcein stained centra at 10dpf?∆P;urp2∆Purp1 mutants . Since these structural changes are subtle, and we do not observe vertebral patterning changes based on calcein staining at earlier time points, we conclude that the vertebral structural changes are not causative in spinal curvature but rather are a consequence of the presence of curves.We have made textual edits to clearly state that we do not see vertebral fusions or missing appendages. We have also added vertebral body length and height aspect ratios to quantify vertebrae shape and find subtle changes in the variance of vertebral body length aspect ratios in 5b. Do you want to base your patterning conclusion on primarily the calcein data as these are closer to the notochord patterning time window. Please anchor this conclusion to a specific time or standard length e.g. 10dpf/5.6mm.We now note the time window when calcein was performed and provide the standard lengths of fish analyzed. As such, our conclusion about vertebral patterning is now anchored to the standard length, as the reviewer suggested:∆P;urp2∆Purp1 mutants at 10 dpf  .”“Staining of juveniles with the vital dye calcein revealed no defects in vertebral patterning or spacing in 6. "At 30 dpf… several mutants exhibited a significant curve in the pre-caudal vertebrae, in addition to a caudal curve . Since pre-caudal curves were rare in mutants at 3-months, this suggested that curve location is dynamic". The frequency of this observation is important. Does it effect all or a fraction of mutants? Can you provide some numbers to anchor these observations? Maybe fractions e.g.. 3 of 4 fish had precaudal curves at 30pdf, and 0 of 10 fish had precaudal curves by 3 mpf?We have now analyzed more 1 mpf fish by µCT and we observe that 5 of 7 exhibit pre-caudal curves:∆P;urp2∆Purp1 mutants .New data showing spinal curves are variable in 1 mpf Figure 2F). Only 2 of 9 fish analyzed by µCT at 3 mpf were scored as exhibiting an “apex of curve” vertebrae in the precaudal vertebrae, and in both of those cases the curve was at the very end of the precaudals. This reinforces our conclusion that curve position is dynamic through growth.These data are also mentioned in our revised text. By contrast to the significant pre-caudal curves at 1 mpf, 3 mpf fish showed predominantly caudal curves . Our results agreed with those previously published for the icm02pkd2l1 mutant line  but we believe it is important to include our data so readers can see side-by-side images of the various mutant conditions using the same skeleton visualization technique.We have now scanned several icm02pkd2l1 mutants essentially appeared wild-type but by 12 mpf they had developed very subtle D/V curves in the pre-caudal vertebrae. They do not exhibit M/L curves at either stage.At 3 mpf, icm02pkd2l1 mutants “kyphosis” to be in line with a previous publication describing these mutants . We have now added new wording in the Discussion about whether or not zebrafish can truly model kyphosis and lordosis, and we now make clear in our Results that the “mutants went on to develop very subtle kyphosis-like curves” rather than “is kyphosis”.We called the phenotype displayed by icm02pkd2l1 mutants do not exhibit any curves until much later in life than ∆P;urp2∆Purp1 and b1436uts2r3 mutants. Inspired by this finding, we aged ∆Purp1 and ∆Purp2 single mutants and found that they go on to develop D/V curves by 12 mpf, with ∆Purp1 mutants being less severe than ∆Purp2 mutants . To summarize all of our findings:It is intriguing that ∆Purp1 and ∆Purp2 single mutants upon aging shows: (1) Urp1 and Urp2 are not entirely redundant in long-term spine maintenance and (2) proper Urp1/Urp2 dose is essential. We have now included these new insights.Phenotypes in b1436uts2r3 mutant spines and reconstructed views from dorsal aspect, including these in new Figure 4Fi and Figure 4—figure supplement 1. We also quantified the degree of lateral curvature . The reviewer’s suggestion is correct – there are minimal side-to-side curves in b1436uts2r3 mutants, highly similar to what we found for ∆P;urp2∆Purp1 double mutants.This is an interesting question. To address it, we imaged more 9. One finding that deserves more discussion is the observation that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? Please consider the following paper. It shows a proprioception system that maintains normal side-to-side posture. A spinal organ of proprioception for integrated motor action feedback.Picton LD, Bertuzzi M, Pallucchi I, Fontanel P, Dahlberg E, Björnfors ER, Iacoviello F, Shearing PR, El Manira A. Neuron. 2021 Apr 7;109(7):1188-1201.e7. doi: 10.1016/j.neuron.2021.01.018. Epub 2021 Feb 11. PMID: 33577748Thank you for pointing out this manuscript. We have now mentioned this potential “uncoupling” in our Discussion and included this citation.Reviewer 21. Figure 3F: might be improved by making the images black and white and possibly inverted. It is not easy to clearly see the vertebrae as is.Thanks for the suggestion, we made this change.


Fmr1y−/).Fragile X syndrome (FXS) is a common single gene cause of intellectual disability and autism spectrum disorder. Cognitive inflexibility is one of the hallmarks of FXS with affected individuals showing extreme difficulty adapting to novel or complex situations. To explore the neural correlates of this cognitive inflexibility, we used a rat model of FXS (Fmr1y−/ and WT littermates over six 10-min exploration sessions in a novel environment—three sessions per day (ITI 10 min). Our recordings yielded 288 and 246 putative pyramidal cells from 7 WT and 7 Fmr1y−/ rats, respectively.We recorded from the CA1 in Fmr1y−/ rats. However, while CA1 pyramidal neurons from WT rats showed experience-dependent changes in firing and spatial tuning between the first and second day of exposure to the environment, these changes were decreased or absent in CA1 neurons of Fmr1y−/ rats. These findings were consistent with increased excitability of Fmr1y−/ CA1 neurons in ex vivo hippocampal slices, which correlated with reduced synaptic inputs from the medial entorhinal cortex. Lastly, activity patterns of CA1 pyramidal neurons were dis-coordinated with respect to hippocampal oscillatory activity in Fmr1y−/ rats.On the first day of exploration of a novel environment, the firing rate and spatial tuning of CA1 pyramidal neurons was similar between wild-type (WT) and Fmr1y−/ rats. Future experiments will focus on this connection as well as the contribution of other neuronal cell types in the hippocampal circuit pathophysiology associated with the loss of FMRP. It would also be interesting to see if hippocampal circuit deficits converge with those seen in other rodent models of intellectual disability.It is still unclear how the observed circuit function abnormalities give rise to behavioural deficits in Fmr1y−/ rats show similar spatial firing properties as those from WT rats but do not show the same experience-dependent increase in spatial specificity or the experience-dependent changes in network coordination. Our findings offer support to a network-level origin of cognitive deficits in FXS.In conclusion, we found that hippocampal place cells from The online version contains supplementary material available at 10.1186/s13229-022-00528-z. FMR1 gene, resulting in its epigenetic silencing and subsequent loss of its protein product FMRP  is the recording session time period, y(t) is the position of the rat at time t, and h is a smoothing factor, which was set to 2.5 cm.The summary algorithm for calculating firing rate for each spatial bin is then given by:The percentage of active bins for each session was calculated by dividing the number of bins in the firing rate map which contained spikes by the number of bins visited. Place fields were defined as areas in the firing rate maps of at least 9 contiguous bins with firing rate > 20% of maximum bin firing rate . In caseTo assess the stability of spatial firing, we calculated the Pearson correlation between firing rate maps of successive recording sessions. This analysis was only conducted on identified cells which exhibited spatial firing, defined as SI > 0.5 bits per spike, in both sessions.http://chronux.org; .Position data from each session were binned into 500-ms epochs, and the velocity for each epoch was calculated. Raw LFP traces (4.8 kHz) were z-scored (mean was subtracted and divided by standard deviation). LFP data analysis was done on all the 4 s periods of activity (> 3 cm/s) following periods of immobility. Time–frequency spectrograms were calculated using Chronux Toolbox , functiectively . Power ehilbert function from the MATLAB Signal Processing Toolbox. Only spikes during bins (500 ms) of strong oscillations (> 2 standard deviations of mean power) were included in this analysis . Both the instantaneous amplitude and the phase time series of a filtered signal were computed from the Hilbert transform, which was obtained by using the analysis , 50. Eve Hz, Medi4 Hz, SloAt the end of the in vivo recording experiments, animals were given an overdose of pentobarbital intraperitoneally  and perfused with 0.9% saline solution followed by a 4% formalin solution. The brain was extracted and stored in 4% formalin for at least seven days prior to any histological analyses. The brains were sliced in 32-µm sections on a freezing microtome at − 20°. These sections were stained with a 0.1% Cresyl violet solution and the tissue section that best revealed the electrode track was imaged using ImageJ software .3, 1.25 NaH2PO4, 25 glucose, 75 sucrose, 7 MgCl2, 0.5 CaCl2). Once perfused, the brain was rapidly removed and 400 μm slices containing the dorsal pole of the hippocampus were cut on an oscillating blade vibratome  in the coronal plane. Slices were then transferred to a submerged chamber containing sucrose-ACSF at 35 °C for 30 min, then at room temperature until needed.Ex vivo brain slices were prepared as previously described . Briefly3, 1.25 NaH2PO4, 25 glucose, 1 MgCl2, 2 CaCl2) at a flow rate of 4–6 mL.min-1 at 31 ± 1 °C) which contained 50 µM picrotoxin to block GABAA receptor-mediated currents. Neurons were visualized under infrared differential interference contrast (IR-DIC) microscopy with a digital camera  mounted on an upright microscope  with a 40 × water-immersion objective lens . Whole-cell patch-clamp recordings were performed with a Multiclamp 700B  amplifier, using recording pipettes pulled from borosilicate glass capillaries  on a horizontal electrode puller . Pipettes were filled with a K-gluconate based internal solution  which gave a 3–5 MΩ tip resistance. Neurons were rejected if: they were more depolarized than − 50 mV, had an access resistance > 30 MΩ, or the access resistance changed by more than 20% during the recording. Cell-attached recordings were performed as above, but without breaking through into the whole-cell configuration.For recording, slices were transferred to a submerged chamber perfused with pre-warmed carbogenated ACSF  were delivered at 10-s intervals at 30 V, 60 V, and 90 V levels from a constant-voltage stimulation box . Recordings were first performed in cell-attached mode to identify cell spike output. Following breakthrough into whole-cell mode, EPSPs were recorded in current-clamp configuration with membrane potential biased to − 70 mV. All recordings were filtered online at 10 kHz with the built-in 4-pole Bessel filter and digitized at 20 kHz . Traces were recorded in pCLAMP 9  and stored on a personal computer. Analysis of electrophysiological data was performed offline using the open-source software package Stimfit . Thp-values reported in the context of LMEs are given by likelihood ratio tests between a model containing the variable or interaction in question and a model without that variable or interaction (a reduced/null model). When significant interactions were indicated by the LME, post hoc tests for between- and within-subjects effects were conducted by comparing estimated marginal means with t-tests with a Tukey correction for multiple comparisons.Statistical modelling routines for the linear mixed effects (LME) models were written and run using RStudio 1.0.153 . Depending on the data distribution, linear mixed models (LMMs) or generalized linear mixed models (GLMMs) were fitted to single unit data metrics using the R package lme4 v1.1–17 . Animal r) are bounded between , and the sampling distribution for highly correlated variables is highly skewed. Therefore, in order to convert the distribution to normal, r values were transformed to (z) values using the Fisher's z transformation:Pearson’s correlation coefficients  x day x session ). This same three-way ANOVA model was also used to analyse oscillatory power in each band. The effects of velocity on power of each oscillatory band of interest . However, given the small size of the distributions (n = 7 for rats for both genotypes), any estimations of distribution type cannot be accurate. Given that fitting an LME model that does not contain random effects would be the same as an ANOVA, we proceeded to use ANOVA for these rat-level analyses, despite the isolated distributions that did not satisfy the assumption of normality. The alternative would involve using nonparametric tests; however, these do not allow for hierarchical structure of repeated factors (session-in-day).The normality of all rat means’ distributions (i.e. group/sessions/metric) was tested using Kolmogorov–Smirnov (KS) tests. Nearly all rat means’ distributions passed the normality test  to the fit of a reduced model that did not contain the interaction term: Phaselock ~ Intercept + Genotype + Session (Model 2). To explore main effects of genotype and session, two separate models were used: Phaselock ~ Intercept + Genotype (Model 3) and Phaselock ~ Intercept + Session (Model 4). Their fit was compared to the null hypothesis that the simplest (intercept-only) model is correct again using log-likelihood ratio (G2 test). For individual comparisons between sessions and genotypes, we used two-proportion z-test [The effects of genotype and session on the percentage of phase locked cells by each oscillatory band Fig.  were anan z-test  with corAs the LME framework for analysis of circular data is still under development, for analysis of oscillatory phase preference Fig. , we firsp < 0.05. Statistical analysis was carried out in SPSS 16.0 (IBM), R v.3.4.4  or MATLAB , and grFmr1y−/) rats showed experience-dependent changes in firing rate, as well as in spatial firing properties. To examine whether the experience-dependent changes in spatial coding were secondary to the experience-dependent changes in firing rate also observed in WT but not Fmr1y−/ rats, we performed a bootstrapping-like down-sampling analysis. We sampled uniformly at random, 30 cells from each day and each genotype, 1000 times, with replacement. The size of subsamples was chosen to approximate the average cell number per rat at each session. Each of the subsamples was constrained to have an average firing rate that is equal to the overall mean firing rate of the WT cell population on day 2 (+ / − 5%). For each subpopulation of each genotype and day, we tested whether other metrics taken at the cell level  were statistically different from their overall population (two-sample t-test except for preferred theta phase). The portion of subpopulations that were statistically different from their overall population in each measure was used as the probability of the measure in question to be driven by changes in mean firing rate for the population tested  formed the distributions plotted in Additional file For each subpopulation, we calculated the mean for each measure. These means n = 000 formeFmr1y−/ cells had to have an average firing rate decrease that is equal to the average firing rate decrease of WT cell population between session 3 and session 4 (+ / − 5%). For the second down-sampling, the subsamples from both WT and Fmr1y−/ cells had to have no change in firing rate (+ / − 5% of WT). As described above, the portion of 1000 subpopulations of each genotype that were statistically different from the overall population distribution was used as the probability of firing rate correlations to be driven by changes in mean firing rate for the population tested  and the first session of day 2 (session 4), we performed two down-sampling analyses. For the first, the subsamples from both WT and N = 7–8/group). For assessment of genotype effect, data were analysed using an LME based approach (see above), with animal and slice identity included as random effects. The p-value was then approximated using the Wald test, with effect size and variation estimated from the LMEs fitted to the data. For synaptic stimulation experiments, either the amplitude of the EPSP or the spike-probability were plotted per slice and compared using a 2-way ANOVA. If a genotype/stimulus interaction was observed, then Sidak post hoc tests were performed (corrected for multiple comparisons). For AIS lengths and morphology analysis, an LME analysis was performed, again using animal and slice identity as random effects with p-value approximated using the Wald test. For dendritic protrusion analysis, the animal average was analysed with Student’s t-test.Data generated from ex vivo brain slices were analysed as described previously . Group sFmr1y−/ and WT rats as they foraged for randomly scattered cereal rewards in an initially novel cylindrical environment for three 10-min sessions (10 min ISI) over each of two consecutive days  = 0.695, p = 0.421), or between days  = 3.982, p = 0.069), but there was a significant decrease in path length across the three sessions within each day  = 51.97, p < 0.0001), with no significant interactions. This decrease in path length across sessions within each day may reflect habituation to the environment and/or decreased motivation to forage for food as the rat becomes more satiated. We also assessed whether the proportion of the environment explored by the rats differed between genotypes or across days and sessions. Rats from both genotypes explored more than 93% of the environment during every session  = 17.03, p = 0.001) whereby the Fmr1y−/ rats visited more of the environment than WT rats. There was also an overall difference between sessions within each day  = 13.46, p < 0.001) with slightly less of the environment being explored on later sessions than earlier sessions each day, but there were no significant differences in exploration across days  = 0.267, p = 0.615), and no significant interactions (p’s > 0.05). As there was a difference between genotypes in the proportion of the environment that was explored during each session, we considered whether this might contribute to any of the differential changes in pyramidal neuron activity and spatial firing properties between groups presented below (see discussion of each finding). Finally, as speed of movement can influence both pyramidal neuron activity and oscillatory activity, we also analysed the amount of time during each session the rats spent moving at different speeds , nor any significant 2-, 3- or 4-way interactions including genotype and velocity bin, indicating that the velocity profile did not differ significantly between genotypes (p’s > 0.05).We recorded from the dorsal CA1 of adult Fmr1y−/ rats that were sufficiently isolated , were classified as pyramidal neurons (spike width > 250 μs and mean firing rate < 5 Hz), and were active in the environment (mean firing rate > 0.1 Hz) , with no significant additional contribution of session-in-day. Exploring the genotype x day interaction further, post hoc Tukey paired comparisons on emmeans comparing Day 1 and Day 2 for each genotype indicated that WT rats had significantly lower mean firing rates on Day 2 than Day 1 (p < 0.0001), while those of Fmr1y−/ rats did not differ significantly between days (p = 0.131). Between genotype post hoc Tukey comparisons on emmeans indicated no significant differences between WT and Fmr1y−/ on either day . Thus, the mean firing rates of WT and Fmr1y−/ CA1 pyramidal cells did not differ overall, but WT cells showed a significant experience-dependent decrease in firing between Day 1 and Day 2, whereas Fmr1y−/ cells did not.CA1 pyramidal neurons have previously been reported to fire at higher rates in novel than in familiar environments , 62 and Fmr1y−/ rats, we calculated their burst probability, which is a property of pyramidal neurons that is controlled by perisomatic and dendritic inhibition [Fmr1y−/ rats . There was no significant difference in burst probability between genotypes on either day . There was also a significant decrease in burst probability across sessions within a day (LME: session-in-day effect p = 0.013), but this did not differ between genotypes (LME: genotype x session-in-day interaction p = 0.916). Additional file To further explore pyramidal neuron activity in WT and hibition . High buhibition , 61. As Fmr1y−/ neurons. It is unlikely that the differential pattern of firing rate and burst probability changes seen across days in WT and Fmr1y−/ rats are secondary to any differences in exploratory behaviour between genotypes, as while more of the environment was explored on both days by Fmr1y−/ rats, there were no changes in exploratory behaviour between Day 1 and Day 2 that could account for the changes in firing rate and burst probability seen in WT cells.Overall, these analyses indicate that both the mean firing rate and the burst probability of WT CA1 pyramidal neurons decreases between Day 1 and Day 2 of exposure to a novel environment, whereas these experience-dependent changes are not seen in Fmr1y−/ and WT rats change over repeated sessions in the novel environment. The spatial tuning of a pyramidal neuron can be quantified and expressed as a number of metrics such as spatial information, sparsity, place field size, and the proportion of the environment in which the neuron fires. Spatial information measures the amount of information a single action potential conveys about the animal’s location in an environment, with higher spatial information reflecting more informative firing [While the mean firing rate of neurons typically decreases with experience in an environment, the spatial tuning of pyramidal neurons has been shown to increase as a function of experience both in mice , 63 and e firing . Sparsite firing ; Place fFmr1y−/ cells. LME models were used to statistically analyse each of these measures of spatial activity across the population of active pyramidal cells in each session . CA1 pyramidal cells in WT rats showed a significant increase in spatial information between the first and second day of exposure to the novel environment. However, cells in Fmr1y−/ rats did not show this increase .The example cells in Fig. Fmr1y−/ cells, again consistent with experience-dependent refinement of spatial tuning in WT rats that is absent in Fmr1y−/ rats . Similar to the pattern for spatial information, there were no significant differences between genotypes on either day for either sparsity  or place field size . The fourth measure of spatial tuning, the proportion of visited pixels in which a cell fired , but no significant genotype x day interaction (p = 0.070), suggesting that by this measure, spatial tuning improved similarly in both genotypes across days. The individual cell and rat mean data for each of these spatial measures are depicted in Additional file Both sparsity Fig. B and plaFmr1y−/ rats. It is unlikely that this pattern of results can be explained by differences in exploratory behaviour between WT and Fmr1y−/ rats. Specifically, while the calculation of spatial information, sparsity and % active pixels are all influenced by the extent of exploration, we would expect this to result in higher spatial information together with lower sparsity and lower % active bins in the Fmr1y−/ rats which explored more of the environment than WT. This is the opposite from what was found on Day 2. The calculation of place field size is independent of visited pixels, and so it too is unlikely to be influenced by the small differences between genotypes in the number of visited pixels. Finally, as there were no differences between the amount of the environment explored on Day 1 and Day 2, it is unlikely that differences in exploration could account for the changes in spatial tuning across days in WT (but not in Fmr1y−/) cells.Taken together, these data indicate that CA1 pyramidal neurons from WT rats exhibit experience-dependent refinement of spatial tuning between the first and second day of exploring a novel environment, but that this refinement is attenuated in Fmr1y−/ cells could be secondary to the decreased mean firing rate of WT cells on Day 2. To address this, we conducted a bootstrapping-like downsampling analysis. We sampled randomly 30 cells from each day and each genotype, 1000 times. The spatial information, sparsity, place field size and % active bins were extracted only for 30-cell subpopulations with average mean firing rate equal to the overall mean firing rate of the WT cell population on day 2 (+ / − 5%). The portion of 30-cell subpopulations (out of 1000) that were statistically different from their overall population in a given measure was used as the probability of that measure to be modulated by mean firing rate for that population. Our downsampling analysis indicated that the experience-dependent increase in the spatial tuning of WT is not secondary to the decrease in mean firing rate . There was a significant main effect of session comparison (LME main effect of comparison p < 0.001), driven by both groups showing lower correlations between days (S3-S4) than between sessions within each day . The interaction between genotype and comparison was also not significant (LME genotype x session comparison p = 0.617). Individual cell data and rat average data are shown in Additional file Another key property of pyramidal neurons that has been linked to synaptic plasticity is the stability of their firing rate maps . InteresFmr1y−/ S3-S4 distributions 1000 times. Only 30-cell subpopulations with average firing rate S3-S4 difference equal to the mean of WT cells were used. The proportion of subpopulations with S3-S4 correlations statistically different from their entire population was used as the probability for the firing rate map correlations between S3 and S4 to be modulated by mean firing rate. We repeated the downsampling, but this time only 30-cell subpopulations with null average firing rate S3-S4 difference were used. We found that the low stability of firing rate maps between S3 and S4 was not due to the decrease of firing rate in WT cells, and that Fmr1y−/ show similar firing rate map stability to WT cells even if there is no difference in their mean firing rate decrease.To address whether the decrease in mean firing rate in WT cells between Day 1 and Day 2 may have contributed to the relatively low stability between S3 and S4, we conducted a similar bootstrapping-like downsampling procedure as described above, whereby we randomly sampled 30 cells from both WT and Fmr1y−/ rats exhibit normal spatial stability in their firing rate maps, both between sessions within the same day and between the first two days of exposure to a novel environment. While this is consistent with stable spatial coding in Fmr1y−/ rats, this finding contrasts with the observed deficits in the experience-dependent improvement in spatial tuning over the two days.These analyses indicate that pyramidal neurons from Fmr1y−/: 48 cells from 12 rats) from the dorsal hippocampus matched to the location of the in vivo recordings. First, we measured the intrinsic excitability of CA1 pyramidal neurons in response to depolarizing current steps , which in WT neurons resulted in repetitive action potential discharge . We have recently shown in the CA1 of juvenile Fmr1y−/ mice that such changes in intrinsic cell excitability are correlated with altered membrane potential, reduced threshold to fire, and longer AIS [Fmr1y−/ rats, we observed no difference in passive properties such as resting membrane potential , input resistance , rheobase current  or action potential threshold . However, the medium afterhyperpolarization (mAHP) was reduced by 13% in Fmr1y−/ CA1 pyramidal neurons compared to WT . Consistent with the similar action potential thresholds measured between genotypes, we observed no difference in the AIS length, as assessed by AnkyrinG immunostaining .Overall, our analyses suggest that loss of FMRP leads to reduced adaptation of CA1 pyramidal neurons to a novel environment across days, maintaining high spike discharge and low spatial tuning independent of experience. A key question is whether these phenotypes result from changes in the intrinsic properties of CA1 pyramidal neurons themselves, alterations in the inputs they receive, or a combination of the two. To address this question, we performed whole-cell patch clamp recordings from adult CA1 pyramidal neurons  inputs from layer 3 of the medial entorhinal cortex (MEC3) onto the distal dendrites in stratum lacunosum-moleculare [stratum radiatum did not show a difference between genotypes in the resulting excitatory postsynaptic potential (EPSP) measured over the range of stimulation intensities tested  = 2.03, p = 0.16). In contrast, there was a robust decrease in the recruitment of EPSPs in CA1 pyramidal neurons from the Fmr1y−/ rats in response to TA stimulation compared to WT rats  = 15.4, p = 0.0002). This was reflected by a similar input–output slope for SC stimulation between genotypes, but a reduced slope for TA inputs in the Fmr1y−/ rats  = 16.12, p = 0.001 Kruskal–Wallis test). We also observed bidirectional effects on short-term plasticity in the different inputs. Specifically, at the SC inputs paired-pulse ratio (PPR) was lower in slices from Fmr1y−/ than from those of WT rats at all stimulation intensities  = 10.6, p = 0.002), whereas at the TA inputs PPR was higher in the Fmr1y−/ rats at the 90 V stimulation intensity  = 3.92, p = 0.024, 90 V: t = 2.47, p = 0.05). These reductions in synaptic input were not associated with gross anatomical changes in CA1 pyramidal neurons  inputs from CA3 neurons in leculare . We haveleculare . Cell atFmr1y−/ and WTs, both in terms of spike probability  = 0.032, p = 0.86) and in the number of cells that produced a spike in any response to any stimulus . In contrast, following TA afferent stimulation, the overall spike probability was not different between genotypes at 30 V and 60 V stimulation . However, it tended towards lower spiking in Fmr1y−/ than in WT rats at 90 V , suggesting a level of compensation, at least at lower stimulation intensities  = 2.276, p = 0.13). Nevertheless, the overall recruitment of individual CA1 pyramidal neurons was reduced by approximately threefold , indicating a degree of information loss. Despite the reduction in CA1 pyramidal neuron recruitment, when present, spikes showed a similar coefficient of variation of spike times between genotypes, in the absence of GABAA receptor mediated inhibition  = 0.23, p = 0.97).To determine whether the increased AP discharge observed in CA1 pyramidal neurons is sufficient to overcome the reduced synaptic strength observed in response to electrical stimulation of the TA pathway, we next asked if spike output was altered. In cell-attached recordings from pyramidal neurons under the same conditions, we performed paired-pulse stimulation at SC and TA synapses to CA1. We observed a similar recruitment of CA1 pyramidal neurons to SC pathways stimulation in ig. F1,15 = 0.032,Fmr1y−/ rat are hyperexcitable, which results from a decrease in medium AHP amplitude. This hyperexcitability is sufficient to overcome the reduced synaptic strength following TA stimulation received by Fmr1y−/ CA1 pyramidal neurons.Together, these data show that CA1 pyramidal neurons in the dorsal hippocampus of the Fmr1y−/ rats, 3-way mixed ANOVAs  indicated that there were no significant differences between genotypes or days, and no genotype x day interactions for theta, slow gamma or medium gamma power  = 0.880, p = 0.367, day F = 0.322, p = 0.581, genotype x day F = 0.666, p = 0.430; Slow gamma: genotype F = 1.988, p = 0.184, day F = 0.120, p = 0.735, genotype x day F = 1.613, p = 0.228; Medium gamma: genotype F = 3.109, p = 0.103, day F = 0.437, p = 0.521, genotype x day F = 3.343, p = 0.092). For theta, there was a significant decrease in power across sessions within a day  = 4.580, p = 0.021) but this did not differ between genotypes  = 0.337, p = 0.717) or across days  = 1.128, p = 0.340). Neither slow nor medium gamma power differed across sessions within each day  = 0.204, p = 0.817); medium gamma: session F = 0.184, p = 0.265). In order to examine whether our findings are dependent on rigid definition of frequency bands, we analysed the power spectrograms across the rats of each genotype for each session  while an animal explores its environment , 69. Thep’s > 0.05), indicating that in this dataset, hippocampal oscillatory power in the frequency ranges we analysed did not differ significantly between genotypes at any velocity.As oscillatory power is affected by speed of movement , 72, we Fmr1y−/ and WT rats across sessions and days . The MVL for theta . However, there was a significant decrease in MVL across sessions within a day (session-in-day effect p = 0.026), with no significant interactions. In contrast, neurons from Fmr1y−/ rats showed significantly less phase locking (shorter MVLs) to slow gamma than those from WT rats , with no differences between days (LME day effect p = 0.537) or sessions within a day (LME session-in-day p = 0.057) and no significant interactions (LME day x genotype p = 0.109). Despite the absence of a significant day x genotype interaction, inspection of the data indicated that the difference between genotypes was larger on Session 1 of Day 1 than in later sessions. Thus, the significant main effect of genotype may have been driven primarily by the Session 1 differences in MVL. To address this possibility, we analysed the data for Day 1 and Day 2 separately. This revealed a significant genotype x session interaction on Day 1 (LME genotype x session p = 0.011), with post hoc tests indicating significant differences between genotypes on sessions 1 and 3 (p < 0.05). On Day 2, there was no significant difference between genotypes (LME genotype effect p = 0.374) and no genotype x session interaction (LME genotype x session interaction p = 0.852). An interpretation of this analysis is that the Fmr1y−/ rats show less phase locking to slow gamma on Day 1, but not on Day 2. For medium gamma , or days (LME p = 0.082), but there was a significant difference between sessions within a day (LME session in day p = 0.046), with no significant interactions.To examine the temporal organization of CA1 pyramidal neuron firing relative to theta, slow gamma and medium gamma oscillations, we computed the mean vector length (MVL) of the phase distribution of spikes for each pyramidal neuron for each of these frequency bands. This provides a measure of how consistently a neuron fires at a specific phase of the oscillation. We first compared the MVLs of all recorded CA1 pyramidal neurons in eta Fig. A, B did Fmr1y−/ rats may be secondary to differences in mean firing rate, we conducted a similar bootstrapping-like downsampling procedure as described previously for spatial metrics and firing rate map correlations. This analysis indicated that the mean firing rate differences between WT and Fmr1y−/ cells on Day 2 cannot explain the genotypic differences in phase locking to slow gamma oscillations . Consistent with the previous analysis, the proportion of neurons  that were significantly phase locked to theta did not differ significantly between genotypes. However, there was a significant effect of session . Additional analyses revealed that theta phase locking was higher in sessions 1 and 3 compared to sessions 5 and 6, for both WT and Fmr1y−/ rats .To further probe the modulation of CA1 spiking activity by oscillatory activity, and to allow direct comparison to previous findings in mice, we next quantified the proportion of recorded CA1 pyramidal neurons that were G2 = 10.92, p = 0.053; genotype G2 = 4.459, p = 0.035; session G2 = 21.19, p < 0.001). Given that the genotype x session interaction approached significance, we compared across sessions for each genotype separately. This revealed that the proportion of slow gamma phase locked neurons was higher in WT that rats in Sessions 1 and 3 of Day 1, but not for any other session . Moreover, a significantly higher proportion of WT neurons were phase-locked in Session 1 than any other session, whereas there were no differences across sessions for Fmr1y−/ rats .For slow gamma Fig. F, the prG2 = 2.178, p = 0.824; genotype G2 = 6.24, p = 0.013; session G2 = 8.42, p = 0.135).Finally, for medium gamma FI there wFmr1y−/ rats was less strongly phase locked to slow gamma oscillations compared to those of WT rats, particularly on Day 1. The overall strength of phase locking across the cell population to medium gamma did not differ significantly between WT and Fmr1y−/ rats, but fewer neurons from Fmr1y−/ rats were significantly phase locked to medium gamma. Thus, the timing of CA1 pyramidal neuron firing relative to both slow and medium gamma oscillations is less consistent in Fmr1y−/ compared to WT rats. In addition, in WT (but not in Fmr1y−/ rats) significantly more neurons were phase-locked to slow gamma in Session 1 on Day 1 (when the environment was completely novel) than in any other session. This provides further evidence of disruption in experience-dependent changes in CA1 neuronal activity in Fmr1y−/ rats. Individual cell data and rat average data are shown in Additional file Collectively, these analyses reveal that the population of CA1 pyramidal neurons from Fmr1y−/ rats exhibit decreased phase modulation by slow gamma oscillations and the fact that inputs from CA3 (reflected in slow gamma power) arrive predominantly during the descending phase of theta, we predicted that Fmr1y−/ CA1 pyramidal neurons would show decreased firing preference for the descending phase of theta compared to WT neurons.Another measure that can be used to characterize the organization of ensemble activity patterns is the timing of CA1 pyramidal neuron spiking relative to the theta and gamma oscillations. Inputs to the CA1 area of hippocampus have specific temporal organization, with inputs from CA3 arriving predominantly during the descending phase of CA1 theta, and inputs from MEC3 neurons primarily during the ascending phase and peak of theta , 74 FigA. MoreovFmr1y−/ and WT rats. Only neurons with significant phase modulation (based on MVL Rayleigh p < 0.05) were included in our analysis. Due to the circular nature of the data, we used the Harrison-Kanji two-factor test for circular data and the Watson-Williams multi-sample test to explore differences between genotypes and days. As can be seen in Fig. Fmr1y−/ rats, pyramidal neurons fired predominantly during the ascending phase of theta on both Day 1 and Day 2 . Analysis of the slow gamma preferred spiking phase , but that Fmr1y−/ neurons fired at an earlier phase of slow gamma than WT neurons , with no difference in phase between Day 1 and Day 2 (main effect of day: p = 0.614), and no significant genotype x day interaction (p = 0.664). Post hoc tests exploring the main effect of genotype further revealed a significant difference in slow gamma phase preference between WT and Fmr1y−/ cells on Day 1 (p < 0.05) but not on day 2 (p = 0.103). Finally, medium gamma phase preference was not significantly different between WT and Fmr1y−/ rats , with no phase differences between days (p = 0.134), and no genotype x day interaction (p = 0.419)  Fig. .Fig. 9ThFmr1y−/ cells were driven by genotypic differences in mean firing rate, we conducted a downsampling analysis as described previously. This showed that theta phase preference differences were not secondary to mean firing rate differences between genotypes. Given the low numbers of cells that were significantly phase locked to gamma oscillations, we did not perform a downsampling analysis for slow and medium gamma phase preference.To explore whether the differences in preferred firing phases relative to theta between WT and Fmr1y−/ pyramidal neurons exhibit differences in their preferred phase discharge compared to their WT counterparts. For theta, this difference is more prominent during the exploration of a novel environment (Day 1), while for slow gamma, the difference persists throughout both days of recordings.Together, these analyses indicate that Fmr1y−/ mice and rats [Fmr1y−/ rats. We also predicted that experience-dependent features of hippocampal cellular activity and spatial coding would be affected in Fmr1y−/ rats, as we and others have previously shown alterations in hippocampal synaptic plasticity [Fmr1y−/ rats display significant impairments in adaptation to repeated presentations of a novel spatial environment. Specifically, while WT neurons exhibit experience-dependent changes in firing rate, bursting, spatial tuning and spike timing relative to theta oscillations across days in a novel environment, these changes are absent in Fmr1y−/ rats. This absence of neurophysiological changes with repeated experience in a novel environment is reminiscent of a core clinical feature of FXS patients; abnormal habituation to novelty [Fmr1y−/ rats also showed less phase locking to gamma oscillations in an experience-independent fashion. Our in vivo findings are supported by ex vivo recordings that provide evidence for single cell hyperexcitability in the face of reduced synaptic inputs from the medial entorhinal cortex. Using ex vivo electrophysiological recordings, we showed that the circuit function abnormalities we observe may be, at least in part, the result of reduced synaptic inputs from MEC3 to CA1. Together, these data provide novel insight into how the absence of FMRP can disrupt neural circuit function that may lead to cognitive difficulties.Individuals with FXS have intellectual disability and ASD, two features in which hippocampal pathophysiology has been implicated , 77, 78.and rats  led us tasticity , 18, 20. novelty , 81. Fmr, but that these changes were absent in Fmr1y−/ rats. The decrease in mean firing rate with experience in a novel environment is thought to be mediated by synaptic plasticity, whereby synaptic inputs to CA1 pyramidal neurons are weaker in a familiar environment than in a novel environment [Fmr1y−/ CA1 pyramidal neurons exhibit increased excitability to compensate for the diminished synaptic inputs from MEC3 , the increased excitability of Fmr1y−/ CA1 pyramidal neurons leads to sustained increase in firing rate. We have previously shown a similar functional effect  in the dorsal hippocampus of a mouse model of FXS [Fmr1y−/ rodents may be species independent, the mechanism by which mice and rats compensate for such change is divergent.We found that the firing rate and burstiness of CA1 pyramidal neurons are modulated by experience in WT rats, as has been shown previously , 62, 82,ironment . Strong EC3 Fig. . We propl of FXS . InteresFmr1y−/ KO mouse models, which had reported inconsistent effects of FMRP loss on CA1 pyramidal cell firing rates. Specifically, Boone et al. [Fmr1y−/ than in WT mice exploring a highly familiar environment. In contrast, no differences in mean firing rate were reported between genotypes in the study by Talbot et al. [Fmr1y−/ rats, which is consistent with the findings of both the Talbot and Arbab studies. Instead, we found that cells in Fmr1y−/ rats failed to show the experience-dependent decrease in firing rate observed in WT rats. This phenotype was likely not observed in the Arbab et al. study because unlike the WT rats in our study, the WT mice in the Arbab et al. study did not show any experience-dependent changes in firing rate between sessions either within or between days. This lack of experience-dependent changes in CA1 pyramidal cell firing rate in a novel environment in WT mice is consistent with a previous study in WT mice [Fmr1y−/ than WT mice in a highly familiar environment, which we suggest may be explained by experience-dependent changes occurring over a more extended period in WT mice (which is lacking in Fmr1y−/ mice), leading to differences between genotypes emerging only after many days of experience in an environment.Our data may provide some reconciliation of previous findings in e et al.  reportedt et al.  in whicht et al.  recorded WT mice  and may  WT mice , 84. The WT mice  result iFmr1y−/ pyramidal neurons. Given that the likelihood of bursting depends largely on the intrinsic excitability of neurons [Fmr1y−/ neurons may also be explained by the homeostatic increase in the excitability of Fmr1y−/ CA1 pyramidal neurons  both on Day 1 and Day 2 in an initially novel environment, with no changes in these parameters across days in either group. They did not observe any differences between genotypes in spatial information. In contrast, neither Boone et al. [Fmr1y−/ mice. Considering these data together, it is possible that spatial tuning deficits in Fmr1y−/ mice do not persist in more familiar environments. While we cannot account for the differences between our rat findings and the mouse findings in a novel environment, both our findings and those of Arbab indicate decreased spatial tuning in Fmr1y−/ animals, albeit with a different dependence on experience within the novel environment. Indeed, the WT mice in the Arbab et al. study [Fmr1y−/ rats.Comparing the current findings to the previous mouse studies, Arbab et al.  reportede et al.  , suggesting some remapping across days in both groups. This degree of remapping between the first two days in an initially novel environment with a total of 30 min exposure on Day 1 is comparable with that recently reported in WT rats [While spatial tuning showed the expected refinement between days 1 and 2 in WT rats, the within-day, between-session stability of firing rate maps of pyramidal neurons did not increase from Day 1 to Day 2 in either WT or  WT rats . HoweverFmr1y−/ rats were as stable as those of WT rats, both within and between days. This is not consistent with the findings of Arbab et al. [Fmr1y−/ mice exhibited lower firing rate map stability both within  and between two 30 min exposures to a novel environment compared to WT mice [Contrary to our predictions, the firing rate maps of b et al.  who repo WT mice . Our dowExperience-dependent increases in spatial tuning of CA1 pyramidal neurons depend on the autophosphorylation of the α-Isoform of the Calcium/Calmodulin-Dependent Protein Kinase II (α-CaMKII) . HoweverFmr1y−/ mice in which stronger theta [Fmr1y−/ rats, echoing the findings from a number of studies reporting that slow gamma power is stronger in the hippocampus of Fmr1y−/ mice [Fmr1y−/ mice [Fmr1y−/ rats [Our LFP recordings from the CA1 pyramidal neuron layer did not reveal any significant differences between genotypes, or any experience-dependent changes in the power of theta (6–12 Hz), low-range gamma (30–45 Hz) or mid-range gamma (55–100 Hz) band activity. Crucially, these results as not dependent on the definition of frequency boundaries we used. Whole spectrogram analyses for every session did not reveal any significant differences either. At face value, these findings contradict previous reports in er theta  and slower theta –23 have er theta . Therefo−/y mice –23. Beyo−/y mice , the par−/y rats , and in −/y rats , which sGiven our ex vivo findings that MEC inputs are diminished in the absence of FMRP, we might have predicted weaker medium gamma power compared to WT. However, the anatomical organization of inputs to the hippocampus leads to large variations in the power of network oscillations between locations within the hippocampus , 69, 93.Fmr1y−/ rats were less strongly phase locked to the cycles of gamma oscillations than those of WT rats. The most robust difference in phase locking between WT and Fmr1y−/ rats were seen in the slow gamma range on Day 1, although the proportion of neurons that were significantly phase locked to medium gamma also differed between genotypes. Furthermore, the preferred firing phase of Fmr1y−/ neurons was earlier during the descending phase of slow gamma compared to WT pyramidal neurons. These results are consistent with a previous study in Fmr1y−/ mice, which reported weaker modulation of pyramidal neuron firing by slow gamma, unstable ensemble coordination in relation to slow gamma (40 Hz), and discharge preference for earlier slow gamma phases [Fmr1y−/ than WT rats suggests not only weaker coupling with extrinsic inputs , but may also reflect local microcircuit dysfunction, as has been shown in the developing neocortex [The spikes of pyramidal neurons in a phases . Less speocortex , 94–96.Fmr1y−/ pyramidal neurons fired preferentially in the early ascending phase of theta on both days and showed no experience-dependent changes. Previous reports suggest that theta phase preference shifts towards the ascending phase during exploration of a novel environment [WT neurons fired preferentially during the late descending phase of theta in the first session in the novel environment, and their activity moved towards the early ascending phase with repeated exposures across the two days. In contrast, ironment , 75, 97.Fmr1y−/ pyramidal neurons to fire during the ascending phase of theta is nearly identical with a previous study in Fmr1y−/ mice [Fmr1y−/ rats. Given the known theta phase segregation of inputs to CA1  for spike output, which is consistent with our previous findings in Fmr1y−/ mice [Fmr1y−/ rats.Interestingly, the preference of −/y mice , and maye theta; ), based −/y mice . TogetheFmr1y−/ pyramidal neurons is, at least in part, due to a persistent decrease of inhibitory tone during the ascending phase of theta in Fmr1y−/ rats, which permits pyramidal neuron discharge during that phase. It would be very interesting to explore the organization of hippocampal activity by theta oscillations further. Using multisite probes that allow better monitoring of neural activity across hippocampal subfield, and allowing animals to explore environments with narrow alleys, phenomena such as theta phase precession can be studied more easily [Given the precise temporal organization of inhibition in the hippocampus , 99 and e easily . Interese easily .Fmr1y−/ rats in the current study did not display the experience-dependent changes in firing and spatial tuning of CA1 place cell activity seen in WT littermates. The failure to update their spatial coding of a novel environment across days may reflect an inability of Fmr1y−/ rats to discriminate subtle spatial and contextual information. In contrast, firing rate map stability between sessions and days does not differ between genotypes. This suggests that, to the extent that firing rate maps reflect recognition of the environment, Fmr1y−/ rats would not be impaired in their ability to recognize that they are in a familiar environment. Our limited behavioural measures (pathlength) in the current study also support this conclusion, as there is a decrease in pathlength both within each day and between days (and it is known that rats tend to explore novel more than familiar environments). However, further behavioural studies are required to establish whether indeed the Fmr1y−/ rats can detect the novelty/familiarity of the environment. This could be done by measuring additional exploratory behaviours known to be associated with environmental novelty, such as rearing, across repeated exposures to a novel environment across days (in a situation in which exploration is not being motivated by scattered treats in the environment), or by testing the animals in a task in which they are required to make different responses for rewards in novel vs familiar environments.Overall, Fmr1y−/ CA1 pyramidal neurons to slow gamma, it is plausible that fine spatial information retrieval is affected in Fmr1y−/ rats, and that underlies their inability to increase their spatial tuning over days. Indeed, previous work has shown that Fmr1y−/ mice exhibit behavioural inflexibility when required to learn new spatial rules that conflict with previous knowledge and that this correlates with abnormally frequent events of strong slow gamma oscillations [Increased phase locking of CA1 pyramidal neurons to slow gamma is believed to be involved in memory retrieval processes , 76, 103llations .Fmr1y−/ CA1 pyramidal neurons would improve their spatial tuning and reduce their mean firing rates to levels comparable to WT littermates. Future work should explore habituation to novelty over longer time scales and increased experience. In addition, comparing activity in a novel environment with that in a very familiar environment would allow more direct comparisons with previous studies.Our recordings span only two days of habituation to an initially novel environment. It is unclear whether, given enough experience, Fmr1y−/ rats. Moreover, none of the measures used to assess firing properties, spatial coding or spike timing within each session depended on having data from the same cells across sessions or days, as only data from “active” cells in a given session were included in these analyses, so they are effectively different (highly overlapping) populations of cells. The only exception to this was in the calculation of spatial firing rate map correlations, where the firing rate maps of individual cells were compared across sessions and days. It is therefore possible that the weaker correlations between days than within days may reflect poorer tracking of cells between days. As there were no differences in firing rate map correlations between genotypes, this limitation does not affect the conclusions of the current study. However, future experiments aimed at assessing stability of spatial firing over multiple days may be better served by using imaging techniques that allow definitive tracking of the same cells across days [A second limitation is that our ability to track the same cells over days may not be as robust as the ability to track cells within a day. To minimize this possibility, spike sorting was conducted on the data from all 6 sessions over the two days combined, and all clusters were visually inspected to ensure that waveforms from a given cluster did not differ markedly from session to session or across days. Nonetheless, it is possible that there was more cluster drift between days than within days. Importantly, there is no reason why poor tracking of cells would differentially affect the data obtained from WT vs oss days .Fmr1y−/ cells, the groups did not differ significantly from one another on either day for these measures. While it is clear from the pattern of the data that the groups did not differ on Day 1, it is possible that the failure to observe significant differences between the groups on Day 2 may have been due in part to being underpowered to detect such a difference. For this reason, we have not drawn any conclusions based on whether or not the groups differed on Day 2, but rather, restricted our discussion to the lack of experience-dependent changes in the Fmr1y−/ cells.A third limitation relates to the observation that while WT cells showed significant decreases in activity and significant increases in spatial coding across days which were not observed in Finally, all of our CA1 recordings were made from neurons approximately midway along the proximal–distal axis between CA3 and subiculum. Given the known spatial tuning gradient along the CA1 proximo-distal axis hippocampus , we shouOverall, our findings offer new insights into circuit pathophysiology associated with the loss of FMRP and how that may lead to abnormal habituation to novelty. It would be interesting to see if hippocampal circuit deficits converge with those seen in other rodent models of ASD/ID. Interestingly, absence of experience-dependent update in spatial tuning has been reported previously in a mouse model of Rett syndrome , while rAdditional file 1. Table S1, Table S2, Fig. S1, Fig. S2, Fig. S3, Fig. S4, Fig. S5, Fig. S6, Fig. S7, Fig. S8, Fig. S9, Fig. S10, Fig. S11 and Fig. S12.Additional file 2. Table S3, Table S4, Table S5, Table S6, Table S7, Table S8 and Table S9.


Large vertical electron detachment energies  confirm the superhalogen identity of these anions. A larger VDE value can be obtained by introducing small M or large M′ central atoms and small halogen ligand atoms. Thus, one isomer of LiCaCl4− possesses the largest VDE value. Besides, when the extra electron is shared by all ligand atoms or three bridging ligand atoms, the isomers have relatively larger VDE values.A series of hetero-binuclear superatom motifs involving chloride/bromide ligands, that is, MM′X They wek+1 for a class of superhalogens, in which M represents the central main group atom, K is the highest valence of M atom, and X is the halogen atom  were reported by Wang et al., and their VDE values were experimentally measured for the first time and theoretically calculated applying the outer valence Green function (OVGF) method, which are consistent with each other well  superhalogen anion n clusters . They cclusters , supersaclusters , ion batclusters , ionic lclusters , liquid clusters , solar cclusters , and so 4−  anions have been proposed and systematically investigated. The considerable VDE values of these anions confirm their superhalogen identity. The geometric features and relative stability of these anions were analyzed. Meanwhile, the correlations between their VDEs and structural features, ligand and central atoms, and extra electron distribution are also revealed. The present investigation predicts a new member of superhalogens and conduces to the development of new strong oxidizing agents.To the best of our knowledge, most hitherto proposed superhalogens are mono- or homo-nuclear. The hetero-nuclear superhalogens involving different central atoms, however, have received very little attention. The investigation on the influence of different ligands on hetero-nuclear superhalogen properties, however, has not been reported yet. In this research, we aim to design a new class of superhalogen anions with two different central atoms using chloride or bromine atoms as ligands. Consequently, the MM′X4−  anions were built by considering all the possible connection between M, M′, and X atoms. Then, all the constructed structures of anions were optimized using the Møller–Plesset perturbation method (MP2)   togetherod (MP2) . Meanwhiod (MP2)  and sing4− anions were calculated applying the outer valence Green function (OVGF) approximation  of the MM′Xximation  with theximation .The above-mentioned calculations were performed using the GAUSSIAN 16 program package . The plo4−anions are depicted schematically in 4− anion has two types of structures, that is, central atoms M and M′ are connected by two or three bridging X atoms. Notably, these two structures are also presented in the superhalogen anions with F ligands  and NaMgBr4−-2 (126.5°), respectively.MM'Br2F5−  . MM'X4−4− anions are gathered in 4−-3 and LiMgCl4−-2 are greater than those of NaMgCl4−-3 and NaMgCl4−-2, respectively. However, the only one exception is the VDE values of isomers LiCaBr4−-3 and NaCaBr4−-3. This is probably due to the different extra electron distribution of these two isomers, which will be discussed in the following. Therefore, the hetero-binuclear superhalogen anions with large VDE values could be constructed by introducing small alkali metal atoms into the system. It is worth noting that the same trend was found for the other hetero-binuclear anions with the F atom, cyanide, and isocyanide as ligands ((1) When the M atom varies from the Li atom to Na atom, the VDE values of the studied anions show a tendency of decreasing in similar structures. For example, the VDE values of the isomers LiMgCl ligands .4− anions are presented in 4−→MMgX4−→MCaX4−. Hence, the hetero-binuclear superhalogen anion MM'X4− could possess a larger VDE value by involving larger alkaline earth metal atoms. Note that it also holds true for the superhalogen anions with other ligands ((2) The largest VDE values for each MM'X ligands .The VDE values of MM'X4− anions can be regarded as MX (M'X3)−; thus, the comparison between MM'X4− anions and their corresponding mononuclear superhalogen anions M'X3− is also necessary. For this reason, the VDE values of M'X3−  anions were also calculated at the same level and are listed in 3− also increase from BeX3− to CaX3−. Besides, the mononuclear anions M'X3− possess lower VDE values than their corresponding hetero-nuclear anions MM'X4− (except for NaBeCl4− and NaMgX4− series). So again, the superhalogen anions could gain larger VDE values by increasing the number of central atoms.4−  The relationship between the VDE values and the ligand atoms is plotted in − 4−, the VDE values of LiM'X4−-3 are always larger than those of LiM'X4−-2. This is probably due to the fact that the extra electron distribution in two isomers is different. To analyze this clearly, the highest occupied molecular orbitals (HOMOs) of some representative MM'X4− isomers are depicted in t atom in LiBeCl4−-2, while localized on the three bridging Xb atoms in LiBeCl4—3, which is a benefit for the extra negative charge dispersion, and thus, LiBeCl4−-3 possesses a larger VDE value. For NaBeX4− anions, the extra electrons of two isomers are all distributed on the terminal X atom. Interestingly, the isomer NaBeX4−-2 in which the extra electron goes on Xt atom has a higher VDE value than isomer NaBeX4—3, which goes on the Xt’ atom  For the two isomers of LiM'Xatom see . As to Nuted see , and, heAs pointed out earlier, the MM'X4−  species has theoretically proposed a series of hetero-binuclear superhalogen anions. The results show that these heteronuclear superhalogen anions could gain larger VDE values by involving a smaller alkali metal atom M, a larger alkaline earth metal atom M′, and a higher electronegative ligand atom X. Thereby, of all the anions studied, an isomer of LiCaCl4− anions possesses the largest VDE value (6.799 eV). Moreover, the extra electron distribution is a very influential factor in the VDE values of structural isomers. For the NaBeX4− anions, the isomers have larger VDE values when the extra electron is distributed on the terminal Xt ligand atom instead of the Xt’ ligand atom. For the other anions, the isomers possess larger VDE values when the extra electron is shared by all ligand atoms or three bridging ligand atoms.Our systematic investigation of the MM′X


Alzheimer’s disease (AD) is a progressive neurodegenerative condition characterized by clinical decline in memory and other cognitive functions. A classic AD neuropathological hallmark includes the accumulation of amyloid-β (Aβ) plaques, which may precede onset of clinical symptoms by over a decade. Efforts to prevent or treat AD frequently emphasize decreasing Aβ through various mechanisms, but such approaches have yet to establish compelling interventions. It is still not understood exactly why Aβ accumulates in AD, but it is hypothesized that Aβ and other downstream pathological events are a result of impaired bioenergetics, which can also manifest prior to cognitive decline. Evidence suggests that individuals with AD and at high risk for AD have functional brain ketone metabolism and ketotherapies (KTs), dietary approaches that produce ketone bodies for energy metabolism, may affect AD pathology by targeting impaired brain bioenergetics. Cognitively normal individuals with elevated brain Aβ, deemed “preclinical AD,” and older adults with peripheral metabolic impairments are ideal candidates to test whether KTs modulate AD biology as they have impaired mitochondrial function, perturbed brain glucose metabolism, and elevated risk for rapid Aβ accumulation and symptomatic AD. Here, we discuss the link between brain bioenergetics and Aβ, as well as the potential for KTs to influence AD risk and progression. Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is expected to affect more than 15 million Americans by the year 2050 . There aImpaired brain metabolism in the form of mitochondrial dysfunction and reduced glucose utilization has also long been recognized as an important AD hallmark. Like Aβ, impaired brain bioenergetics can occur long prior to AD clinical symptoms  and mechAn estimated 20–40% of cognitively unimpaired older adults have elevated cerebral Aβ, meeting the definition of “preclinical AD” . These iAlthough a small proportion of AD diagnoses are familial, greater than 97% of all diagnoses are late onset, sporadic AD . Recent The role for Aβ in normal physiology is not well understood. It is expressed in low concentration in multiple peripheral tissues and the brain throughout the lifespan. In the brain, Aβ monomers are thought to be expressed in response to neuroinflammation as part of the brain’s innate immune system  causing Aβ is a peptide, commonly of 40 (Aβ40) or 42 (Aβ42) amino acids in length in its primary forms , derivedThe majority of APP is processed in the “non-amyloidogenic” pathway where cleavage by α-secretase produces a secreted APPα (sAPPα) fragment that is released into extracellular space and an 83 amino acid C-terminal fragment (CTF83) that remains at the plasma membrane. CTF83 is cleaved by γ-secretase to form p3  that is released into extracellular space and an APP intracellular domain (AICD) peptide that enters the cytoplasm. The truncated Aβ peptide, p3, in AD has not been well studied and recent work suggests that p3 can aggregate in AD and that α-secretase cleavage of APP may not truly be “non-amyloidogenic” . Cleavagvia multiple pathways including efflux and localized protein degradation. Approximately 50% of all brain Aβ clearance occurs via efflux with 25% directly transporting across the blood brain barrier and 25% entering cerebro-spinal fluid (CSF) for reabsorption into venous circulation  . GeneticAlthough several genes have been linked as risk factors for sporadic AD, allelic expression of Apolipoprotein E ε4 (APOE ε4) is thought to be the most impactful genetic risk factor. Approximately 9–23% of humans express at least one APOE ε4 allele , a prevaAberrant APP post-translational modification and trafficking are also observed in patients with sporadic AD  which alMitochondria are the primary sites for cellular energy metabolism. APP, its processing, and mitochondria exhibit a complex, dynamic relationship where dysregulation of either APP or mitochondria exert detrimental influential effects upon the other in a vicious cycle. Here we briefly review this relationship as this topic has been previously reviewed in detail .Mitochondrial bioenergetics are influenced by dysregulated mitochondrial import of APP and Aβ. APP localized to the mitochondria is thought to inhibit mitochondrial protein import  and altein vitro studies suggest that inhibiting glycolysis or mitochondrial respiration downregulates non-amyloidogenic APP processing pathways  is an eating pattern characterized by high fat, very low carbohydrate, and adequate protein intake. Americans typically consume roughly half of their energy intake as carbohydrate  which code novo glucose (gluconeogenesis) to maintain glucose homeostasis , which we have shown can be nutritionally dense , seeks tMedium-chain triglycerides (MCTs) are comprised of glycerol backbones with aliphatic fatty acid tails that are 6–12 carbons in length and are more readily absorbed than long-chain triglyceride (LCT) . Unlike MCTs are naturally occurring in coconut oil, coconut products, palm kernel oil, and whole dairy products and marketed products can be derived from any one of these foods or synthetically made. Since MCTs are directly absorbed in the gut rather than undergoing emulsification in bile salts, large doses of MCT can cause GI distress. Emulsification of MCTs is suggested to optimize absorption, GI tolerance, and ketone production  which havia the portal vein (Exogenous sources of ketones are another way to induce systemic ketosis without dietary macronutrient manipulation . Though tal vein . R-1,3-Btal vein , resultiPreliminary studies suggest that KTs may benefit cognition in patients with AD and MCI . Many naThere is evidence that KTs may beneficially affect Aβ in both transgenic AD-model and non-transgenic mice as well as in humans at risk for AD. Compared to a standard chow diet, APP/V717I transgenic mice fed a 43-day KD had a 25% reduction in brain Aβ levels . SimilarThe reason that Aβ is dysregulated in the pathological process leading up to symptomatic AD is still not well understood. Several AD etiological hypotheses suggest that impaired bioenergetics could explain this phenomenon. For instance, the mitochondrial cascade hypothesis for Alzheimer’s disease suggests that AD pathology, including dysregulation of Aβ, is due to impaired mitochondrial bioenergetics . AD is aKTs may bolster overall brain metabolism by providing an alternative fuel substrate when glucose metabolism is impaired, potentially exerting beneficial effects on APP processing. In an attempt to explain decreased glucose uptake observed in AD, it is commonly hypothesized that neuronal death and decreased synaptic activity result in decreased glucose demand , even thThere are several mechanisms by which KTs may beneficially influence bioenergetics through mitochondrial metabolism of ketone bodies. The majority of AcAc and BHB are produced in the liver mitochondria, transport across the BBB, and enter mitochondria of the brain CNS where they undergo ketolysis for ATP production in the TCA cycle and electron transport chain . In the 2O2, a species of ROS (KTs may also regulate Aβ by mitigating the damaging effects of ROS. ROS are common byproducts of normal metabolic functions that play an important role in cellular physiology at homeostatic concentration but cause oxidative stress in excessive concentration . Excessis of ROS . Whethers of ROS . In precs of ROS , which hs of ROS . The pots of ROS . It is as of ROS , possibls of ROS . BHB alss of ROS . KTs mays of ROS  and may s of ROS . High les of ROS , therefo+/NADH ratio in the brain (KTs have also been shown to potentiate anti-inflammatory pathways which may protect against Aβ pathology by modulating neuroinflammation. Inflammation sensed by microglia and astrocytes in the CNS stimulates neuronal expression of inflammatory cytokines and Aβ . There ahe brain , which hhe brain . BHB mayhe brain  and to dhe brain . It is she brain  and, in he brain . Of notehe brain . The KD he brain .Elevated glucose, insulin resistance, and type 2 diabetes induce chronic inflammation and oxidative stress and are increasingly linked to elevated risk for AD . Human sin vitro models of mitochondrial distress and AD transgenic mouse models, though some positive preliminary data in humans at risk for AD do exist. Humans with metabolic risk factors or preclinical AD due to elevated Aβ also exhibit impaired brain bioenergetics and susceptibility for Aβ accrual; and thus, are prime candidates to study prevention strategies that may physiologically change AD pathology. Given the current evidence highlighted in this narrative, KTs warrant investigation into their value for regulating Aβ in at risk individuals. Successful rescue of brain bioenergetics and reduction of Aβ in these individuals could potentially prevent downstream manifestation of other AD hallmarks such as tau, neurodegeneration, and cognitive impairment.A culmination of work across various cellular and living models suggests that KTs may be valuable for improving brain bioenergetics and modulating Aβ. Much of this work has been conducted using MT conceived, developed the manuscript, and took responsibility for the final content of the manuscript. DS, JK, JB, and RS provided critical revision for important intellectual content. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.