BACKGROUND:Missense mutations in the HSPB8 gene, encoding the small heat shock protein B8, cause distal hereditary motor neuropathy (dHMN) or an axonal form of Charcot-Marie-Tooth disease (CMT subtype 2L). Mice expressing mutant Hspb8 (Lys141Asn) mimic the human disease, whereas mice lacking Hspb8 show no overt phenotype. We aimed to design an RNA interference treatment strategy that rescues the mutant HSPB8 neuronal and muscle phenotype in patient-derived motor neurons and in a knock-in mouse model of CMT2L/dHMN. METHODS:We optimized RNA interference sequences targeting both human HSPB8 and mouse HspB8 transcripts with the aim to alleviate disease symptoms. We used human induced pluripotent stem cells and the Hspb8 knock-in mouse model. We designed lenti- and adeno-associated viral vectors that contained the short-hairpin RNA constructs. We performed expression and microscopy studies, magnetic resonance imaging, behaviour analysis and electrophysiology. RESULTS:In CMT2L patient-derived induced pluripotent stem cells differentiated towards motor neurons, reducing the HSPB8 expression with a short-hairpin RNA (shRNA), directed towards the 3' untranslated region (3'UTR), ameliorated the morphology and fragmentation of mitochondria. The AAV9-mediated treatment of the 3'UTR shRNA construct, under neuron-specific regulation, in Hspb8 knock-in mice showed inconclusive results towards functional improvement upon expression studies, magnetic resonance imaging and neuropathological findings. CONCLUSIONS:Given the limited beneficial effect of the treatment, the RNA interference-mediated reduction of HSPB8/Hspb8 expression might not be the best therapeutic strategy to treat dHMN/CMT2L, unless a higher viral load and earlier treatment can be applied to the mouse model.

The present study aims to investigate whether begging calls elicit specific auditory responses in non-parenting birds, whether these responses are influenced by the hormonal status of the bird, and whether they reflect biparental care for offspring in the European starling (Sturnus vulgaris). An fMRI experiment was conducted to expose non-parenting male and female European starlings to recordings of conspecific nestling begging calls during both artificially induced breeding and non-breeding seasons. This response was compared with their reaction to conspecific individual warbling song motifs and artificial pure tones, serving as social species-specific and artificial control stimuli, respectively. Our findings reveal that begging calls evoke a response in non-parenting male and female starlings, with significantly higher responsiveness observed in the right Field L and the Caudomedial Nidopallium (NCM), regardless of season or sex. Moreover, a significant seasonal variation in auditory brain responses was elicited in both sexes exclusively by begging calls, not by the applied control stimuli, within a ventral midsagittal region of NCM. This heightened response to begging calls, even in non-parenting birds, in the right primary auditory system (Field L), and the photoperiod induced hormonal neuromodulation of auditory responses to offspring’s begging calls in the secondary auditory system (NCM), bears resemblance to mammalian responses to hunger calls. This suggests a convergent evolution aimed at facilitating swift adult responses to such calls crucial for offspring survival.

Avian innate nestling begging calls are similar to human infant cries in the behavioral response they elicit. However, it remains unknown whether the auditory processing of innate begging calls changes in seasonal songbirds from non-breeding to breeding season when hormonal neuromodulation of the auditory forebrain occurs. An fMRI experiment was set up to expose male and female European starlings ( Sturnus vulgaris ) to recordings of seasonal conspecific nestling begging calls in the breeding and non-breeding season. This response was compared with their response to conspecific warble motifs and artificial pure tones, both proven seasonally invariable at least in the male starling’s neural response. Our results demonstrate significant seasonal variation in auditory forebrain responses exclusively elicited by begging calls and not by the applied control stimuli. Right Field L and the Caudomedial Nidopallium (NCM) seemed, irrespective of season or sex, more sensitive in response to begging than to control stimuli. A seasonal differential response specifically to begging calls was found in both sexes in a ventral midsagittal region of NCM. Our findings thereby support the functional fine-tuning of vocal communications between sender and receiver in a breeding context for innate vocalizations and are in line with the bi-parenting behavior in this species.

Seasonality in songbirds includes not only reproduction but also seasonal changes in singing behavior and its neural substrate, the song control system (SCS). Prior research mainly focused on the role of sex steroids on this seasonal SCS neuroplasticity in males. In this review, we summarize the advances made in the field of seasonal neuroplasticity by applying in vivo magnetic resonance imaging (MRI) in male and female starlings, analyzing the entire brain, monitoring birds longitudinally and determining the neuronal correlates of seasonal variations in plasma hormone levels and song behavior. The first MRI studies in songbirds used manganese enhanced MRI to visualize the SCS in a living bird and validated previously described brain volume changes related to different seasons and testosterone. MRI studies with testosterone implantation established how the consequential boost in singing was correlated to structural changes in the SCS, indicating activity-induced neuroplasticity as song proficiency increased. Next, diffusion tensor MRI explored seasonal neuroplasticity in the entire brain, focusing on networks beyond the SCS, revealing that other sensory systems and even the cerebellum, which is important for the integration of sensory perception and song behavior, experience neuroplasticity starting in the photosensitive period. Functional MRI showed that olfactory, and auditory processing was modulated by the seasons. The convergence of seasonal variations in so many sensory and sensorimotor systems resembles multisensory neuroplasticity during the critical period early in life. This sheds new light on seasonal songbirds as a model for unlocking the brain by recreating seasonally the permissive circumstances for heightened neuroplasticity.

Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing toward an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic-pituitary-gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas, an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity.

Traditionally, research unraveling seasonal neuroplasticity in songbirds has focused on the male song control system and testosterone. We longitudinally monitored the song behavior and neuroplasticity in male and female starlings during multiple photoperiods using Diffusion Tensor and Fixel-Based techniques. These exploratory data-driven whole-brain methods resulted in a population-based tractogram confirming microstructural sexual dimorphisms in the song control system. Furthermore, male brains showed hemispheric asymmetries in the pallium, whereas females had higher interhemispheric connectivity, which could not be attributed to brain size differences. Only females with large brains sing but differ from males in their song behavior by showing involvement of the hippocampus. Both sexes experienced multisensory neuroplasticity in the song control, auditory and visual system, and cerebellum, mainly during the photosensitive period. This period with low gonadal hormone levels might represent a 'sensitive window' during which different sensory and motor systems in the cerebrum and cerebellum can be seasonally re-shaped in both sexes.

Traditionally, research unraveling seasonal neuroplasticity in songbirds has focused on the male song control system and testosterone. We longitudinally monitored the song and neuroplasticity in male and female starlings during multiple photoperiods using Diffusion Tensor and Fixel-Based techniques. These exploratory data-driven whole-brain methods resulted in a population-based tractogram uncovering microstructural sexual dimorphisms in the song control system and beyond. Male brains showed microstructural hemispheric asymmetries, whereas females had higher interhemispheric connectivity, which could not be attributed to brain size differences. Only females with large brains sing but differ from males in their song behavior by showing involvement of the hippocampus. Both sexes experienced multisensory neuroplasticity in the song control, auditory and visual system, and the cerebellum, mainly during the photosensitive period. This period with low gonadal hormones might represent a ‘sensitive window’ during which different sensory and motor systems in telencephalon and cerebellum can be seasonally re-shaped in both sexes.

Human speech and bird song are acoustically complex communication signals that are learned by imitation during a sensitive period early in life. Although the brain areas indispensable for speech and song learning are known, the neural circuits important for enhanced or reduced vocal performance remain unclear. By combining in vivo structural Magnetic Resonance Imaging with song analyses in juvenile male zebra finches during song learning and beyond, we reveal that song imitation accuracy correlates with the structural architecture of four distinct brain areas, none of which pertain to the song control system. Furthermore, the structural properties of a secondary auditory area in the left hemisphere, are capable to predict future song copying accuracy, already at the earliest stages of learning, before initiating vocal practicing. These findings appoint novel brain regions important for song learning outcome and inform that ultimate performance in part depends on factors experienced before vocal practicing.

Background: Activation and dysregulation of innate, adaptive and resident immune cells in response to damage determine the pathophysiology of demyelinating disorders. Among the plethora of involved cells, microglia/macrophages and astrocytes play an important role in the pathogenesis of demyelinating disorders. The in-depth investigation of the spatio-temporal profile of these cell types in vivo may inform about the exact disease state and localization as well as may allow to monitor therapeutic modulation of the components of the neuroinflammatory response during the course of multiple sclerosis (MS). In this study, we aimed to non-invasively decipher the degree and temporal profile of neuroinflammation (TSPO - [18F]DPA-714 PET) in relation to selected magnetic resonance imaging (MRI) parameters (T2 maps) in the cuprizone (CPZ)-induced model of demyelination. Methods: C57Bl6 (n=30) mice were fed with a standard chow mixed with 0.2% (w/w) CPZ for 4 (n=10; demyelination) and 6 weeks (n=10; spontaneous remyelination). The degree of neuroinflammation at de- and remyelination was assessed by [18F]DPA-714 PET, multi-echo T2 MRI, autoradiography and immunohistochemistry. Results: CPZ-induced brain alterations were confirmed by increase of T2 relaxation times in both white and grey matter after 3 and 5 weeks of CPZ. Peak [18F]DPA-714 was found in the corpus callosum (CC, white matter), the hippocampus (HC, grey matter) and thalamus (grey matter) after 4 weeks of CPZ treatment and declined after 6 weeks of CPZ. Ex vivo autoradiography and dedicated immunofluorescence showed demyelination/remyelination with corresponding increased/decreased TSPO levels in the CC and hippocampus, confirming the spatial distribution of [18F]DPA-714 in vivo. The expression of TSPO microglia and astrocytes is time-dependent in this model. Microglia predominantly express TSPO at demyelination, while the majority of astrocytes express TSPO during remyelination. The combination of PET- and MRI-based imaging biomarkers demonstrated the regional and temporal development of the CPZ model-associated neuroinflammatory response in grey and white matter regions. Conclusions: The combination of [18F]DPA-714 PET and T2 mapping may allow to further elucidate the regional and temporal profile of inflammatory signals depending on the myelination status, although the underlying inflammatory microenvironment changes. A combination of the described imaging biomarkers may facilitate the development of patient-tailored strategies for immunomodulatory and neuro-restorative therapies in MS.

Adult neuroplasticity in the song control system of seasonal songbirds is largely driven by photoperiod-induced increases in testosterone. Prior studies of the relationships between testosterone, song performance and neuroplasticity used invasive techniques, which prevent analyzing the dynamic changes over time and often focus on pre-defined regions-of-interest instead of examining the entire brain. Here, we combined (i) in vivo diffusion tensor imaging (DTI) to assess structural neuroplasticity with (ii) repeated monitoring of song and (iii) measures of plasma testosterone concentrations in thirteen female photosensitive starlings (Sturnus vulgaris) who received a testosterone implant for 3 weeks. We observed fast (days) and slower (weeks) effects of testosterone on song behavior and structural neuroplasticity and determined how these effects correlate on a within-subject level, which suggested separate contributions of the song motor and anterior forebrain pathways in the development of song performance. Specifically, the increase in testosterone correlated with a rapid increase of song rate and RA volume, and with changes in Area X microstructure. After implant removal, these variables rapidly reverted to baseline levels. In contrast, the more gradual improvement of song quality was positively correlated with the fractional anisotropy values (DTI metric sensitive to white matter changes) of the HVC-RA tract and of the lamina mesopallialis, which contains fibers connecting the song control nuclei. Thus, we confirmed many of the previously reported testosterone-induced effects, like the increase in song control nuclei volume, but identified for the first time a more global picture of the spatio-temporal changes in brain plasticity.

While no definitive cure for Alzheimer's disease exists yet, currently available treatments would benefit greatly from an earlier diagnosis. It has previously been shown that Magnetization transfer contrast (MTC) imaging is able to detect amyloid β plaques in old APP/PS1 mice. In the current study we investigated if MTC is also able to visualize early amyloid β (Aβ) induced pathological changes. In a cross-sectional study, a comparison was made between the MT ratio of wild type (WT) and APP/PS1 mice at 2, 4, 6, 8 and 24 months of age. We observed an increased MT-ratio in the cortex of 24 month old APP/PS1 mice as compared to WT mice. However, when comparing the MT-ratio of the cortex of WT mice with the MT-ratio of the APP/PS1 mice at 2, 4, 6 or 8 months of age, no significant changes could be observed. In contrast to the cortex, we consistently observed a decreased MT-ratio in the splenium of 4, 6 and 8 month old APP/PS1 mice as compared to age-matched WT mice. Lastly, the decreased MT-ratio in the splenium of APP/PS1 mice correlated to the Aβ plaque deposition, astrogliosis and microgliosis. This MT-ratio decrease did however not correlate to the myelin content. Combined, our results suggest that MTC is able to visualize early Aβ-induced changes in the splenium but not the cortex of APP/PS1 mice.

Conventional MRI is frequently used during the diagnosis of multiple sclerosis but provides only little additional pathological information. Proton MRS (1H‐MRS), however, provides biochemical information on the lesion pathology by visualization of a spectrum of metabolites. In this study we aimed to better understand the changes in metabolite concentrations following demyelination of the white matter. Therefore, we used the cuprizone model, a well‐established mouse model to mimic type III human multiple sclerosis demyelinating lesions. First, we identified CX3CL1/CX3CR1 signaling as a major regulator of microglial activity in the cuprizone mouse model. Compared with control groups (heterozygous CX3CR1+/− C57BL/6 mice and wild type CX3CR1+/+ C57BL/6 mice), microgliosis, astrogliosis, oligodendrocyte cell death and demyelination were shown to be highly reduced or absent in CX3CR1−/− C57BL/6 mice. Second, we show that 1H‐MRS metabolite spectra are different when comparing cuprizone‐treated CX3CR1−/− mice showing mild demyelination with cuprizone‐treated CX3CR1+/+ mice showing severe demyelination and demyelination‐associated inflammation. Following cuprizone treatment, CX3CR1+/+ mice show a decrease in the Glu, tCho and tNAA concentrations as well as an increased Tau concentration. In contrast, following cuprizone treatment CX3CR1−/− mice only showed a decrease in tCho and tNAA concentrations. Therefore, 1H‐MRS might possibly allow us to discriminate demyelination from demyelination‐associated inflammation via changes in Tau and Glu concentration. In addition, the observed decrease in tCho concentration in cuprizone‐induced demyelinating lesions should be further explored as a possible diagnostic tool for the early identification of human MS type III lesions. Copyright © 2015 John Wiley & Sons, Ltd.

Non-invasive measures of well-known pathological hallmarks of multiple sclerosis (MS) such as demyelination, inflammation and axonal injury would serve as useful markers to monitor disease progression and evaluate potential therapies. To this end, in vivo localized proton magnetic resonance spectroscopy (1H-MRS) provides a powerful means to monitor metabolic changes in the brain and may be sensitive to these pathological hallmarks. In our study, we used the cuprizone mouse model to study pathological features of MS, such as inflammation, de- and remyelination, in a highly reproducible manner. C57BL/6J mice were challenged with a 0.2% cuprizone diet for 6-weeks to induce demyelination, thereafter the mice were put on a cuprizone free diet for another 6weeks to induce spontaneous remyelination. We employed in vivo 1H-MRS to longitudinally monitor metabolic changes in the corpus callosum of cuprizone-fed mice during the demyelination (weeks 4 and 6) and spontaneous remyelination (week 12) phases. The MRS spectra were quantified with LCModel and since the total creatine (tCr) levels did not change over time or between groups, metabolite concentrations were expressed as ratios relative to tCr. After 4 and 6weeks of cuprizone treatment a significant increase in taurine/tCr and a significant reduction in total N-acetylaspartate/tCr, total choline-containing compounds/tCr and glutamate/tCr could be observed compared to mice under normal diet. At week 12, when almost full remyelination was established, no statistically significant metabolic differences were present between the control and cuprizone group. Our results suggest that these metabolic changes may represent sensitive markers for cuprizone induced demyelination, axonal injury and inflammation. To the best of our knowledge, this is the first longitudinal in vivo 1H-MRS study that monitored biochemical changes in the corpus callosum of cuprizone fed mice.