Role of nitric oxide and endocannabinoids in synaptic plasticity in the perirhinal cortex and in visual recognition memory

Introduction and aims of the research Nitric oxide (NO) and endocannabinoids (eCBs) are major retrograde messengers, involved in synaptic plasticity (long-term potentiation, LTP, and long-term depression, LTD) in many brain areas (including hippocampus and neocortex), as well as in learning and memory processes. NO is synthesized by NO synthase (NOS) in response to increased cytosolic Ca2+ and mainly exerts its functions through soluble guanylate cyclase (sGC) and cGMP production. The main target of cGMP is the cGMP-dependent protein kinase (PKG). Activity-dependent release of eCBs in the CNS leads to the activation of the Gαi/o-coupled cannabinoid receptor 1 (CB1) at both glutamatergic and inhibitory synapses. The perirhinal cortex (Prh) is a multimodal associative cortex of the temporal lobe, critically involved in visual recognition memory. LTD is proposed to be the cellular correlate underlying this form of memory. Cholinergic neurotransmission has been shown to play a critical role in both visual recognition memory and LTD in Prh. Moreover, visual recognition memory is one of the main cognitive functions impaired in the early stages of Alzheimer’s disease. The main aim of my research was to investigate the role of NO and ECBs in synaptic plasticity in rat Prh and in visual recognition memory. Part of this research was dedicated to the study of synaptic transmission and plasticity in a murine model (Tg2576) of Alzheimer’s disease. Methods Field potential recordings. Extracellular field potential recordings were carried out in horizontal Prh slices from Sprague-Dawley or Dark Agouti juvenile (p21-35) rats. LTD was induced with a single train of 3000 pulses delivered at 5 Hz (10 min), or via bath application of carbachol (Cch; 50 μM) for 10 min. LTP was induced by theta-burst stimulation (TBS). In addition, input/output curves and 5Hz-LTD were carried out in Prh slices from 3 month-old Tg2576 mice and littermate controls. Behavioural experiments. The spontaneous novel object exploration task was performed in intra-Prh bilaterally cannulated adult Dark Agouti rats. Drugs or vehicle (saline) were directly infused into the Prh 15 min before training to verify the role of nNOS and CB1 in visual recognition memory acquisition. Object recognition memory was tested at 20 min and 24h after the end of the training phase. Results Electrophysiological experiments in Prh slices from juvenile rats showed that 5Hz-LTD is due to the activation of the NOS/sGC/PKG pathway, whereas Cch-LTD relies on NOS/sGC but not PKG activation. By contrast, NO does not appear to be involved in LTP in this preparation. Furthermore, I found that eCBs are involved in LTP induction, but not in basal synaptic transmission, 5Hz-LTD and Cch-LTD. Behavioural experiments demonstrated that the blockade of nNOS impairs rat visual recognition memory tested at 24 hours, but not at 20 min; however, the blockade of CB1 did not affect visual recognition memory acquisition tested at both time points specified. In three month-old Tg2576 mice, deficits in basal synaptic transmission and 5Hz-LTD were observed compared to littermate controls. Conclusions The results obtained in Prh slices from juvenile rats indicate that NO and CB1 play a role in the induction of LTD and LTP, respectively. These results are confirmed by the observation that nNOS, but not CB1, is involved in visual recognition memory acquisition. The preliminary results obtained in the murine model of Alzheimer’s disease indicate that deficits in synaptic transmission and plasticity occur very early in Prh; further investigations are required to characterize the molecular mechanisms underlying these deficits

Hyperspectral analysis applied to micro-Brillouin maps of amyloid-beta plaques in Alzheimer’s disease brains

A recent investigation on the architecture and chemical composition of amyloid-β (Aβ) plaques in ex vivo histological sections of an Aβ-overexpressing transgenic mouse hippocampus has shed light on the infrared light signature of cell-activation related biomarkers of Alzheimer’s disease. A correlation was highlighted between the biomechanical properties detected by Brillouin microscopy and the molecular make-up of Aβ plaques provided by FTIR spectroscopic imaging and Raman microscopy (with correlative immunofluorescence imaging) in this animal model of the disease. In the Brillouin spectra of heterogeneous materials such as biomedical samples, peaks are likely the result of multiple contributions, more or less overlaid on a spatial and spectral scale. The ability to disentangle these contributions is very important as it may give access to discrete components that would otherwise be buried within the Brillouin peak envelope. Here, we applied an unsupervised non-negative matrix factorization method to analyse the spontaneous Brillouin microscopy maps of Aβ plaques in transgenic mouse hippocampal sections. The method has already been proven successful in decomposing chemical images and is applied here for the first time to acoustic maps acquired with a Fabry–Perot Brillouin microscope. We extracted and visualised a decrease in tissue rigidity from the core through to the periphery of the plaque, with spatially distinct components that we assigned to specific entities. This work demonstrates that it is possible to reveal the structure and mechanical properties of Aβ plaques, with details visualized by the projection of the mechanical contrast into a few relevant channels

Caregivers of Patients with Hematological Malignancies within Home Care: A Phenomenological Study

The role of caregivers in homecare settings is relevant to the patient\u2019s wellbeing and quality of life. This phenomenon is well described in the literature for the oncological setting but not specifically for that of hematological malignancies. The aim of this study was to explore the experience of primary caregivers of patients with hematological malignancies within home care. We conducted a phenomenological study based on interviews with 17 primary caregivers of hematological patients. Analysis of the contents led to the identification of five main themes. Perhaps, the innovative aspects of this study can be summarized in three points: This service was demonstrated to fulfil the ethical aspects of providing the patient with a dignified accompaniment to the end of life. Secondly, the efficiency of the service and the benefit are directly dependent on the caregivers\u2019 wellbeing, so knowledge of the dynamics and emotions involved can lead to the development and implementation of programs for hematological malignancies. Lastly, a collaborative caregivers\u2013professionals relationship can improve a sense of accomplishment for all parties involved, lessening the family\u2019s frustration related to not having done their best. Home care brings significant benefits for both the patient and the caregivers and fulfils the ethical obligation of providing the patient dignified end-of-life care

Low concentrations of the solvent dimethyl sulphoxide alter intrinsic excitability properties of cortical and hippocampal pyramidal cells

Dimethylsulfoxide (DMSO) is a widely used solvent in biology. It has many applications perhaps the most common of which is in aiding the preparation of drug solutions from hydrophobic chemical entities. Recent studies have suggested that this molecule may be able to induce apoptosis in neural tissues urging caution regarding its introduction into humans, for example as part of stem cell transplants. Here we have used in vitro electrophysiological methods applied to murine brain slices to examine whether a few hours treatment with 0.05% DMSO (a concentration regarded by many as innocuous) alters intrinsic excitability properties of neurones. We investigated pyramidal neurones in two distinct brain regions, namely area CA1 of the hippocampus and layer 2 of perirhinal cortex. In the former there was no effect on resting potential but input resistance was decreased by DMSO pre-treatment. In line with this action potential count for any level of depolarizing current stimulus was reduced by ~25% following DMSO treatment. Ih-mediated “sag” was also increased in CA1 pyramids and action potential waveform analysis demonstrated that DMSO treatment moved action potential threshold towards resting potential. In perirhinal cortex a decreased action potential output for various depolarizing current stimuli was also seen. In these cells action potential threshold was unaltered by DMSO but a significant increase in action potential width was apparent. These data indicate that pre-treatment with this widely employed solvent can elicit multifaceted neurophysiological changes in mammalian neurones at concentrations below those frequently encountered in the published literature

Time-dependent reduction of calcium oscillations in adipose-derived stem cells differentiating towards adipogenic and osteogenic lineage

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can dif-ferentiate into various cell types, including osteocytes and adipocytes. Due to their ease of har-vesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation has been proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentia-tion-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing sponta-neous oscillations and the frequency of the oscillation (measured as interpeak interval – IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as con-trols. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both, adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their fre-quency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a re-duction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and dif-ferentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth

Hippocampal synaptic and membrane function in the DBA/2J-mdx mouse model of Duchenne muscular dystrophy

Dystrophin deficiency is associated with alterations in cell physiology. The functional consequences of dystrophin deficiency are particularly severe for muscle physiology, as observed in Duchenne muscle dystrophy (DMD). DMD is caused by the absence of a 427 kDa isoform of dystrophin. However, in addition to muscular dystrophy symptoms, DMD is frequently associated with memory and attention deficits and epilepsy. While this may be associated with a role for dystrophin in neuronal physiology, it is not clear what neuronal alterations are linked with DMD. Our work shows that CA1 pyramidal neurons from DBA/2J-mdx mice have increased afterhyperpolarization compared to WT controls. All the other electrotonic and electrogenic membrane properties were unaffected by this genotype. Finally, basal synaptic transmission, short-term and long-term synaptic plasticity at Schaffer collateral to CA1 glutamatergic synapses were unchanged between mdx and WT controls. These data show that the excitatory component of hippocampal activity is largely preserved in DBA/2J-mdx mice. Further studies, extending the investigation to the inhibitory GABAergic function, may provide a more complete picture of the functional, network alterations underlying impaired cognition in DMD. In addition, the investigation of changes in neuronal single conductance biophysical properties associated with this genotype, is required to identify the functional alterations associated with dystrophin deficiency and clarify its role in neuronal function

Imbalance in the response of pre- and post-synaptic components to amyloidopathy

Alzheimer’s disease (AD)-associated synaptic dysfunction drives the progression of pathology from its earliest stages. Aβ species, both soluble and in plaque deposits, have been causally related to the progressive, structural and functional impairments observed in AD. It is, however, still unclear how Aβ plaques develop over time and how they progressively affect local synapse density and turnover. Here we observed, in a mouse model of AD, that Aβ plaques grow faster in the earlier stages of the disease and if their initial area is > 500 µm2; this may be due to deposition occurring in the cloud part of the plaque. In addition, synaptic turnover is higher in the presence of amyloid pathology and this is paralleled by a reduction in pre- but not post-synaptic densities. Plaque proximity does not appear to have an impact on synaptic dynamics. These observations indicate an imbalance in the response of the pre- and post-synaptic terminals and that therapeutics, alongside targeting the underlying pathology, need to address changes in synapse dynamics

Estrogen and testosterone secretion from the mouse brain

Estrogen and testosterone are typically thought of as gonadal or adrenal derived steroids that cross the blood brain barrier to signal via both rapid nongenomic and slower genomic signalling pathways. Estrogen and testosterone signalling has been shown to drive interlinked behaviours such as social behaviours and cognition by binding to their cognate receptors in hypothalamic and forebrain nuclei. So far, acute brain slices have been used to study short-term actions of 17β-estradiol, typically using electrophysiological measures. For example, these techniques have been used to investigate, nongenomic signalling by estrogen such as the estrogen modulation of long-term potentiation (LTP) in the hippocampus. Using a modified method that preserves the slice architecture, we show, for the first time, that acute coronal slices from the prefrontal cortex and from the hypothalamus maintained in aCSF over longer periods i.e. 24 h can be steroidogenic, increasing their secretion of testosterone and estrogen. We also show that the hypothalamic nuclei produce more estrogen and testosterone than the prefrontal cortex. Therefore, this extended acute slice system can be used to study the regulation of steroid production and secretion by discrete nuclei in the brain

Viscoelasticity of amyloid plaques in transgenic mouse brain studied by Brillouin microspectroscopy and correlative Raman analysis

Amyloidopathy is one of the most prominent hallmarks of Alzheimer’s disease (AD), the leading cause of dementia worldwide, and is characterized by the accumulation of amyloid plaques in the brain parenchyma. The plaques consist of abnormal deposits mainly composed of an aggregation-prone protein fragment, β-amyloid 1-40/1-42, into the extracellular matrix. Brillouin microspectroscopy is an all-optical contactless technique that is based on the interaction between visible light and longitudinal acoustic waves or phonons, giving access to the viscoelasticity of a sample on a subcellular scale. Here, we describe the first application of micromechanical mapping based on Brillouin scattering spectroscopy to probe the stiffness of individual amyloid plaques in the hippocampal part of the brain of a β-amyloid overexpressing transgenic mouse. Correlative analysis based on Brillouin and Raman microspectroscopy showed that amyloid plaques have a complex structure with a rigid core of β-pleated sheet conformation (β-amyloid) protein surrounded by a softer ring-shaped region richer in lipids and other protein conformations. These preliminary results give a new insight into the plaque biophysics and biomechanics, and a valuable contrast mechanism for the study and diagnosis of amyloidopathy