Reduced neuronal density in the CA1 anterodorsal hippocampus of the mdx mouse

Accepted Manuscript Title: Reduced neuronal density in the CA1 anterodorsal hippocampus of the mdx mouse model of duchenne muscular dystrophy Author: Rubén Miranda, Serge Laroche, Cyrille Vaillend PII: DOI: Reference:

S0960-8966(16)30030-X http://dx.doi.org/doi: 10.1016/j.nmd.2016.08.006 NMD 3234

To appear in:

Neuromuscular Disorders

Received date: Revised date: Accepted date:

22-1-2016 22-6-2016 10-8-2016

Please cite this article as: Rubén Miranda, Serge Laroche, Cyrille Vaillend, Reduced neuronal density in the CA1 anterodorsal hippocampus of the mdx mouse model of duchenne muscular dystrophy, Neuromuscular Disorders (2016), http://dx.doi.org/doi: 10.1016/j.nmd.2016.08.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reduced

neuronal

density

in

the

CA1

anterodorsal

hippocampus of the mdx mouse model of Duchenne muscular dystrophy Rubén MirandaI a, Serge Larochea, Cyrille Vaillenda.

a

Neuroscience Paris-Saclay Institute (Neuro-PSI), UMR 9197, Université Paris Sud,

CNRS, Université Paris Saclay, Orsay, France.

Email addresses: Rubén Miranda ([email protected]); Serge Laroche ([email protected]); Cyrille Vaillend ([email protected])

*Correspondence: Rubén Miranda ([email protected])

I

Present address: Department of Psychobiology, Universidad Complutense de Madrid. Edificio 'La Almudena' (Room #0405), Rector Royo Villanova s/n, 28040 (Madrid), Spain. Phone: (+34) 91 394 6138

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HIGHLIGHTS  Brain dystrophin deficiency reduces CA1 hippocampal neuronal density  Reduction in neuronal density varies along hippocampal antero-posterior axis  Mechanisms may rely on neurodegeneration processes and/or impaired neurogenesis  Altered neuronal density may contribute to cognitive deficits in muscular dystrophy

ABSTRACT Duchenne muscular dystrophy (DMD) is associated with non-progressive cognitive dysfunction including hippocampal-dependent memory deficits. Loss of the cytoskeleton-associated dystrophin protein in central inhibitory synapses, associated with consequent alterations in GABAergic function and synaptic plasticity, has been proposed as a primary mechanism responsible for cognitive impairments. However, several lines of evidence suggest a multifactorial etiology involving alternative signaling pathways, some of which could affect neuronal survival. To determine whether changes in neuronal density in the hippocampus could contribute to the emergence of memory deficits, we undertook an unbiased stereological estimation of neuron number in the anterodorsal CA1 region of the hippocampus of the dystrophindeficient mdx mouse model of DMD. We found a significant reduction (~34%) in the number of pyramidal neurons, with an heterogeneous magnitude of genotype differences along the hippocampal antero-posterior axis. This extends previous knowledge of brain morphofunctional alterations induced by dystrophin loss and suggests that putative mechanisms involved in neurogenesis and/or neuron survival might contribute to the emergence of hippocampal-dependent learning and memory deficits in DMD.

KEYWORDS: Dystrophin; Hippocampus; Neuronal density; mdx mice; Duchenne muscular dystrophy

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Running head: Neuronal loss in dystrophin-deficient mice

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INTRODUCTION Duchenne Muscular Dystrophy (DMD) is an X-linked recessive syndrome characterized by progressive muscle degeneration and non-progressive cognitive deficits. It is caused by mutations in the dmd gene that disrupt the expression of the 427-KDa dystrophin protein (Dp427), which normally links a complex of intrinsic membrane proteins to actin in both muscle and brain cells, with cell-specific interactions with membrane receptors and/or ion channels [1]. Cognitive dysfunction in DMD ranges from mild deficits in verbal skills and memory performance to intellectual disability [2]. The absence of Dp427 in brain structures involved in cognitive functions, such as the hippocampus, neocortex, cerebellum and amygdala, has been linked to learning and memory deficits. Post-mortem brain alterations reported in DMD patients include ventricular enlargement, cortical atrophy and cortical dendritic abnormalities, suggesting altered brain development [3]. However, other neuroimaging and functional studies revealed glucose hypometabolism in hippocampus, cerebellum and sensorimotor cortex, altered inorganic phosphate to ATP ratio, nonspecific EEG disturbances, grey matter alterations and decreased local synchronization of spontaneous activity in motor cortex. This suggests that altered brain metabolism and synaptic function also contribute to cognitive dysfunction [4-6], in line with the normal expression of dystrophin in central synapses. Accordingly, the dystrophin-deficient mdx mouse model of DMD shows selective learning and memory deficits associated with impaired central GABA A receptor clustering and altered synapse ultrastructure and plasticity (reviewed in [7]). However, the lack of dystrophin in mdx mice may also alter neuron proliferation, survival and/or differentiation [8,9] and putative changes in the organization and/or function of dystrophin-associated proteins such as dystroglycan could also perturb neuronal migration [1]. This hypothesis is also supported by several neuroanatomical

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studies in mdx mice pointing to the presence of architectural reorganizations in neuronal and/or interneuronal networks in cortical and hippocampal areas [10-12]. A significant part of the learning and memory deficits in DMD patients [13] and mdx mice [14] are thought to depend on hippocampal dysfunction. In mdx mice, cummulative evidence from molecular to behavior and therapeutic approaches suggests that the CA1 hippocampus is particularly affected following dystrophin loss (see [7] for a review). Although the loss of Dp427 from pyramidal cells may alter neuronal function in all CA1-4 hippocampal subfields [11], the CA1 dorsal hippocampus is known to be especially vulnerable to hypoxic insults, which may selectively damage neurons in this subfield while sparing neurons in CA3 and dentate gyrus [15]. In mdx mice CA1 hippocampal neurons are more sensitive to hypoxia compared to wild-type controls [16] and activation of NMDA receptor is enhanced [17], which might induce cascading effects on neuronal integrity and survival in CA1. To date, however, hippocampaldependent cognitive deficits in mdx mice have been mostly attributed to alterations in synaptic density, ultrastructure and plasticity in CA1 [14,18,19]. In the present study, we therefore undertook a stereological evaluation of neuron density in the anterodorsal CA1 region of the hippocampus in adult mdx and littermate control mice, to determine whether changes in neuronal number in this specific region could also contribute to the emergence of hippocampal-dependent cognitive deficits in this model.

MATERIALS AND METHODS Experiments were conducted in 8-month old male mice of the C57BL/10ScSn-dmdmdx/J (mdx; n=4) mutant line and littermate controls (WT; n=4) bred and genotyped in our laboratory [14]. Experiments were conducted blind to the genotype and in accordance with the European Communities Council Directive of 24 November 1986 (CEE 86/609)

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and following the guidelines of our animal facility approved by direction of veterinary services (Direction des Services Vétérinaires, DSV-France, agreement # B91-471-104). Mice were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and intracardially perfused with a 4% paraformaldehyde solution in 0.1M phosphate buffer, pH 7.3-7.4. Brains were cryoprotected in 30% sucrose and frozen in 2-methylbutane (Roth, Karlsruhe, Germany) at -30ºC. Coronal 30μm-thick serial sections from the anterodorsal hippocampus (Bregma -0.94 to -2.30) [20] were stored at -80ºC. One every sixth sections was stained with cresyl violet, mounted with Eukitt (EMS Co. Ltd, Washington, USA) and coverslipped. The first section was randomly chosen within the first six sections. For hippocampal volume estimation, sections were photographed at x4 with a Sony DFW-X700 digital camera (Sony Co., Tokyo, Japan) coupled to Olympus BX60 light microscope (Olympus Optical Co., Hamburg, Germany) (Fig. 1A). Crosssectional hippocampal areas were outlined (http://rsb.info.nih.gov/ij/) and volume was determined as the sum of the traced areas multiplied by the distance between sampled sections (180 μm). Neuron counting was performed with an Olympus BX51 microscope equipped with an interactive computer system comprising a high-precision motorized microscope stage and a 0.5-m resolution microcator (HeidenhainVZR401). Planapochromatic 10x dry and 100x oil-immersion lens were used to draw regions of interest and to count neurons, respectively. The CAST stereological software (Olympus, Denmark) controlled stage movements and interactive counting grids (rectangular unbiased disector frames). Neuron number was estimated using the optical fractionator method [21]. Briefly, the total number of neurons (N) in the anterodorsal hippocampus of a given mouse was calculated by multiplying the reciprocals of three sampling fractions by the total particle count (Q-) obtained with the optical disectors (Eq. 1). The mean section thickness was measured with the microcator during the counting

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procedure in at least five of the selected sections at various random places, in order to estimate the fraction of the section thickness (tsf). The measured final section thickness was 9 m, and we therefore used an optical disector height of 5 m, thus leaving an upper and guard zone of 2 m. The area sampling fraction (asf) was the ratio a/A of the counting frame area (a) by the product of the distances between the optical disector positions in the x and y directions within the sectional plane of each section (A): a(frame) /A(x,y step). The dimension of the a(frame) was 352.14 m2 and the x and y step sizes from A(x,y step) were 80 x 80 m. An average of 203 cell profiles was counted in 137 disectors per mouse in the CA1 pyramidal layer of the anterodorsal hippocampus, using the nucleus’ equatorial plane as the counting unit (Fig. 1B-C).

RESULTS AND DISCUSSION The number of CA1 pyramidal neurons was assessed within a specific range of coordinates comprising the anterodorsal hippocampus [18]. Neuron density was significantly reduced in mdx compared to WT mice (p<0.005; Fig. 2A). However, the volume of this given portion of the anterodorsal hippocampus was comparable between genotypes (p>0.05; Fig. 2B), suggesting that the change in neuronal density in CA1 had minimal impact on the volume of the anterodorsal hippocampus. As shown in Fig. 2C, neuronal density was not constant along the antero-posterior axis of the dorsal hippocampus, but progressed following an inverted U-shaped curve in the anteroposterior direction (F(8,48)=11.053; p<0.001), reaching maximal values in sections 5-6 (distance from Bregma -1.62/-1.79) in both genotypes. Neuron density was globally reduced in mdx mice (Genotype effect, F(1,6)=17.237; p<0.001). However, the magnitude of genotype differences was heterogeneous along the antero-posterior axis and more accentuated in sections 2, 6 and 7 (distance from Bregma: -1.11, -1.79 and -

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1.96, respectively) of the anterodorsal hippocampus (all tests, p<0.05), while differences did not reach significance at other coordinates.

Here we demonstrate that brain dystrophin deficiency in mdx mice is associated with a reduced number of CA1 pyramidal cells in the anterodorsal hippocampus, which may contribute to the physiopathological defects underlying cognitive dysfunction in DMD. DMD patients may display reduced brain weight, ventricle dilation, cortical atrophy, heterotopias, gliosis, dendritic abnormalities and reduced neuronal density [3]. However, this was observed in a limited number of patients, suggesting variable outcomes depending on age, genotype and motor/cognitive profiles. Moreover, analyses of DMD brains did not include unbiased stereological counting and were performed in a restricted number of brain regions excluding for instance the hippocampal formation.

In the mdx mouse, 1H-magnetic resonance spectroscopy (1H-MRS) has been previously used in vivo and in vitro to detect N-acetyl aspartate (NAA), which is considered as a biochemical marker of central neurons as decreases in NAA signal in various neuropathologies are thought to represent irreversible neuronal loss and/or compromised neuronal metabolism [22]. As NAA levels were unaltered in mdx mice, including in the hippocampus, a lack of neuronal necrosis in the dystrophic brain was concluded [23]. However, NAA levels due to neuron loss are technically difficult to detect in the hippocampus, due to the relative small size and specific location of this brain structure [22], and a lack of significant change in NAA levels in mdx mice could occur if neuronal necrosis was moderate or restricted to specific subfields. Here we found that the density of CA1 pyramidal neurons is reduced by ~34% in the

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anterodorsal hippocampus but the quantitative reductions indeed vary along the hippocampal antero-posterior axis.

The reduced neuron density that we report here may seem at variance with previous estimations of hippocampal neuron density that did not reveal any significant change in this model [18,24]. In our former study [18], however, the physical disector method provided a relative neuronal density [25] in a smaller portion of the anterodorsal hippocampus. This portion corresponds to sections 3 to 5 in the present study (-1.28 to 1.62 from Bregma), in which we also found no significant genotype differences. In Tuckett et al. [24], neuron density was estimated at a single random, and undisclosed level, of the hippocampus and given that genotype differences are not uniformly expressed along the longitudinal axis of the hippocampus direct comparisons with our previous or present results are not possible. Likewise, straightforward comparisons with other estimations of neuron density in the wild-type mouse hippocampus are limited due to the variability of quantification methods and/or anatomical boundaries among studies. Thus, in a reference study by Jinno and Kosaka [26] the neuron density was estimated using the optical disector without fractionator scheme and the data reported as a number of neurons per unit of volume representing a “relative” or local estimation, which contrasts with our optical fractionator global estimation of the total number of neurons in a given region [21]. Moreover, estimation of neuron number in this study was

based

on

the

quantification

of

immunohystochemichally

characterized

glutamatergic neurons and this was performed within specific dorsal, middle and ventral portions of the hippocampus that differ from the anatomical boundaries of the anterodorsal hippocampus defined in our study.

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The present results suggest that there are two patches along the longitudinal axis of the anterodorsal hippocampus that undergo reduction in neuronal density in mdx mice, while genotype differences were not significant within the medial part of the sampled region (-1.28 to -1.62 from Bregma). Although this seems in line with a previous study [18], the origin of this variance along the longitudinal axis of the anterodorsal hippocampus remains unclear. Besides the widely admitted functional differentiation between the dorsal and ventral parts of the hippocampus [27,28], recent evidence demonstrate that CA1 pyramidal cells also present different functional properties along the hippocampal longitudinal axis, as characterized by both gradual and discrete transitions in connectivity patterns and by changes in NMDA receptor expression, size of place fields, vulnerability to ischemia and gene expression patterns [29]. The existence of multiple subtypes of CA1 pyramidal cells forming distinct molecular domains and subdomains along the longitudinal axis might reflect presence of distinct local microcircuits connected to specific subcortical regions and involved in separate cognitive and emotional functions [30]. However, it remains as an open question for future studies to uncertain the putative functional significance of the present changes in neuronal density occurring at specific levels of the anterodorsal hippocampus longitudinal axis in mdx mice, through a detailed characterization of the molecular nature and connectivity of these specific patches of CA1 pyramidal neurons.

Although the cognitive and behavioral deficits in mdx mice may primarily result from the loss of dystrophin in central inhibitory synapses, our present results suggest that additional changes in neuron density might also contribute to brain dysfunction. Alterations in neuronal density and/or spatial clustering have also been reported in cortical areas of mdx mice and proposed as a mechanism underlying local networks

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dysfunction in this model [10,11,31]. It has been suggested that a normal number of neurons is initially generated in mdx mice, while a reduced survival capacity could induce progressive neuronal loss [32]. Accordingly, increases in inorganic phosphate to phosphocreatine ratio, increased intracellular brain pH and altered glucose metabolism have been reported in mdx mice, which could affect cell survival [33]. Interestingly, the loss of neuronal nitric oxide synthase in dystrophic muscles and the consequent reduction in systemic circulation of nitric oxide have been associated with increased cell proliferation and suppressed neuronal differentiation in the dentate gyrus of adult mdx mice [8], suggesting that muscle degeneration could indirectly affect hippocampal neuron integrity. Selective changes in neuron density, spatial distribution and activity have also been reported in cortical areas involved in the control of motor function in both DMD patients [6] and mdx mice [34], but it remains unclear whether they reflect a brain deterioration secondary to muscle wasting or a primary cause for specific motor impairments with a central origin. Nevertheless, the non-progressive nature of many of the cognitive deficits observed in patients, in contrast with the progressive nature of the muscular degeneration, suggests an important independence of the distinct physiopathological mechanisms. In mdx mice, reduced cell density and altered cell morphology were detected in cortico-spinal and brainstem neurons, while the number of pyramidal neurons in sensorimotor areas appears to be conversely increased and associated with dendritic rearrangements [12]. Widespread alterations in the spatial distribution of cortical interneurons were also reported in anterior cingulate cortex and dorsal hippocampus, which may perturb firing synchrony, neuron inhibitory modulation, excitatory/inhibitory balance and synapse plasticity [10,11,18,19], with a potential impact on neuronal survival of specific populations of hippocampal cells [35].

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Alternatively, changes in neuronal cell distribution and/or density may also reflect compensatory mechanisms to counterbalance the altered GABAergic function due to dystrophin loss in inhibitory synapses. Indeed, in the dorsal hippocampus the loss of dystrophin has been associated with an increase in parvalbumin interneuron density across the CA1-CA3 and dentate gyrus subfields [11] and of inhibitory synapses in CA1 proximal radiatum [18]. It is still unknown whether such alterations appeared early during development or have emerged progressively during adulthood. Early developmental compensatory plasticity may potentially occur in all brain structures that normally express brain dystrophin and part of the observed brain defects may be secondary to inefficient functional network connectivity between structures involved in common behavioral functions. A main feature of the behavioral phenotype in mdx mice is the presence of enhanced defensive responses and impaired fear memory [36,37]. Enhanced fearfulness could be detrimental for the capacity to associate a context to a fearful event, which normally involves cooperative processes between amygdala and hippocampus [38]. Likewise, morphofunctional alterations in sensorimotor areas could contribute to abnormal amygdala-dependent fear responses [39]. Understanding how the disrupted development, plasticity or function of a brain area impacts the function of other connected brain structures, or whether disrupted inputs could lead to neuronal degeneration in these structures, might be a major challenge to unravel the neurobiology of DMD (e.g., [40]).

Although GABAergic dysfunction is currently viewed as a primary mechanism responsible for the cognitive deficits due to dystrophin loss, several lines of evidence suggest a multifactorial etiology involving several alternative signaling pathways, some of which could affect neuronal survival. Pioneer studies suggested alterations of

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intracellular calcium homeostasis, which has been associated with enhanced susceptibility to hypoxia-induced loss of synaptic transmission in hippocampal area CA1 in mdx mice [16,41]. Oxidative stress might also contribute to neuronal damage due to increases in antioxidant activity of superoxide dismutase enzyme and altered lipid and protein peroxidation [42]. Dystrophin deficiency has also been associated with an upregulation of specific proteins involved in neuron survival, such as the nerve growth factor (NGF) and its receptors [43]. Although primarily interpreted as a factor contributing to the angiogenic response associated with brain-blood barrier alterations, abnormal NGF levels could also alter neuronal growth, differentiation and survival during prenatal development and impair postnatal neurogenesis [43,44]. Furthermore, decreased levels in brain-derived neurotrophic factor (BDNF), a regulator of neuronal survival, fast synaptic transmission, and activity-dependent synaptic plasticity, have been found in the striatum of mdx mice and associated with reduced striatal volumes. However, as no alterations were detected in the hippocampus of mdx mice, a direct involvement in hippocampal neuron loss is unlikely [45]. Interestingly, the hippocampal expression of the neurotrophic factor prosaposin (PS) is reduced in mdx mice, suggesting that the reduced hippocampal neuronal density could be linked to an impaired neuroprotective action of PS [46]. Finally, as recently observed in the superior cervical ganglion of mdx mice, dystrophin loss may modulate the expression of genes involved in neuron survival and differentiation [9]. It remains important to determine if the same can occur in brain structures that underlie cognitive functions, which warrants the study of alternative neuropathological pathways underlying brain dysfunction in DMD.

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Conclusions This study extends our previous knowledge of the brain morphofunctional alterations induced by dystrophin loss by demonstrating for the first time the existence of an important decrease in CA1 pyramidal neuron density in the anterodorsal hippocampus of dystrophin-deficient mdx mice, which support the hypothesis that some neurodegenerative processes and/or impaired neurogenetic development may contribute to the development of cognitive deficits in DMD.

CONFLICT OF INTEREST STATEMENT 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.

CONTRIBUTORS RM and CV designed the study, acquired data, wrote and reviewed the manuscript; SL participated to the interpretation of data and reviewed the manuscript. All authors approved the final version of the manuscript.

FUNDING This work was supported by the AFM (Association Française contre les Myopathies), France, [Grant # DdT1 2006 to C.V.]; the Centre National de la Recherche Scientifique (France); and the University Paris-Sud (France).

ACKNOWLEDGMENTS The authors are grateful to Emmanuel Brouillet (Molecular Imaging Research Center, Neurodegenerative Diseases Laboratory, Fontenay-aux-Roses, France) for his help and

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advice in stereological counting. The authors also thank P. Leblanc-Veyrac and N. Samson for mouse breeding and care, and M. Guegan for her collaboration in the histology.

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FIGURE LEGENDS

Equation 1. Optical fractionator estimator for total neuron density. The section sampling fraction (ssf) represents the proportion of microscopical sections taken from the entire set of serially sectioned and sampled brain region. Analyses were done using 1 every 6 sections and therefore the ssf corresponded to 1/6. The area sampling fraction (asf) corresponds to the proportion of sectional areas investigated within the sampled sections. The thickness sampling fraction (tsf) captures the part of the investigated cross-sectional area of the sampled sections.

N 





Q 

1 ssf



1 asf



1 tsf

Figure 1. Methods. (A) Series coronal sections at x1.25 magnification stained with cresyl violet and used for volume and neuron density quantification. Sections are numbered in antero-posterior direction and mouse brain atlas diagrams show the limits of the anterodorsal hippocampus (-0.94 and -2.30 from Bregma). (B) Low magnification (x10) image of the anterodorsal hippocampus showing delimitation of the CA1 pyramidal-layer area used for neuron quantification. (C) Example of CA1 pyramidal neurons (x100) (triangles) lying within a counting frame (disector) formed by solid and dashed lines representing exclusion and inclusion lines, respectively. Neurons profiles were counted if they crossed inclusion lines or laid within the counting frame.

Figure 2. Results. (A) Number of pyramidal neurons in the anterodorsal CA1 hippocampal area represented for each mouse. Horizontal bars show the mean neuron density for each genotype (t-test, p<0.005). (B) Individual anterodorsal hippocampal

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volumes. Mean group volume is represented by horizontal bars (t-test, p>0.05). (C) Neuron density expressed as mean ± SEM at successive Bregma coordinates along the antero-posterior axis of the CA1 hippocampus. Genotype differences were determined using an ANOVA (Distance from Bregma used as a repeated measure). *p<0.05, Tukey post-hoc test. WT: white circles; mdx: black circles.

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