Polysialic acid (PSA) is a linear homopolymer formed of chains of 2,8-linked sialic acid. Found predominantly attached to the neural cell adhesion molecule, PSA acts to reduce cell-cell adhesion during development. It is also found in some areas of the adult central nervous system (CNS) associated with persistent neuroplasticity. Preliminary data from our laboratory indicated an inverse relationship between PSA expression and the formation of perineuronal nets (PNNs), specialised extracellular matrix structures with a role in limiting plasticity in the adult CNS. The primary aims of this thesis were to investigate this relationship in more detail, using in vitro models of PNN formation and in vivo. Also, to evaluate whether lentiviral vector-mediated PSA expression can enhance locomotor recovery and neuroplasticity in a rodent model of spinal cord injury. PNNs were heterogeneously distributed throughout the grey matter of the rat cervical spinal cord, and increased in numbers down the dorsoventral axis. Induced expression of PSA in the spinal cord of either naïve or injured rats did not alter the number or density of PNNs. Similarly, enzymatic removal of PSA from the surface of cultured embryonic neurons did not affect the formation of the PNNs. In a rodent model of cervical spinal cord injury, induced PSA expression resulted in an improvement in hindlimb, but not forelimb, locomotor function compared to animals injected with control virus. Interestingly, this was not associated with an increased density of serotonin or synaptophysin-labelled boutons in the areas of induced PSA expression. Taken together, the data presented in this thesis suggests that while induced PSA expression may contribute to improved locomotor function in a model of cervical spinal cord injury, this is not due to a reduction in the density or number of PNNs in the spinal cord.

PURPOSE: To characterize the distribution of white matter neurons (WMNs) positive for perineuronal nets (PNNs) in the adult monkey. WMNs are a mixed population of excitatory and inhibitory neurons. They have an important role in axon guidance during cortical development, but their role in the adult brain is less understood. In vitro and in vivo experiments provide evidence that WMNs are incorporated into cortical circuitry. The majority of investigations in the adult, however, have focused on regional variations in overall density, or on characterization of morphological and neurochemical subtypes. The present study was motivated by the observation that some WMNs exhibit PNNs in adult monkey. Since PNNs are associated with plasticity in younger animals, their occurrence with some WMNs might be functionally significant. METHODS: PNNs were visualized, at the light microscopic level, by WFA staining in three adult macaque brains. Density of WFA positive WMNs was scored at three anterior-posterior levels (frontal, mid-hemispheric, and occipital), and compared with overall density of WMNs, as visualized by immunocytochemistry for NeuN. Quantitation of WFA+ neurons and neuron morphology were analyzed via light microscopy. Soma size and appearance, and dendritic length were recorded and measured. RESULTS: On the basis of soma size and proximal dendritic shape, several types of WFA+ WMNs were provisionally identified, consistent with previous reports in the literature. Subpopulation densities were of highest density in mid-cortical areas and lowest quantities at occipital, matching previous studies. Morphological measurements suggested a heterogeneous neuron population through soma measurements and dendrite orientation. Soma sizes exhibited a range of circularity and size (10 µm – 30 µm). Dendrites were stained beyond the “proximal” area, including intermediate areas beyond the first branch, and up to 500 µm. CONCLUSIONS: A small population of WMNs are coated by PNNs in adult monkey. On the basis of morphology, these might be further subdivided, but combined studies with other markers would be needed. Future studies might investigate age- or pathology-related changes in the density and subtypes of WMNs that express PNNs in human or nonhuman primates. We speculate that these WMNs might have functional specializations, perhaps similar to the plasticity effects documented for PNNs in early development.

BACKGROUND: Schizophrenia is a debilitating disorder that has a profound impact on quality of life due to the presence of both cognitive deficits and psychotic symptoms. Despite having significant global economic and social costs and a worldwide prevalence of 1%, schizophrenia is still not well understood. Research has been making strides in uncovering the pathophysiology and the etiology that drive this disease, ranging from genetic abnormalities, disrupted circuitry, changes in microarchitecture, to impaired synaptic connectivity. Evidence suggests that disrupted-in-schizophrenia-1 (DISC1) driven genetic disturbances in fast-spiking parvalbumin (PV) neurons and their surrounding perineuronal nets (PNNs) likely contribute to schizophrenia etiology as they are part of the microcircuits required for working memory, a cognitive function that has been consistently impaired in schizophrenic patients. OBJECTIVE: To identify the neuroanatomical changes in PV neurons and surrounding PNNs in the superficial and deep layers of the prelimbic and infralimbic prefrontal cortex of a rat DISC-1 knockout model. METHODS: 19 DISC1-KO male rats and 15 wildtype rats were treated with saline or MK-801. They were sacrificed between P268-269 and brains were extracted and separated at the corpus callosum. After fixing and preserving, the brains were sliced then stained to visualize parvalbumin and perineuronal nets with immunohistochemistry. Slices were imaged and analyzed for PV, PNN, and PV+PNN counts in the superficial and deep regions of the prelimbic and infralimbic cortices. Averages counts within each group were taken and analyzed via 2-way ANOVAs for each brain region and dependent variable. RESULTS: DISC1-KO rats displayed the following trending changes: decreased PV cells in deep layers of infralimbic and decreased PNNs throughout the prelimbic cortex. MK-801 appears to increase the number of unsheathed PV cells in the superficial layers of prelimbic and infralimbic cortex. It decreased the number of PNNs in the prelimbic of wildtype animals but not in the DISC1-KO cohort. MK-801 moderately increased PV counts in DISC1-KO. CONCLUSIONS: This DISC1-KO model is a promising model of schizophrenia as we see the same directionality of decreases in PV and PNN as post mortem human studies. Furthermore, MK-801 is seen to have an increasing trend effect on PV cells, which should be considered when interpreting findings in future studies that look at these markers.

Chondroitin sulfate proteoglycans (CSPGs) are found in specialized brain extracellular matrix structures termed perineuronal nets (PNNs). The chondroitin sulfate chains of these CSPGs are thought to have a strong effect on neuroplasticity, along with development, injury, and diseased states of the brain. Wisteria floribunda agglutinin (WFA) is a plant lectin used to identify PNN via staining; the pattern of this staining is changed upon schizophrenia. As such, one powerful method of probing the identity of the CS chains of PNNs and addressing what changes in CS identity occur during schizophrenia is to characterize the features of the CS which bind to the lectin. Methods for characterization of WFA-CS binding and their biological relevance were developed and evaluated. Commercially available CS was used to probe the binding affinity of the agglutinin to various regions of CS via hemagglutination inhibition assays and affinity gradient elution of CS bound to WFA. The size, sulfation extent, and fragment location in the CS chain from these eluates were determined using HILIC-LC-MS. As commercial sources can be used to elucidate the binding specificity of WFA, but not the actual relevant binding partner of WFA within the brain, PNN CS extractions were performed with a modified method aimed at reducing the timescale at which PNN CS can be obtained so as to allow similar experimentation on CS directly from PNN. The results pave the way for further determination of WFA-CS binding.

Modifications of perineuronal nets to regulate plasticity Heleen Merel van 't Spijker Perineuronal nets (PNNs) are macromolecular structures formed by neurons after closure of critical periods of plasticity. During development, the central nervous system (CNS) goes through critical periods of plasticity; a period when substantial changes occur to adapt to the environment, during which many synapses are formed and also discarded. When a region of the CNS has finished its development and reached an efficient neuronal circuit, the capacity for plasticity needs to be reduced to preserve the formed circuit. PNNs are formed around neurons during this period of reduced plasticity. PNNs consist of a backbone of hyaluronan, bound by chondroitin sulfate proteoglycans (CSPGs). Here, I present my studies on the possible modifications of PNNs to regulate plasticity. Firstly, I have investigated the potential use of 4-methylumbelliferone (4-MU) to reduce PNN formation in vivo. 4-MU reduces the formation of hyaluronan. Since hyaluronan is the backbone of PNNs, I hypothesized 4-MU treatment would reduce PNN formation. For this study, I developed a method to orally administer 4-MU to rats. Subsequently, I investigated whether 4-MU treatment can improve recovery of rats after spinal cord injury, both with behavioural tests and with immunohistochemistry. Secondly, I have investigated a new binding partner of PNNs, neuronal pentraxin 2 (Nptx2). Nptx2 is secreted by neurons and regulates AMPA receptor diffusion. Nptx2 knockout mice show a prolonged critical period of plasticity in the visual cortex. Here, I have identified Nptx2 as a new binding partner of PNNs. Nptx2 is found in isolated PNN protein preparations and is removed from the surface of neurons by digestion of PNNs with chondroitinase ABC. I also determined Nptx2 facilitates PNN formation in vitro. Addition of Nptx2 to the medium of cortical neurons leads to an increase of neurons that start to form PNNs, as well as an increase in size and density of PNNs. These findings indicate Nptx2 may be used as a modulator of PNNs. Thirdly, I investigated the interaction between Nptx2 and PNNs. I developed a sandwich ELISA to determine which glycan chains from PNNs bind to Nptx2. Nptx2 binds to chondroitin sulfate E and hyaluronan. To investigate the binding properties of Nptx2, I performed quartz crystal microbalance with dissipation monitoring for Nptx2 films. Furthermore, I developed crystals of purified Nptx2 and hyaluronan for x-ray crystallography. The here presented results provide new insights in potential approaches to modulate PNN formation. Both lines of research provide a further understanding of the factors which regulate PNNs and may allow for the development of treatments for PNN related disorders.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19644号 / 人健博第36号 / 新制||人健||3(附属図書館) / 32680 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 三谷 章, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

In der vorliegenden Dissertation wurden Neurone untersucht, die von einer speziellen Form der extrazellulären Matrix, dem perineuronalen Netz (PN), umgeben sind. Neurone mit einem PN zeichnen sich durch eine geringe Vulnerabilität bei neurodegenerativen Erkrankungen aus. Da das PN mit hoher Affinität Eisen bindet, war zu klären, ob das PN den Eisenhaushalt der Neurone beeinflusst und diese mit einer protektiven Eigenschaft gegenüber Eisen-induzierten oxidativen Stress ausstattet. Es wurde die Eisenkonzentration und der Gehalt an Eisentransport- und Eisenspeicherproteinen von Neuronen mit PN und Neuronen ohne PN in der Ratte untersucht. Dabei kamen quantitative Methoden wie die ortsaufgelöste Ionenstrahlmikroskopie und die Objektträger-basierte Laser Scanning Zytometrie sowie Western Blot Analysen und quantitative Real-Time-PCR zum Einsatz. Die Untersuchungen zeigen, dass Neurone, die mit einem PN umgeben sind, eine höhere Konzentration an Eisen sowie Eisentransport- und Eisenspeicherproteinen besitzen als Neurone ohne ein PN. Das PN könnte so den Eisenhaushalt der Neurone beeinflussen und diese mit einer protektiven Eigenschaft gegenüber Eisen-induziertem oxidativen Stress ausstatten.

Thesis advisor: John P. Christianson / Trauma and exposure to extreme stressors greatly increases a person’s vulnerability to developing mental illnesses like post-traumatic stress disorder (PTSD). Patients with PTSD often have impaired fear and safety learning, and despite the fact that women are more than twice as likely to develop PTSD, much of the research on this disorder has relied on the use of male subjects. This paper will review potential contributors to the sex differences seen in PTSD and fear-related learning. Our group has found that female rats show greater fear discrimination abilities than their male counterparts, but show no difference in levels of safety learning. Analysis of specialized extracellular matrix structures called perineuronal nets (PNNs) revealed that females displayed a much higher density of PNNs in the basolateral amygdala (BLA) than males, but not in the prefrontal cortex (PFC). / Thesis (BS) — Boston College, 2017. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Psychology.

Submitted by Irvana Coutinho (irvana@ufpa.br) on 2013-01-25T14:29:10Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ AcuidadeVisualMatriz.pdf: 2171256 bytes, checksum: ce1733a75cf4138644c2b48ef3377b5b (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-01-25T17:00:07Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ AcuidadeVisualMatriz.pdf: 2171256 bytes, checksum: ce1733a75cf4138644c2b48ef3377b5b (MD5) / Made available in DSpace on 2013-01-25T17:00:07Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ AcuidadeVisualMatriz.pdf: 2171256 bytes, checksum: ce1733a75cf4138644c2b48ef3377b5b (MD5) Previous issue date: 2012 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O objetivo do presente trabalho e analisar a influencia do enriquecimento ambiental sobre a acuidade visual e a distribuição das redes perineuronais (RPNs) no córtex visual primário de camundongos submetidos a privação monocular durante o período critico pos-natal. Camundongos suíços albinos fêmeas foram submetidos a sutura da pálpebra direita no 10o dia pos-natal (M, n=16), enquanto que os animais do grupo binocular não foram submetidos a nenhum procedimento cirúrgico (B, n=16). Ao completarem 21 dias, os animais foram subdivididos em: ambiente padrão e ambiente enriquecido, constituindo os grupos M.AP, M.AE, B.AP e B.AE. Após três meses, os animais foram submetidos ao teste de acuidade visual, perfundidos e secções coronais de seus cérebros processadas para histoquímica da lectina Wisteria floribunda e posterior quantificação através do método estereologico do fracionador óptico. Os animais do grupo B.AP apresentaram acuidade visual de 0.48 ciclos/grau, enquanto que aqueles alojados em ambiente enriquecido (B.AE) apresentaram um melhor desempenho do teste, atingindo 0.996 ciclos/grau. A acuidade visual foi significantemente menor nos animais submetidos a privação monocular (M.AP 0.18 ciclos/grau; M.AE 0.4 ciclos/grau). Os resultados estereologicos revelaram que o ambiente enriquecido aumenta o numero de RPNs tipo 1 e de RPNs total nas camadas supragranular e granular em ambos os hemisférios nos camundongos submetidos a privação monocular (ANOVA dois critérios, p<0.05), sendo que essa diferença na camada granular e decorrente principalmente do aumento das redes perineuronais da matriz extracelular no hemisfério direito. Na camada infragranular, os animais do grupo M.AE apresentaram um aumento apenas no numero de RPNs tipo 1. / The aim of the present study is to analyze the influence of enriched environment on the visual acuity and on the distribution of perineuronal nets (PNNs) in the primary visual cortex of albino mice that underwent monocular deprivation during the critical period of postnatal development. Mice at 10th postnatal day, were monocular deprived through right eye-lid sutured (M, n = 16) and the control group animals were not submitted to any cirurgical procedures (B, n = 16). After weaning, on postnatal day 21, animals were subdivided in: standard environment (AP) and enriched environment (AE), constituting the following groups: M.AP, M.AE, B.AP and B.AE. After 3 months, animals were submitted to grating visual acuity tests, perfused and coronal sections of their brains processed for Wisteria floribunda agglutinin to posterior stereological quantification through optical fractionator method. B.AP animals present visual acuity of 0.48 cycles/degree, while those raised in enriched environment (B.AE) present a better performance at visual test, reaching 0.996 cycles/degree. Animals with monocular deprivation had significantly lower visual acuity (M.AP 0.18 cycles/degree; M.AE 0.4 cycles/degree). Stereological quantifications revealed that enriched environment increases type 1 and the total number of perineuronal nets at supragranular and granular layers in both hemispheres of deprived animals (ANOVA, two-ways, p < 0.05) and this difference at granular layer is due to an increase of perineuronal nets mainly at the right hemisphere (ipsilateral to the monocular deprivation). At infragranular layer, M.AE animals presented an increase only at the number of type 1 PNNs in both hemispheres.

Stroke is a leading cause of neurological disability, often resulting in long-term motor impairments due to damage to the striatum and/or motor cortex. While both humans and animals show spontaneous recovery following stroke, little is known about how the injury location affects recovery and what causes recovery to plateau. This information is essential in order to improve current rehabilitation practice and develop new therapies to enhance recovery. In this thesis, we used endothelin-1 (ET-1), a potent vasoconstrictor, to produce focal infarcts in the forelimb motor cortex (FMC), the dorsolateral striatum (DLS) or both the FMC and DLS in male Sprague-Dawley rats. In the first experiment, the spontaneous recovery profile of animals was followed over an 8-week period using multiple behavioural tasks assessing motor function and limb preference to identify how recovery varies depending on injury location. Infarct volumes were measured to determine the association between injury and behavioural outcome. All three groups had significant functional impairments on the Montoya staircase, beam traversal, and cylinder tests following stroke, with the combined group having the largest and most persistent impairments. Importantly, spontaneous recovery was not simply dependent on lesion volume but on the lesion location and the behavioural test employed. In the second experiment, we focused on a potential cellular mechanism thought to underlie post-stroke plasticity and functional recovery. In a separate cohort of animals, we assessed how plasticity-limiting perineuronal nets (PNNs) and associated parvalbumin-positive (PV) GABAergic interneurons change following similar ET-1 strokes as in the prior experiment. A significant reduction in the density of PNNs was observed in the perilesional cortex of animals that received a cortical-only or combined stroke but not a striatal-only injury. Although there were no significant differences in the density of PV interneurons between sham and stroked groups, a significant negative correlation existed between cortical infarct volume and the density of PV interneurons in the perilesional cortex. Taken together these results demonstrate that lesion location influences motor recovery and neuroplastic changes following stroke. This supports the idea that a “one size fits all” approach for stroke rehabilitation may not be effective and treatment needs to be individualized to the patient.