Dendritic Spines: Synaptogenesis and Synaptic Pruning for the Developmental Organization of Brain Circuits.

Synaptic overproduction and elimination is a regular developmental event in the mammalian brain. In the cerebral cortex, synaptic overproduction is almost exclusively correlated with glutamatergic synapses located on dendritic spines. Therefore, analysis of changes in spine density on different parts of the dendritic tree in identified classes of principal neurons could provide insight into developmental reorganization of specific microcircuits.The activity-dependent stabilization and selective elimination of the initially overproduced synapses is a major mechanism for generating diversity of neural connections beyond their genetic determination. The largest number of overproduced synapses was found in the monkey and human cerebral cortex. The highest (exceeding adult values by two- to threefold) and most protracted overproduction (up to third decade of life) was described for associative layer IIIC pyramidal neurons in the human dorsolateral prefrontal cortex.Therefore, the highest proportion and extraordinarily extended phase of synaptic spine overproduction is a hallmark of neural circuitry in human higher-order associative areas. This indicates that microcircuits processing the most complex human cognitive functions have the highest level of developmental plasticity. This finding is the backbone for understanding the effect of environmental impact on the development of the most complex, human-specific cognitive and emotional capacities, and on the late onset of human-specific neuropsychiatric disorders, such as autism and schizophrenia.

Cortical interneurons in schizophrenia - cause or effect?

GABAergic cortical interneurons are important components of cortical microcircuits. Their alterations are associated with a number of neurological and psychiatric disorders, and are thought to be especially important in the pathogenesis of schizophrenia. Here, we reviewed neuroanatomical and histological studies that analyzed different populations of cortical interneurons in postmortem human tissue from patients with schizophrenia and adequately matched controls. The data strongly suggests that in schizophrenia only selective interneuron populations are affected, with alterations of somatostatin and parvalbumin neurons being the most convincing. The most prominent changes are found in the prefrontal cortex, which is consistent with the impairment of higher cognitive functions characteristic of schizophrenia. In contrast, calretinin neurons, the most numerous interneuron population in primates, seem to be largely unaffected. The selective alterations of cortical interneurons are in line with the neurodevelopmental model and the multiple-hit hypothesis of schizophrenia. Nevertheless, a large number of data on interneurons in schizophrenia is still inconclusive, with different studies yielding opposing findings. Furthermore, no studies found a clear link between interneuron alterations and clinical outcomes. Future research should focus on the causes of changes in the cortical microcircuitry in order to identify potential therapeutic targets.

In the eye of the beholder - how course delivery affects anatomy education.

BACKGROUND: The COVID-19 pandemic caused major shifts in students' learning strategies as well as teaching environments that profoundly affected the delivery of anatomy courses in medical schools. The Department of Anatomy at the University of Zagreb School of Medicine had a unique experience where the anatomy course in 2019/2020 was first taught in-person before transferring to an online course delivery, while the inverse happened in 2020/2021. The core curriculum, course material and examination criteria were the same in both academic years. The aim of the study was to determine whether course delivery affected students' perceptions of the course and whether it impacted students' engagement and success. METHODS: The students' perceptions of the course were assessed via an anonymous course survey (student evaluation of teaching, SET). The questions in the SET assessed the usefulness of teaching modalities rather than students' satisfaction. Most questions were in the form of statements to which students responded with their level of agreement on a five-point Likert scale. Differences between responses in 2019/2020 and 2020/2021 were analyzed using the Mann-Whitney test. Effect size was estimated using Cliff's delta and association between responses was assessed using Spearman's r coefficient. RESULTS: Students' perceptions were significantly affected by changes in course delivery. Students' success and engagement were higher in 2019/2020 when in-person teaching preceded online teaching. Furthermore, students' views on course organization and the usefulness of continuous assessment were more positive in 2019/2020. Finally, students' perceptions of the usefulness of online materials and activities were more positive in 2019/2020. All comparisons between the two academic years were statistically significant (P ≤ 0.0001 for all comparisons, Mann-Whitney test). CONCLUSIONS: Students' perceptions of the anatomy course were dependent on the teaching environment they were exposed to at the beginning of the course. A transfer from in-person to online course delivery was more successful than vice-versa. This has important implications for structuring hybrid courses in medical education in the future.

The anatomy lesson of the SARS-CoV-2 pandemic: irreplaceable tradition (cadaver work) and new didactics of digital technology.

AIM: To compare the efficacy of different components of online and contact anatomy classes as perceived by medical students. METHODS: An anonymous course evaluation survey was conducted at the end of the academic year 2019/2020. The organization of classes due to the SARS-CoV-2 pandemic provided our students with a unique opportunity to compare online and contact classes. Students' responses were analyzed according to the type of obtained data (ratio, ordinal, and categorical). RESULTS: The response rate was 95.58%. Approximately 90% of students found anatomical dissection and practical work in general to be the most important aspect of teaching, which could not be replaced by online learning. During online classes, students missed the most the interaction with other students, followed by the interaction with student teaching assistants and teaching staff. Very few students found contact lectures useful, with most students reporting that they could be replaced with recorded video lectures. In contrast, recorded video lectures were perceived as extremely helpful for studying. Regular weekly quizzes were essential during online classes as they gave students adequate feedback and guided their learning process. Students greatly benefitted from additional course materials and interactive lessons, which were made easily available via e-learning platform. CONCLUSIONS: Anatomical dissection and interaction during contact classes remain the most important aspects of teaching anatomy. However, online teaching increases learning efficiency by allowing alternative learning strategies and by substituting certain components of contact classes, thus freeing up more time for practical work.

Axon morphology of rapid Golgi-stained pyramidal neurons in the prefrontal cortex in schizophrenia.

AIM: To analyze axon morphology on rapid Golgi impregnated pyramidal neurons in the dorsolateral prefrontal cortex in schizophrenia. METHODS: Postmortem brain tissue from five subjects diagnosed with schizophrenia and five control subjects without neuropathological findings was processed with the rapid Golgi method. Layer III and layer V pyramidal neurons from Brodmann area 9 were chosen in each brain for reconstruction with Neurolucida software. The axons and cell bodies of 136 neurons from subjects with schizophrenia and of 165 neurons from control subjects were traced. The data obtained by quantitative analysis were compared between the schizophrenia and control group with the t test. RESULTS: Axon impregnation length was consistently greater in the schizophrenia group. The axon main trunk length was significantly greater in the schizophrenia than in the control group (93.7 ± 36.6 µm vs 49.8 ± 9.9 µm, P = 0.032). Furthermore, in the schizophrenia group more axons had visibly stained collaterals (14.7% vs 5.5%). CONCLUSION: Axon rapid Golgi impregnation stops at the beginning of the myelin sheath. The increased axonal staining in the schizophrenia group could, therefore, be explained by reduced axon myelination. Such a decrease in axon myelination is in line with both the disconnection hypothesis and the two-hit model of schizophrenia as a neurodevelopmental disease. Our results support that the cortical circuitry disorganization in schizophrenia might be caused by functional alterations of two major classes of principal neurons due to altered oligodendrocyte development.

THE ANATOMY OF OROFACIAL INNERVATION.

The whole human body receives rich sensory innervation with only one exception and that is the brain tissue. The orofacial region is hence no exception. The head region consequently receives a rich network of sensory nerves making it special because the two types of sensory fibres, visceral and somatic overlap, especially in the pharynx. Also, different pain syndromes that affect this region are rather specific in comparison to their presentation in other body regions. With this review article we wanted to show the detailed anatomy of the peripheral sensory pathways, because of its importance in everyday body functions (eating, drinking, speech) as well as the importance it has in pathological conditions (pain syndromes), in diagnostics and regional analgesia and anaesthesia.

Integration of complementary biomarkers in patients with first episode psychosis: research protocol of a prospective follow up study.

In this project, we recruited a sample of 150 patients with first episode of psychosis with schizophrenia features (FEP) and 100 healthy controls. We assessed the differences between these two groups, as well as the changes between the acute phase of illness and subsequent remission among patients over 18-month longitudinal follow-up. The assessments were divided into four work packages (WP): WP1- psychopathological status, neurocognitive functioning and emotional recognition; WP2- stress response measured by saliva cortisol during a stress paradigm; cerebral blood perfusion in the resting state (with single photon emission computed tomography (SPECT) and during activation paradigm (with Transcranial Ultrasonography Doppler (TCD); WP3-post mortem analysis in histologically prepared human cortical tissue of post mortem samples of subjects with schizophrenia in the region that synaptic alteration was suggested by WP1 and WP2; WP4- pharmacogenetic analysis (single gene polymorphisms and genome wide association study (GWAS). We expect that the analysis of these data will identify a set of markers that differentiate healthy controls from patients with FEP, and serve as an additional diagnostic tool in the first episode of psychosis, and prediction tool which can be then used to help tailoring individualized treatment options. In this paper, we describe the project protocol including aims and methods and provide a brief description of planned post mortem studies and pharmacogenetic analysis.

The Protracted Maturation of Associative Layer IIIC Pyramidal Neurons in the Human Prefrontal Cortex During Childhood: A Major Role in Cognitive Development and Selective Alteration in Autism.

The human specific cognitive shift starts around the age of 2 years with the onset of self-awareness, and continues with extraordinary increase in cognitive capacities during early childhood. Diffuse changes in functional connectivity in children aged 2-6 years indicate an increase in the capacity of cortical network. Interestingly, structural network complexity does not increase during this time and, thus, it is likely to be induced by selective maturation of a specific neuronal subclass. Here, we provide an overview of a subclass of cortico-cortical neurons, the associative layer IIIC pyramids of the human prefrontal cortex. Their local axonal collaterals are in control of the prefrontal cortico-cortical output, while their long projections modulate inter-areal processing. In this way, layer IIIC pyramids are the major integrative element of cortical processing, and changes in their connectivity patterns will affect global cortical functioning. Layer IIIC neurons have a unique pattern of dendritic maturation. In contrast to other classes of principal neurons, they undergo an additional phase of extensive dendritic growth during early childhood, and show characteristic molecular changes. Taken together, circuits associated with layer IIIC neurons have the most protracted period of developmental plasticity. This unique feature is advanced but also provides a window of opportunity for pathological events to disrupt normal formation of cognitive circuits involving layer IIIC neurons. In this manuscript, we discuss how disrupted dendritic and axonal maturation of layer IIIC neurons may lead into global cortical disconnectivity, affecting development of complex communication and social abilities. We also propose a model that developmentally dictated incorporation of layer IIIC neurons into maturing cortico-cortical circuits between 2 to 6 years will reveal a previous (perinatal) lesion affecting other classes of principal neurons. This "disclosure" of pre-existing functionally silent lesions of other neuronal classes induced by development of layer IIIC associative neurons, or their direct alteration, could be found in different forms of autism spectrum disorders. Understanding the gene-environment interaction in shaping cognitive microcircuitries may be fundamental for developing rehabilitation and prevention strategies in autism spectrum and other cognitive disorders.

A Quantitative Golgi Study of Dendritic Morphology in the Mice Striatal Medium Spiny Neurons.

In this study we have provided a detailed quantitative morphological analysis of medium spiny neurons (MSNs) in the mice dorsal striatum and determined the consistency of values among three groups of animals obtained in different set of experiments. Dendritic trees of 162 Golgi Cox (FD Rapid GolgiStain Kit) impregnated MSNs from 15 adult C57BL/6 mice were 3-dimensionally reconstructed using Neurolucida software, and parameters of dendritic morphology have been compared among experimental groups. The parameters of length and branching pattern did not show statistically significant difference and were highly consistent among groups. The average neuronal soma surface was between 160 µm2 and 180 µm2, and the cells had 5-6 primary dendrites with close to 40 segments per neuron. Sholl analysis confirmed regular pattern of dendritic branching. The total length of dendrites was around 2100 µm with the average length of individual branching (intermediate) segment around 22 µm and for the terminal segment around 100 µm. Even though each experimental group underwent the same strictly defined protocol in tissue preparation and Golgi staining, we found inconsistency in dendritic volume and soma surface. These changes could be methodologically influenced during the Golgi procedure, although without affecting the dendritic length and tree complexity. Since the neuronal activity affects the dendritic thickness, it could not be excluded that observed volume inconsistency was related with functional states of neurons prior to animal sacrifice. Comprehensive analyses of tree complexity and dendritic length provided here could serve as an additional tool for understanding morphological variability in the most numerous neuronal population of the striatum. As reference values they could provide basic ground for comparisons with the results obtained in studies that use various models of genetically modified mice in explaining different pathological conditions that involve MSNs.

Neocortical calretinin neurons in primates: increase in proportion and microcircuitry structure.

In this article we first point at the expansion of associative cortical areas in primates, as well as at the intrinsic changes in the structure of the cortical column. There is a huge increase in proportion of glutamatergic cortical projecting neurons located in the upper cortical layers (II/III). Inside this group, a novel class of associative neurons becomes recognized for its growing necessity in both inter-areal and intra-areal columnar integration. Equally important to the changes in glutamatergic population, we found that literature data suggest a 50% increase in the proportion of neocortical GABAergic neurons between primates and rodents. This seems to be a result of increase in proportion of calretinin interneurons in layers II/III, population which in associative areas represents 15% of all neurons forming those layers. Evaluating data about functional properties of their connectivity we hypothesize that such an increase in proportion of calretinin interneurons might lead to supra-linear growth in memory capacity of the associative neocortical network. An open question is whether there are some new calretinin interneuron subtypes, which might substantially change micro-circuitry structure of the primate cerebral cortex.

Spatio-temporal extension in site of origin for cortical calretinin neurons in primates.

The vast majority of cortical GABAergic neurons can be defined by parvalbumin, somatostatin or calretinin expression. In most mammalians, parvalbumin and somatostatin interneurons have constant proportions, each representing 5-7% of the total neuron number. In contrast, there is a threefold increase in the proportion of calretinin interneurons, which do not exceed 4% in rodents and reach 12% in higher order areas of primate cerebral cortex. In rodents, almost all parvalbumin and somatostatin interneurons originate from the medial part of the subpallial proliferative structure, the ganglionic eminence (GE), while almost all calretinin interneurons originate from its caudal part. The spatial pattern of cortical GABAergic neurons origin from the GE is preserved in the monkey and human brain. However, it could be expected that the evolution is changing developmental rules to enable considerable expansion of calretinin interneuron population. During the early fetal period in primates, cortical GABAergic neurons are almost entirely generated in the subpallium, as in rodents. Already at that time, the primate caudal ganglionic eminence (CGE) shows a relative increase in size and production of calretinin interneurons. During the second trimester of gestation, that is the main neurogenetic stage in primates without clear correlates found in rodents, the pallial production of cortical GABAergic neurons together with the extended persistence of the GE is observed. We propose that the CGE could be the main source of calretinin interneurons for the posterior and lateral cortical regions, but not for the frontal cortex. The associative granular frontal cortex represents around one third of the cortical surface and contains almost half of cortical calretinin interneurons. The majority of calretinin interneurons destined for the frontal cortex could be generated in the pallium, especially in the newly evolved outer subventricular zone that becomes the main pool of cortical progenitors.

Dynamics of optic canal and orbital cavity development revealed by microCT.

PURPOSE: Numerous studies have attempted to clarify the exact anatomy and variations of the optic canal with non-conclusive results due to its close proximity to many vulnerable structures. We sought to determine the dynamics of growth and development of these structures on fetal skulls, which will help us to better understand of gender and age-dependent variations, as well as fatal malformations. METHODS: Fifteen previously macerated fetal frontal and sphenoid bones were analyzed and the diameters of optic canal, and distance of orbit from frontomaxillary suture to frontozygomatic suture were measured using 3D reconstruction images obtained by micro-CT. RESULTS: Average diameter of the optic canal in 300 mm fetus was measured to be 1,546 ± 36 µm, in 400 mm fetus 2,470 ± 123 µm and in 500 mm fetus 3,757 ± 203 µm. This trend indicates a linear enlargement of optic canal during the fetal period. During the same time period, diameter of the orbit enlarges from 12,319 ± 559 µm in 300 mm fetus to 19,788 ± 736 µm in 500 mm fetus. Growth curve is significantly lower in comparison with the same curve in optic canal data. We also calculated the ratio of orbit diameter and optic canal diameter between those groups which decreased from a value of 7.9 ± 0.4 for 300 mm fetus to 5.3 ± 0.2 for 500 mm fetus. CONCLUSION: Dynamics of optic canal and orbital cavity development is different in early and late fetal period. Diameters of those structures are in better correlation with the fetal length.