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It is widely accepted that neurogenesis in humans ends around the middle of gestation. However, the subventricular zone of some focal telencephalic domains continues with neuron production throughout the last trimester. We evaluated Nissl, Golgi and immunohistochemically stained human slides from the Zagreb Neuroembryological Collection to determine if late neurogenesis might also occur in the major proliferative zones of the telencephalon. Between 24-26 postconceptional weeks (pcw) a massive stream of densely packed cells was found leaving the ganglionic eminence (GE), a progenitor domain of the ventral telencephalon that is a major source of cortical GABAergic neurons. Between 32-36 pcw, the GE decreased in size but was clearly distinguishable, and a stream of non-radially oriented MAP-2 positive migratory-like cells still extended towards the dorsal telencephalon. Additionally, volumetric analysis on MRI images was performed to evaluate the developmental pattern of the GE in period from 13 to 34 pcw and found a persistent GE throughout the entire analyzed period. This suggests that the GE maintains its proliferative capabilities up to the end of gestation and that a significant production of cortical GABAergic neurons continues beyond the proliferative period of principal glutamatergic neurons. We propose that protracted neurogenesis from the GE contributes to the disproportional increase in the number of calretinin neurons, the most numerous GABAergic neuron population in primates. We further suggest that late fetal production of neurons is not restricted to neurons within the dentate gyrus and rostral migratory stream.

Evolutionary increase in complexity of cortical GABAergic network leads to significant variability in places of origin and routes of migration for GABAergic neurons. Here we present data from postmortem human and monkey fetal brain tissue processed for classical histological and immunohistochemical methods, with aim to identify their places of origin and their migratory routes. Our data confirmed that the ganglionic and septal eminence are the most important sources of GABAergic neurons. From these sources, they tangentially migrate through the subventricular and intermediate zone of the dorsal telencephalon (pallium). During the middle fetal period, significant production of GABAergic neurons also occurs in the pallial proliferative zones. The vast majority of the progenitors as well as migratory neurons from aforementioned sources strongly expressed GAD65 but not GAD67. Through early and middle fetal period numerous tangentially migrating cells within ganglionic eminence and lateral migratory stream were calretinin expressing cells. Intensive GAD67 expression was observed in a population of migratory cells that accumulates in the basal telencephalon and migrate to the pallium through the marginal zone and the layer under the cortical plate. These cells originate from, until now undescribed specific parts of the proliferative zones in the rostro-dorsal and caudal part of the medial telencephalic wall, proliferative zones between medial and lateral ganglionic eminence, hypothalamus and reticular thalamic nucleus. During middle fetal period they also originated from the proliferative zone at the top of the temporal lobe from where they enter into the marginal zone. Somatostatin positive migratory cells were densely packed in part of the zones where strong GAD67 reactive cells are found. Around mid-gestation, GAD65 positive small migratory like cells increased in number through marginal zone, forming 5-10 cells thick densely packed sublayer. By the end of middle trimester, lateral migratory stream was still massive and ganglionic eminence maintained its size. These data strongly support the view that important fraction of cortical GABAergic neurons is produced in the last trimester of gestation. Extended production, new sources and migratory routes of cortical GABA-ergic neurons participate to increased diversity of this cell population in the human cerebral cortex, but also makes them more prone to pathological alterations during development.

GABAergic neurons (GABAn) represent 20% of cortical neurons and are a highly diverse group that can be classified using molecular markers: the parvalbumin-expressing neurons account for about 40% of total cortical GABAn population in rodents, around 30% are somatostatin-expressing and 25% are calretinin-expressing neurons. Most of calbindin-expressing GABAn are also somatostatin positive, but significant proportion of somatostatin neurons does not co- express calbindin. However, the level of overlap between the aforementioned interneuron subpopulations is still controversial since some studies have shown additional co- expression between above mentioned markers. In this study we performed systematic assessment of their number and laminar position in frontal, parietal and occipital cortical region to assess proportion of different subclasses and the level of overlap between calretinin neurons and the remaining three subpopulations. A comprehensive qualitative analysis of double- labeled immunofluorescent histological sections showed that there were no major rostro-caudal differences in the number and laminar distribution of calbindin, parvalbumin and somatostatin neurons, while calretinin neurons were more abundant in occipital region. No overlap between calretinin and other major interneuron populations was observed. Parvalbumin, somatostatin and calretinin neurons were evenly distributed within a cortical column, while calbindin neurons were more numerous in upper cortical layers. In conclusion, the laminar distribution of GABAergic interneurons does not differ substantially between rostral and caudal cortical regions, however, calretinin neurons are generally more abundant in caudal than in rostral regions. Data also pointed on the significant difference in proportion and distribution of GABAn subtypes between rodents and primate cerebral cortex.

We analyzed changes in dendritic morphology and spine density on associative layer IIIc cortical projecting neurons and large layer V subcortical projecting pyramidal neurons to establish age- related changes within microcircuitries of the human prefrontal cortex (Brodmann area 9). Postmortem human brain tissue of adults was processed using the rapid Golgi method in two age groups: 38 – 64 years (n = 8) and 72 – 91 years, (n = 7). Neuropathological findings were unremarkable in all analyzed brain specimens. From each layer, the basal dendritic arbor and side dendritic branches from 10 – 15 well-impregnated pyramidal neurons per subject were three- dimensionally reconstructed using Neurolucida software. Soma size, total dendritic length, total segment number, individual segment length and spine density were quantitatively analyzed. Regarding layer V neurons, no significant differences were observed between adults and the elderly, either for dendritic morphology or for the spine density. The interindividual differences in the elderly group were however higher than in adults. Regarding associative layer IIIc pyramidal neurons, the mean values of spine density, on both side branches and basal dendrites, were 20–25% lower in the elderly than in adults (p = 0.07). In two aged cases the spine density was around mean level of adult and in the remaining aged subjects values were lower than in all adult subjects. These data show that the dendritic morphology and synaptic connectivity of the major classes of principal neurons in higher order associative areas are largely preserved in aging, while the connectivity of associative cortico-cortical layers is more prone to regression.

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 influence of cannabis use on the occurrence, clinical course and the treatment of the first psychotic episode (FEP) is well documented. However, the exact link is still not clearly established. The aim of this article is to review and report the noticed increase in the number of hospitalizations of young people with a clinical appearance of severe psychotic decompensation following cannabis consumption and to show the clinical challenges in treatment of the FEP. The case study describes the clinical course of a five selected patients with a diagnosis of the FEP and positive tetrahydrocannabinol (THC) urine test who were hospitalized in a similar pattern of events. They all have a history of cannabis consumption for at least 6 years in continuity and were presented with severe psychomotor agitation, disorganisation, confusion and aggression at admission. Although the chosen drug to treat all patients was atypical antipsychotic and benzodiazepines, the course of the disorder and the clinical response to therapy were noticeably different in each patient. The clinical presentation of FEP in cannabis users can be atypical and highly unpredictable from mild psychotic symptoms to severe substance intoxication delirium. In clinical practice clinicians treating new onset psychosis need to be watchful for cannabis and synthetic cannabinoids induced psychosis. Pharmacotherapeutic interventions include prompt and adequate use of the benzodiazepine, second-generation antipsychotic, and mood-stabilizers. Further research in the pharmacotherapy of cannabis-induced psychosis is required.

Th e most essential element in the management of patient’s pain problem is an understanding of normal function of the system. Diff erent individuals receiving identical noxious stimulation feel pain in diff erent ways and react at diff erent levels of suff ering. Orofacial disorders are usually accompanied with pain felt in the mouth, jaws and the face and thus can aff ect the quality of life of the suff erer dramatically. Some of the most prevalent and debilitating pain conditions arise from the structures in nervated by the trigeminal system (head, face, masticatory musculature, temporomandib ular joint and associated structures), making the trigeminal nerve the most important cra nial nerve for pain. Although generally considered part of the somatosensory system, the trigeminal sensory system has unique anatomy of the pathways for orofacial sensations, involving the trigeminal ganglion and its associated nuclei within the brainstem. Th e glos sopharyngeal and vagus nerves contribute also to somatic pain processing within orofacial region through trigeminal nuclei system, but their general aff erent innervation is mainly related with processing visceral sensations through solitary tract nucleus. Th ere is a minor contribution from the facial nerve to general somatic innervation, and its contribution to visceral pain processing needs clarifi cation. Although the sensory innervation to the ear and pharynx may appear fairly well defi ned, there is considerable overlap and ambiguity in the somatic and visceral innervation in the oropharynx, nasopharynx, Eustachian tube and middle ear. Th us, both pain processing systems, one passing through trigeminal nuclei and the other through solitary nucleus could transmit pain sensations from these regions. Th e orofacial region has certain peculiarities in comparison to fundamental pattern of innervation and information processing throughout the body. Consideration of the anatomy of craniofacial innervation can provide useful insights in the understanding of the unique pathophysiology of orofacial pain so that appropriate individual therapy can be designed

Abnormalities in oligodendrocytes lead to altered myelination in schizophrenia, according to most recent studies. The degree of myelination affects axon impregnation in Golgi staining. Therefore, the aim of this study is to compare the axon impregnation on rapid Golgi between schizophrenic and control subjects. We analyzed sections of the prefrontal cortex containing Brodmann area 9 in five schizophrenic and five control subjects. The sections were stained using the rapid Golgi method and the axons of randomly selected pyramidal neurons of layer III and V were reconstructed using Neurolucida 4 software. The axon impregnation lengths were then compared between the schizophrenic and control groups. Our results showed an increase in the length of axonal staining of the pyramidal neurons in the prefrontal cortices of schizophrenic subjects. The length of the stained axon main trunk was 132.5 ± 63.5 µm in the schizophrenic group and 64.8 ± 20.2 µm in the control group. The difference was shown to be statistically significant (p-value on Student’s t-test was

The anterior and posterior ethmoidal foramina that open into canals entering the cranial cavity are situated on the medial orbital wall. Since there may be a variable number of accessory ethmoidal foramina and their anatomy appears to be dependent on studied population, we are presenting a study of frequency and morphological characteristics of posterior and additional ethmoidal canals in adult Croatian population. In this study 439 skulls from the Zagreb skull collection were examined in order to confirm the existence of the posterior ethmoidal canal that opens in the anterior cranial fossa and the additional ethmoidal canal that opens in ethmoidal cells. Length and width of both canals were analyzed on computerized tomography scans using Analyze 8.1. software. The posterior ethmoidal canal was found in 86% of skulls. In 10% of skulls the posterior and the additional ethmoidal canals were found. In 4% of skulls we found only the additional ethmoidal canal. On skulls that had the additional ethmoidal canal the posterior ethmoidal canal was shorter and narrower. Variations in communications between orbital cavity and anterior cranial fossa, as well as ethmoidal and sphenoid sinuses could be related to increased need for vascular and nerve supply. Moreover, knowing anatomical variations of the posterior ethmoidal canal is crucial for development of safe surgical and therapeutic guidelines both in orbital and cranial regions. Based on observed communications, we suggest the revision of commonly used nomenclature for the anterior and posterior, as well as additional ethmoidal canals.

Although the basic principles of cortical development are conserved across mammals, evolutionary increased complexity of cortical GABAergic network leads to significant variability in origin and migration of GABAergic neurons when primates are compared to rodents. GABAergic cortical network in primates is particularly distinguished by tremendous increase in the proportion of calretinin neurons which contribute to half of GABAergic neuron population and are two times more numerous than other two major subpopulations, parvalbumin and calbindin/somatostatin. Therefore, it could be expected that the evolution is changing developmental rules to enable substantial changes that appear inside primate GABAergic network. To establish place of origin and migratory routes of different GABAergic neuron subpopulations in primates, we examined postmortem human and monkey fetal brain tissue during early fetal period (8-12 postconceptional weeks in human and embryonic day 47-56 in cynomologus monkey) processed immunohistochemicaly for markers of GABAergic neurons (GAD65 and GAD67) as well as for molecular markers of three major subpopulations. Data confirmed that, as in all mammals described so far, the most important source of GABAergic neurons during the early fetal period is the ganglionic eminence. The vast majority of progenitors in the ganglionic eminence express GAD65 (but not GAD67). After leaving the ganglionic eminence tangentially migrating cells continue to express GAD65, emerge laterally on the cortico-striatal border and form main tangential migratory stream in the subventricular/intermediate zone of the lateral telencephalic wall. Intensive GAD67 expression is observed by progenitors in the proliferative zones in rostro- dorsal and ventro-caudal part of the medial telencephalic wall. These so far undescribed proliferative regions are a source of GABAergic neurons which accumulate in the basal telencephalon and continue to migrate through the marginal zone and the layer below the cortical plate (primordial subplate). Part of the GAD67+ migrating cells in this superficial stream originates also from the preoptic area and hypothalamic proliferative zone which are in continuation with GAD67+ proliferative zones of medial telencephalon. Data also showed that already during the early fetal period the majority of GABAergic cells that originate from the ganglionic eminence and migrate via deeper migratory stream express calretinin, while GAD67+ proliferative zones of the medial telencephalic wall are the main source of somatostatin cells that migrate via the superficial stream. Our results suggest that primate specific features of GABAergic neuronal development are present already during the early fetal period. Two main subpopulations of GABAergic cortical neurons, somatostatin and calretinin, have clearly distinguishable origin and migratory routes, and significant production of calretinin subpopulation occurs earlier than in rodents. In addition to their dorsal production later on, this significant early production also accounts for tremendous increase in the proportion of calretinin neurons in primates.

In this study postmortem human and monkey fetal brain tissue processed by classical neurohistological and immunohistochemical methods was analyzed. Comparing changes in molecular characteristics and distribution of tangentially migrating cells in the human and monkey telencephalon during the first half of gestation, places of their origin and tangential migration pathways were proposed. Data confirmed that the ganglionic eminence is the most important source of GABAergic neurons that migrate tangentially to the pallium, and that significant production of GABA-ergic neurons occurs in the dorsal proliferative zones during the middle fetal period. These cells predominantly express GAD65, but there is also a migratory population that predominantly expresses GAD67 and enters the pallium through the basal telencephalon. During the early fetal period, these cells originate from preoptic area and hypothalamic proliferative zones, and from so far undescribed proliferative zones of rostro-dorsal and ventro-caudal part of the medial telencephalic wall. During the middle fetal period, the proliferative zone around the temporal horn of lateral ventricle enlarges, and gives GABAergic neurons for limbic structures and GABA-ergic neurons that migrate through the marginal zone. Data also suggested that during early fetal period, two major subpopulations of cortical GABA-ergic neurons have different place of origin and different migratory routes. GAD67 positive cells migrate through the marginal zone and the layer under the cortical plate and express somatostatin, while GAD65 positive cells migrate inside lateral migratory stream through the subventricular zone and express calretinin.This suggests that in primate significant production of calretinin neurons occurs earlier than in rodents. Populations of non-GABA-ergic cells also migrate tangentially. Calretinin positive cells from anterior part of diencephalon and ventro-medial part of ganglionic eminence migrate tangentially to the pallium and form primordial cortical plate at the end of embryonic and beginning of early fetal period. During early fetal period, calbindin positive Cajal-Retzius like cells originate from the choroid plexus and coupling proliferative zone and migrate tangentially through marginal zone, especially intensive in the fornix and retrosplenium during the middle fetal period. In addition, calbindin expressing pyramidal cells that form the precursor of hippocampal plate also migrate tangentially.

The principal neurons in associative areas of the human frontal cortex show intensive dendritic outgrowth and elongation during the perinatal period. In this study, we quantitatively analyzed the dendritic tree development and spine formation on the rapid Golgi impregnated layer III pyramidal neurons in prospective motor, Broca´s, premotor and prefrontal areas ranging from the 32nd postconceptional week until the 3rd postnatal month in human. At the 32nd postconceptional week the pyramidal neurons already show typical morphological features: the triangular cell body shape, as well as the prominent apical dendrite reaching the marginal zone with the bifurcating terminal tuft. The total number of basal and oblique dendrites is already reached at that stage. After basic morphology is established the two sharply segregated stages will follow: the first stage is characterized mainly by the outgrowth of new segments and the second by dendritic elongation. On average, neurons in primary regions have the same level of maturation as neurons in high order associative areas. Within the areas analyzed, large differences in the level of maturation among neurons were observed with some of the neurons already at 60% of adult values. Our data do not support the traditional view of hierarchical development from primary to high order areas, but rather suggest cross-areal asynchronous maturation of the neurons inside the cortico-cortical network in the early period of infancy. Such maturation pattern may represent a neurobiological basis in the sequential development of the initial cognitive functions that are already present in newborns and young infants.

Pain is a complex sensory state based on the integration of a variety of nociceptive inputs processed centrally through many parallel and overlapping neural systems. The traditional anatomical concept implies that nociceptive information is dominantly used to generate and regulate perception of pain through one major sensory pathway. It becomes recognized that experiencing the affective component of the pain is at least as important as perception. Also, nociceptive information is strongly influencing brain centers for regulating homeostasis. So, understanding neuroanatomical organization of central processing of nociceptive information is of great clinical importance. There is an attempt to simplify this complex set of interacting networks to a core set of brain regions or a generalizable pain signature. Herewith we wish to give a short overview of recent advances by presenting principles about neuroanatomical organization for processing various aspects of nociceptive inputs.

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.

Presented data in the monkey and human suggest more protracted production of cortical GABAergic neurons when compared to principal, glutamatergic neurons. Data also support the view that production occurs in both, cortical and subcortical (GE) proliferative zones. Such protracted proliferation could significantlycontribute for an increased proportion of primate cortical GABAergic neurons.

Previous studies have demonstrated that in primates, cortical GABAergic neurons (cxGABAn) are first generated exclusively by the ganglionic eminence (GE), resembling spatial pattern in origin of cxGABAn in rodents. Then, during the second trimester of gestation cxGABAn are massively produced in the newly evolved outer subventricular zone of the pallium. This massive production of cxGABAn by pallium progenitors reported in primates could be an evolutionary answer to provide cxGABAn to a complex and expanded cortex. How the GE contribute to cxGABAn production during this second trimester of gestation remains to be established. For this purpose we investigate the development of the primate GE during the stage when pallial production of cxGABAn occurs. Our data indicate that there is no decrease in the proliferative capability of the GE up to midgestation both in human and monkey when cxGABAn are generated in the pallium. In the rhesus monkey no decrease was observed in a stream of GAD65-containing tangentially migrating cells arising from the GE up to the embryonic day (E)75. In addition, a pool of GAD/Ki65 labeled progenitors was still observed in the GE. Golgi impregnated sections from human fetuses aged 22-26 post- conceptual week (pcw), illustrated clearly streams of densely packed cells leaving the GE toward the dorsal telencephalon. At 32-36 pcw streams of migratory-like cells extended in ventro-dorsal direction from the GE at the position of cortico-striatal border. At this developmental stage, the GE displayed decreased size but was still clearly distinguishable. Many of these nonradially oriented migratory cells leaving the GE were labeled for MAP-2 and calretinin. These data suggests that the GE conserves its proliferative properties to generate cxGABAn during pallial production of cxGABAn. Furthermore, the GE reaches a maximum peak of cxGABAn production at the beginning of the second half of gestation, when the vast majority of principal cortical glutamatergic neurons have already been generated. In keeping with this observation, volumetric analyses of GE from in vivo MRI images of 15 human fetuses aged 13-32 pcw demonstrates a maximum size of the GE around 18-20 pcw and persistence of GE through the entire third trimester of gestation. These data suggest a protracted period for cxGABAn production from the GE in human fetuses. Therefore, a pallial production but also subpallial protracted neurogenesis are a hallmark of cxGABAn development in primates.

In this study we have quantitatively analyzed development of dendritic tree of large layer IIIC (L3N) and layer V pyramidal neurons (L5N) in the human prefrontal cortex (PFC) impregnated by Golgi method in period from 17 week of gestation (wg) up to third month postnatal. At 17wg both classes of neurons were immature showing similar dendritic morphology. Period between 17-26wg was characterized by protrusion of primary dendrites in basal and apical part of dendritic tree, without change in the length and in the number of segments. After 26wg no further increase in number of primary dendrites could be observed. The phase of rapid growth of basal dendritic tree begins with an outgrowth of new dendritic segments (stage1), followed by their large elongation without significant formation of new dendrites (stage2). Stage1 occurred between 26-32wg for L5N and between 36wg-1m for L3N. Stage2 occurred between 32wg-1m for L5N and newborn-3m for L3N. By the 3rd postnatal month L5N attained 80% and layer L3N 60% of the total adult size of their dendritic trees and both classes of neurons already displayed an adult-like overall morphological dendritic phenotype. Phase of intensive dendritic growth coincides with the massive ingrowth of afferent fibers into the cortical plate ; around 26wg for L5N with thalamo-cortical, and around 36wg for L3N with cortico-cortical axons entrance. Such an early functioning neural network may represent a neurobiological basis for initial cognitive functions already present in newborns and young infants.

Cortical hem, a putative signaling centre at the interface of the prospective hippocampus and the choroid plexus, is a significant source of Cajal-Retzius cell population that invade cortical marginal zone. Analyzing Nissl stained sections of human fetuses during the period from 8 to 14 postconceptional weeks (pcw), we observed, in the medial telencephalic wall, a stream of cells extending from the proliferative zones of cortical hem to the marginal zone (MZ) of the prospective archicortex. Golgi staining showed that this stream is composed of small unipolar migratory like cells. At corresponding ages in the macaque monkey fetuses (embryonic day/E 47-55), many cells in the proliferative zones of cortical hem were labeled for calbindin (CB). From this zone, a stream of unipolar migratory like CB-containing cells extended to the subpial position. In addition, at E47 few CB-containing neurons were observed in the thick MZ of the prospective archicortex. They were in apparent continuity with the stream of migratory like CB-containing cells extending from proliferative zone of cortical hem. At E55, CB-containing cells in the prospective hippocampus were organized in clusters. The clusters of CB-containing cells close to the proliferative zones were formed of immature cells, while more distant clusters included cells with typical pyramidal neuron body shape. During this period, GAD-containing migrating cells were mostly observed in the lateral wall, only very few were present in the medial telencephalic wall. Therefore, our results suggest that in primates the cortical hem gives rise to populations of non-GABA-ergic calbindin neurons, some of them destined to the hippocampus.

Cortical hem, a putative signaling centre at the interface of the prospective hippocampus and the choroid plexus, is a significant source of Cajal-Retzius cell population that invade cortical marginal zone. Analyzing Nissl stained sections of human fetuses during the period from 8 to 14 postconceptional weeks (pcw), we observed, in the medial telencephalic wall, a stream of cells extending from the proliferative zones of cortical hem to the marginal zone (MZ) of the prospective archicortex. Golgi staining showed that this stream is composed of small unipolar migratory like cells. At corresponding ages in the macaque monkey fetuses (embryonic day/E 47-55), many cells in the proliferative zones of cortical hem were labeled for calbindin (CB). From this zone, a stream of unipolar migratory like CB-containing cells extended to the subpial position. In addition, at E47 few CB-containing neurons were observed in the thick MZ of the prospective archicortex. They were in apparent continuity with the stream of migratory like CB-containing cells extending from proliferative zone of cortical hem. At E55, CB-containing cells in the prospective hippocampus were organized in clusters. The clusters of CB-containing cells close to the proliferative zones were formed of immature cells, while more distant clusters included cells with typical pyramidal neuron body shape. During this period, GAD-containing migrating cells were mostly observed in the lateral wall, only very few were present in the medial telencephalic wall. Therefore, our results suggest that in primates the cortical hem gives rise to populations of non-GABA-ergic calbindin neurons, some of them destined to the hippocampus.

Background: The middle ear contains three auditory small serious ossicles, which are, in order from the eardrum to the inner ear: malleus, incus, and stapes. Studies have shown that ossicles ossify endochondrally as a parts of Meckel´s cartilage, that are attached to the jaw. Anatomy of ossicles in adult humans was described in different studies. In a contrary to the adult studies there are only few dana available about ossicles dimensions during fetal life. The aim of our study was to investigate the dynamic of growth process of ossicles in fetal skulls. Materials and methods: In this experiment we used 30 ossicles from bone collection of the Department of Anatomy, University of Zagreb. Bones were scanned by microCT and for bone volume analysis CTAn software was used. Bones were divided in three groups based on fetus length: 300, 400, and 500mm. Results: Average incus and malleus bone volume in all groups was increased in accordance with the fetal linear growth. Average stapes bone volume in 500mm fetus length showed a dynamic growth. In other two groups bone volume was without significant change. Conclusion: In conclusion, we showed that stapes increased in size when fetus gained 500mm, while incus and malleus enlarges linearly during whole fetal period.

The source of cortical GABA-ergic neurons has unique features in human and non-human primates (Petanjek et. al, Front Neuroanatom 2009 ; 3:26.). In contrast to other mammals, where vast majority if not all are born in ganglionic eminence (GE) and migrate to dorsal telencephalon, in primates about 2/3 of cortical GABA-ergic neurons originate from the local proliferative zones. We have analyzed frontal sectioned slices of the human fetal telencephalon during second half of gestation impregnated by Golgi method. During the period from 22 to 26 postconceptional weeks (pcw) numerous nonradialy oriented migratory like unipolar cells have been observed all around telencephalon, most densely packed in the subventricular and intermediate zones. Also, a large and very densely packed stream of migratory like cells leaving well pronounced GE in direction of dorsal telencephalon was observed at cortico-striatal border. At stage of 32-36 pcw there was still a significant number of nonradially migratory like cells in the dorsal telencephalon, while the GE decreased in size, but was clearly distinguishable. Also, a stream of migratory like cells was extending in ventro-dorsal direction and continuing out of the GE at the position of cortico-striatal border. During whole period many nonradially oriented migratory like cells in dorsal telencephalon, as in the stream leaving GE, were MAP-2 and calretinin positive. These observations suggest that in the human brain significant production of subpopulations of cortical GABA-ergic neurons occurred much beyond proliferative period for principal glutamatergic neurons and extend even to early postnatal period.

GABA development

Introduction: The function of the orbit is to protect and support the eye. It is a four-sided pyramidal cavity of the skull. Seven bones conjoin to form the orbital structure. The orbits are aligned so that the medial walls are parallel and the lateral walls are vertical. The arc from the medial to lateral wall in each orbit is 45°. The ordinary dimensions of the adult orbit are: height of orbital margin – 40 mm, width of orbital margin – 35 mm. The optic canal is one of the paired openings in the sphenoid bone that transmits the optic nerve, opthtalmic artery and central retinal vein. It connects the anterior cranial fossa and the orbit. There have been several attempts to clarify the exact anatomy and variations of the optic canal, since procedures such as endoscopic optic nerve decompression have variable success rates. The orbital apex is a congested area and orbital surgeons should have an exact knowledge of the relations and sizes of each structure. For a better understanding of those structures in adults, it is crucial to investigate their exact fetal evolution. Our aim was to investigate, for the first time, the dynamic of growth and development of those structures on fetal skulls. Material and methods: In this experiment, we used 12 fetal frontal and sphenoid bones from bone collection of the Department of Anatomy at the University of Zagreb. Ethical approval was obtained from the Research Ethics Committee of the University of Zagreb. The bones were analyzed and following groups were established (based on the length of fetus): (1) 300 ± 20 mm, (2) 400 ± 20 mm and (3) 500 ± 20 mm, four skulls per group. The diameters of optic canals and orbits from frontomaxillar suture to frontozygomatic suture were measured by microCT. Results: The average diameter of the optic canal in the 300 mm fetus was 1570 µm, in 400 mm fetus 2436 µm and in the 500 mm fetus 3812 µm. Standard deviations in all groups are around 50 µm, which represents 2% of the mean value. There was a slight difference in fetus weight among the groups. In the first group, the optic canal diameters were practically the same (±35 µm), while their weight varied substantially. The same trend occurred in the second group. In the third group, the lightest fetus had the largest optic canal. In the same period, the orbit diameter enlarged from 13290 µm to 19657 µm, showing strict linear enlargement. We also calculated the ratio of the orbit diameter and optic canal diameter: it was 8, 5 in the first group, and 7 and 5 in the second and the third group respectively. Conclusion: The development of both the optic canal and the orbit varies during fetal periods ; there are representative differences between intervals. The ratio of those diameters in different time periods is significant and shows that orbit grows faster in the earlier fetal period, while the optic canal enlarges later. We also show that the diameters of those structures correlate better with fetus length then weight.

Introduction: The development of human skull has been the subject of many studies so far, since this forms the basis for understanding the structure of adult skull and the development of new surgical approaches to intracranial structures. However, there is little literature related to the development of the sphenoid bone during fetal and neonatal periods. The sphenoid bone and its large wings in particular are a place of great clinical interest because of the vicinity of the middle cerebral artery and the trigeminal nerve. There are also studies reporting meningiomas and fractures of the sphenoid bone. In this study, we traced the development of large wings and Turkish saddle of the sphenoid bone by microCT scanning. Material and methods: In the study we included a total of 17 isolated sphenoid bones of fetuses and newborns from 160 to 520mm length from the bone collection of the Department of Anatomy at the University of Zagreb Medical School. Ethical approval was obtained from the Research Ethics Committee of the University of Zagreb. The bones consisted of separate large wings and a central portion with Turkish saddle and small wings. The bones were scanned by microCT, and we recorded the length, width and weight of the large sphenoid wing as well as the distance between the foramen rotundum and foramen ovale. In the central part, we measured the width of dorsum sellae, the width of medial clinoid sequels and the antero-posterior diameter of the Turkish saddle. Results: The development of large sphenoid wings is not linear, but rather represented by a curve. The period of the largest increase in length and width of large wings occurs when the fetus measures from 300 to 430mm, when the change of intensity is more pronounced in length growth. At the same time the greatest changes in weight of large sphenoid wings occur in the period when the fetus measures from 400 to 500mm. The growth rate of the distance between foramen rotundum and foramen ovale shows smaller variations in the intensity of changes. Changes of basic dimensions of Turkish saddle were most intensive in the period from 400 to 500mm fetus size, which is best visible in the antero-posterior diameter. Comparing the absolute increase of the parameters described in the aforementioned period, we found that the largest increase occured in the weight of large sphenoid wings and that it is nearly tenfold greater than the increase in length or width of large sphenoid wings. Conclusion: Analyzing the results, we concluded that the development of sphenoid bone is not characterized by a constantly equal increase, and that the period of greatest intensity of development differs with regard to specific parameters. We found that the most rapid development of large sphenoid wings in length and width precedes the fastest increase of their weight. We also found that the highest intensity of growth in large sphenoid wings occurs earlier than in the central part of sphenoid bone. We conclude that the development of sphenoid bone is a complex process that is not parallel and equal to the development of the entire skull and also not uniform in all of its constituent parts. This study of the sphenoid bone development allows us to predict the size and position of certain structures of neurocranium, thus contributing to better understanding of the structure of the skull and, consequently, better planning and development of new surgical approaches.

Gamma-Aminobutyric acidergic (GABAergic) neurons that correspond mainly to local circuit neurons play a crucial role in regulating the activity of cortical neuronal networks and the complex interactions among principal neurons. These functions depend on a very heterogeneous population of neurons, whose number and diversity may increase during mammalian evolution. Interestingly dysfunction and cell death of several specific types of GABAergic neurons is a hallmark of various psychiatric and neurological disorders such as schizophrenia, autism and epilepsies to which defect in genesis or migration of these neurons can be a predisposing factor. Understanding primate specific developmental mechanisms that regulate generation, migration and maturation of cortical GABA neurons, is a crucial step in elucidating such pathologies. However, there are few such studies in monkeys and humans, whereas they are numerous in lower mammals. In rodents, cortical GABAergic neurons are generated exclusively in the subcortical proliferative zone of the ventral telencephalon, the ganglionic eminence (GE), and migrate tangentially to the dorsal telencephalon to reach their target cortical layer. We reported (Petanjek et al. 2009, Cerebral Cortex 19:249-62) that in contrast to rodents, in cynomolgus monkey cortical GABAergic neurons are generated massively in the proliferative zones of the dorsal telencephalon, in addition to the GE. This neurogenesis occurs very early during brain development in both dorsal and ventral telencephalon but with distinct temporal profiles. GABAergic neuron progenitors labeled for transcription factor Mash1 and GABA synthesizing enzyme GAD65 were present mainly in the GE at embryonic days (E) 47-55, and in the entire dorsal telencephalon at E64-75. These progenitors within the dorsal telencephalon are generated locally rather than in the GE. In the human embryonic forebrain (Letinic et al. 2002, Nature 417:645-9) retroviral labeling in organotypic slice cultures also demonstrated the existence of two distinct lineages of neocortical GABAergic neurons. One lineage expresses Dlx1/2 and Mash1 transcription factors, represents 65% of neocortical GABAergic neurons in humans, and originates from Mash1-expressing progenitors of the neocortical proliferative zones of the dorsal telencephalon. The second lineage, characterized by the expression of Dlx1/2 but not Mash1, forms in human brain only around 35% of the GABAergic neurons and originates from the GE of the ventral forebrain, in comparison to rodent where all GABAergic neurons originate in GE. The dorsal telencephalic origin of cortical GABA-ergic neurons is a unique feature of human and non-human primates and appears to be, in the primate lineage, an early evolutionary modification which provided increased number and variety of GABA-ergic neurons to an expanding neocortex.

That human cognitive and emotional development continues through adolescence is a general fact accepted among developmental psychologists and in past decade supported by extensive data of functional brain development. These findings have been taken as indicators of protracted synaptic pruning or/and protracted dendritic growth. However, as per our knowledge, there are no available data of cortical circuitry development that can support this assumption. The aim of this study was to examine adolescent and post-adolescent dendritic growth and dendritic spine density on key elements of circuitry involved in processing of higher cognitive functions, the layer IIIC pyramidal neurons in dorsolateral prefrontal cortex (Brodmann’s area 9). We used available quantitative data of dendritic morphology and dendritic spine density obtained from 25 specimen of Zagreb neuroembryological collection. Data obtained per subject were used as a row data for statistical analysis between following groups: childhood (2-8 years), adolescence (9-18 years), early adulthood (19-30 years), adult (31-65 years) and aging (66 years+). Except intermediate segments, length and branching pattern of dendrites has not changed significantly. However, we found massive statistically significant decrease in dendritic spine density that occurred during adolescence, but also continued through early adulthood for all segment type analyzed. In parallel, the length of intermediate segments decreased for about 20%. Some differences in pattern of dendritic spine density between different segment types were seen. Dendritic spine density in adulthood was highest in the most distal segments of apical oblique dendrites, which showed also largest and most protracted decrease in spine density compared to proximal oblique and basal dendrites. Distal dendrites are known to be predominantly innervated by intracortical and associative cortico-cortical fibers. The maturation of layer IIIC pyramidal neurons might represent a biological basis for protracted cognitive development and, as such, may be highly influenced by education and social environment. Besides biomedical, educational and social implications, these results also point to highly protracted environmentally driven plasticity in associative cortex that influence circuitry development, suggesting mechanisms how and for how long the environment reshapes synaptic circuitries that builds biological basis of individuality.

Gamma-Aminobutyric acidergic (GABAergic) neurons that correspond mainly to local circuit neurons play a crucial role in regulating the activity of cortical neuronal networks and the complex interactions among principal neurons. These functions depend on a very heterogeneous population of neurons, whose number and diversity may increase during mammalian evolution. Interestingly dysfunction and cell death of several specific types of GABAergic neurons is a hallmark of various psychiatric and neurological disorders such as schizophrenia, autism and epilepsies to which defect in genesis or migration of these neurons can be a predisposing factor. Understanding primate specific developmental mechanisms that regulate generation, migration and maturation of cortical GABA neurons, is a crucial step in elucidating such pathologies. However, there are few such studies in monkeys and humans, whereas they are numerous in lower mammals. In rodents, cortical GABAergic neurons are generated exclusively in the subcortical proliferative zone of the ventral telencephalon, the ganglionic eminence (GE), and migrate tangentially to the dorsal telencephalon to reach their target cortical layer. We reported (Petanjek et al. 2009, Cerebral Cortex 19:249-62) that in contrast to rodents, in cynomolgus monkey cortical GABAergic neurons are generated massively in the proliferative zones of the dorsal telencephalon, in addition to the GE. This neurogenesis occurs very early during brain development in both dorsal and ventral telencephalon but with distinct temporal profiles. GABAergic neuron progenitors labeled for transcription factor Mash1 and GABA synthesizing enzyme GAD65 were present mainly in the GE at embryonic days (E) 47-55, and in the entire dorsal telencephalon at E64-75. These progenitors within the dorsal telencephalon are generated locally rather than in the GE. In the human embryonic forebrain (Letinic et al. 2002, Nature 417:645-9) retroviral labeling in organotypic slice cultures also demonstrated the existence of two distinct lineages of neocortical GABAergic neurons. One lineage expresses Dlx1/2 and Mash1 transcription factors, represents 65% of neocortical GABAergic neurons in humans, and originates from Mash1-expressing progenitors of the neocortical proliferative zones of the dorsal telencephalon. The second lineage, characterized by the expression of Dlx1/2 but not Mash1, forms in human brain only around 35% of the GABAergic neurons and originates from the GE of the ventral forebrain, in comparison to rodent where all GABAergic neurons originate in GE. The dorsal telencephalic origin of cortical GABA-ergic neurons is a unique feature of human and non-human primates and appears to be, in the primate lineage, an early evolutionary modification which provided increased number and variety of GABA-ergic neurons to an expanding neocortex.

Endothelium is a thin layer of cells that coats the inner surface of the cardiovascular system and provides a link between the circulating blood and the vessel wall. Due to variety of functions and wide distribution in the body, endothelium can be thought of as an organ by itself. Endothelial dysfunction (ED) is a term which describes the state of damaged endothelium with related loss of function. ED consists of a wide range of pathophysiologic states, in span of localised intimal damage to global, persistent, inadequate endothelial activation important for development of numerous clinically different pathologic states. Major mechanisms of ED include: oxidative stress, endothelial NO synthase (eNOS) uncoupling, inflammatory pathways activation, differential expression of vascular endothelial growth factor, intracellular sorbitol accumulation. ED is a hallmark of impaired vascular integrity from which chronic complications of diabetes mellitus type 2 (DMT2) stem from. ED starts to appear in pre-diabetic state and worsens proportionally with duration of DMT2. Assessment of endothelial function in patients with risk of cardiovascular disease (CVD) development is of great importance because it enables us to detect vascular abnormalities and efficiency of therapeutic approach. Increasing quantities of therapeutic modalities have been shown to improve endothelial dysfunction, which has important implications for the treatment of patients at risk of developing chronic complications related to DMT2., Endotel je tanki sloj stanica koji oblaže unutarnje površine kardiovaskularnog sustava te služi kao poveznica između cirkulirajuće krvi i krvnih žila. S obzirom na raznolikost funkcija i široku rasprostranjenost u tijelu, na endotel se može gledati i kao na zaseban organ. Endotelna disfunkcija je pojam koji opisuje stanje oštećenog endotela te posljedično njegovu smanjenu funkciju. Sastoji se od niza patofizioloških stanja, počevši od lokalne ozljede intime do globalne, dugotrajne i neadekvatne endotelne aktivacije koja dovodi do brojnih patoloških stanja. Glavni mehanizmi nastanka endotelne disfunkcije uključuju povećan oksidativni stres, disfunkciju endotelne NO-sintaze (eNOS), upalna stanja, poremećeni izražaj vaskularnog endotelnog faktora rasta (VEGF) te unutarstanično nakupljanje sorbitola. ED je glavno obilježje poremećenog vaskularnog integriteta, iz kojeg se razvijaju kronične komplikacije diabetesa mellitusa tipa 2 (DMT2). Praćenje funkcionalnosti endotelnih stanica od velike je važnosti kod pacijenata s rizikom od razvitka kardiovaskularnih bolesti jer nam omogućuje praćenje stanja vaskularnog sustava te učinkovitosti terapije. Konstantan razvoj i povećanje broja terapijskih pristupa koji vode k poboljšanju ED, također su od velike važnosti u prevenciji, odgodi nastanka ili terapiji kroničnih komplikacija DMT2.

Both diabetes mellitus and neurodegenerative diseases have become major health challenges worldwide. Diabetes mellitus is now known to be a major risk factor for cognitive decline, dementia and Parkinson’s disease. It has also been shown that the risk of cognitive decline and neurodegeneration is not only seen in diabetic patients, but it is also increased in patients with prediabetes and metabolic syndrome. Chronic hyperglycemia suppresses substantia nigra dopaminergic neuronal firing and decreases dopamine turnover, as well as induces chronic brain insulin resistance, ultimately leading to impaired brain insulin signaling. Recent studies have focused on the role of insulin and impaired insulin signaling as a link between diabetes and neurodegenerative diseases. Impairments in brain insulin signaling mechanisms, together with hyperglycemia, might induce the molecular and biochemical changes such as dysfunction of the mitochondria, oxidative damage, and inflammatory responses, ultimately leading to pathological lesions seen in neurodegenerative diseases, as well as in diabetes mellitus. In addition, microvascular changes are also a critical factor involved in the etiology of diabetes mellitus and neurodegenerative diseases. Taken together, the complex systemic effects of diabetes mellitus on neurodegenerative diseases require further investigation., Šećerna bolest i neurodegenerativne bolesti postale su velikim zdravstvenim izazovima današnjice. Šećerna bolest poznata je kao glavni faktor rizika za kognitivne poremećaje, demencije i Parkinsonovu bolest. Rizik za razvoj neurodegenerativnih bolesti i kognitivnih poremećaja nije samo povećan kod šećerne bolesti, već i kod pacijenata s predijabetesom ili metaboličkim sindromom. Kronična hiperglikemija potiskuje “paljenje” dopaminergičkih neurona substantije nigre i smanjuje omjer dopamina i njegovih metabolita, te inducira kroničnu inzulinsku rezistenciju u mozgu i dovodi do promijenjene signalizacije inzulina u mozgu. Nedavne studije usmjerene su na ulogu inzulina i promijenjenu signalizaciju inzulina kao poveznicu između dijabetesa i neurodegenerativnih bolesti. Oštećenja u mehanizmima signalizacije inzulina u mozgu, zajedno s hiperglikemijom, mogu izazvati molekularne i biokemijske promjene kao što su disfunkcija mitohondrija, oksidativni stres, i upalni odgovori, što u konačnici dovodi do patoloških lezija u neurodegenerativnim bolestima, kao i do šećerne bolesti. Osim navedenih promjena, mikrovaskularne promjene ključni su čimbenik u etiologiji šećerne bolesti i neurodegenerativnih bolesti. Stoga, s obzirom na vrlo kompleksnu pozadinu ovih procesa, složeni sistemski učinci šećerne bolesti na razvoj neurodegenerativnih bolesti zahtijevaju daljnje istraživanje.