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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.

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.

The study investigates the possibility, extent, and persistence of structural or connectivity changes in the rat hippocampus after moderate perinatal hypoxia. The distribution, qualitative and quantitative changes in parvalbumin-positive neurons (PV) and the perineuronal nets (PNN) were researched. For this purpose, 18 Wistar Han (RccHan: WIST) rat pups were randomly subjected to hypoxia (partial pressure of oxygen-pO2 73 mmHg ; atmosphere pressure-pATM 350 mmHg) or normoxia (pO2 159 mmHg ; pATM 760 mmHg) for two hours at the first postnatal day. At the age of 3.5 months, the rats were sacrificed, their brains were isolated and used for immunohistochemistry and analysis of PV and PNN of the hippocampi (bregma: levels -2.92 mm ; -3.36 mm ; -4.56 mm) using OlympusBX60 and Neurolucida 10 software. Qualitative analysis revealed differences in the co-expression pattern of PNNs’ constituents (aggrecan, versican, and neurocan) and differences in PNN and PV areal/layer distribution between hypoxia and normoxia groups. Quantitative analysis revealed a significantly higher density of PNN and PV, especially in the CA1 area of the hippocampus of mature animals subjected perinatally to hypoxia. These results align with the changes in behavior such as significant hyperactivity and a slower pace of learning in rats after perinatal hypoxia, published previously by our group. Further research is needed to disclose the underlying sequence of molecular events and the injury- plasticity relations that lead to the permanent hippocampal and behavioral alteration in rats after moderate perinatal hypoxia. Funding: project CSF-IP-2019-04-3182 and project GA KK01.1.1.01.0007 by the European Union through the European Regional Development Fund.

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.

Recent research found that understanding alterations of the brain extracellular matrix (ECM) is crucial for determining the pathophysiological processes occurring after hypoxic brain injury. Perineuronal nets (PNNs), a form of condensed ECM, are of particular interest in the adult brain. In this study we analyzed the anterior cingulate cortex (ACC) from three adult human brains using double labelling immunofluorescence. The ECM marker Wisteria floribunda agglutinin (WFA) was combined with the neuronal nuclear antigen (NeuN) and four interneuron markers – parvalbumin (PV), calretinin (CR), calbindin (CB) and somatostatin (SOM). NeuN/WFA double labelled slides were quantified using Neurolucida software. Qualitative analysis revealed that WFA+ condensed ECM was present around NeuN+ cells, confirming that these were indeed PNNs. Furthermore, PNNs were found surrounding certain pyramidal and PV+ cells, but not surrounding CR+, CB+ or SOM+ cells. Quantitative analysis revealed that 6.17% ± 0.67% of NeuN+ cells had WFA+ PNNs. PNNs were most abundant in layer III (10.94%), followed by layers V (5.07%) and VI (4.78%). Faintly stained PNNs were found in the superficial white matter (7.11%). PNNs were only sporadically found in layer II and were never present in layer I. In conclusion, PNNs in the human ACC have a specific laminar distribution and are predominantly found surrounding certain pyramidal cells and PV+ interneurons. This has important implications for the functional and laminar organization of the microcircuitry in the human brain, which is critical for understanding the underlying pathophysiology of hypoxic brain injury as well as of disorders such as schizophrenia and bipolar disorder.

Hippocampal sclerosis (HS) is the commonest histopathological finding in patients with drug- resistant mesial temporal lobe epilepsy (MTLE). The type 1 HS is the most prevalent type characterized by severe loss of pyramidal neurons, parvalbumin (PV) interneurons, and gliosis in the CA1 and CA4 fields. The extracellular matrix (ECM) is recognized as an important regulator of excitability and synaptic plasticity, especially in its highly condensed pericellular form of perineuronal nets (PNN). As experimental rodent models suggest that the different ECM components and PNN may have a role in epileptogenesis this study aimed to analyze and correlate PNN, glycosylation pattern with Wisteria floribunda agglutinin (WFA) and other ECM constituents, and expression of Nuclear-neuronal marker (NeuN), and parvalbumin (PV), with clinical findings of 65 patients surgically treated for pharmacoresistant MTLE caused by HS. Apart from the reduced number and impoverished morphology of pyramidal neurons and PV interneurons in HS, we found a changed distribution of the specific glycosylation pattern recognized by WFA. The proteoglycans, versican, and aggrecan show significantly changed expression patterns, whereas other ECM constituents as fibronectin, chondroitin-sulfate proteoglycan-56, and neurocan were slightly altered or showed no apparent difference. We also found reduced WFA specific glycosylation of the PNN around pyramidal neurons in all CA fields (CA4-CA1) as well as around PV-immunoreactive interneurons and simultaneously increased WFA specific glycosylation of the diffuse ECM in all CA fields. These findings suggest that PNN reduction and change of the glycosylation neuropil profile in the MTLE due to HS may cause the increase of the excitability of pyramidal hippocampal neurons, thus contributing to the development of drug- resistant epilepsy, at the same time offering a potential therapeutic target.

We designed a non-invasive model in newborn rats to determine the hallmarks of possible permanent structural and behavioral changes in the brain after moderate perinatal hypoxia. The 82 Wistar Han (RccHan: WIST) rats were randomly subjected to hypoxia (pO2 73mmHg/2h) or normoxia at the first postnatal day. The increase of hypoxia-inducible factor-1α and decreased cytochrome-C-oxygenase expression in treated animals' brains, detected by Western blot, confirmed an acute moderate lesion. The results of the open-field, hole-board, social- choice, and T-maze tests, applied at the 30 - 45th and 70-85th postnatal days, displayed significant hyperactivity and a slower pace of learning in rats subjected to perinatal hypoxia. At 3.5 months of age, the histochemical examination revealed a significantly increased number of specific extracellular matrix-perineuronal nets and parvalbumin-expressing interneurons in the medial and retrosplenial cingulate cortex of these animals. Conclusively, moderate perinatal hypoxia in the rat causes lifelong cellular and connectivity organization changes in the cingulate cortex and related behavioral and cognitive alterations. This non-invasive hypoxia in the rat successfully and complementary models the frequently unrecognized moderate perinatal brain injury in fetuses in humans and may enhance future research of new diagnostic and therapeutic strategies for perinatal brain injuries.

Somatostatin positive cells represent a major population of GABAergic neurons in the human cerebral cortex. Using double labelling immunofluorescence and RNAscope in situ hybridization we characterized the somatostatin cell population in the human prefrontal cortex (PFC). We analyzed histological slides from five adult specimens and evaluated the results in two regions of the PFC – Brodmann areas 9 and 14. There were no observable qualitative differences between the two areas and, therefore, the results presented are applicable to both regions of the PFC. After thorough qualitative analysis, we found that somatostatin cells in the supragranular layers were small (soma diameter of approximately 10 μm), had a high expression of somatostatin mRNA and a relatively low expression of somatostatin peptide. Furthermore, almost all of the supragranular somatostatin cells colocalized with calbindin, indicating that somatostatin and calbindin positive cells represent a single cell population in the supragranular layers. In the infragranular layers, somatostatin cells were large (soma diameter of approximately 20 μm) with complex dendritic morphology, typically had a lower expression of somatostatin mRNA and a high expression of somatostatin peptide, and did not express calbindin protein. All somatostatin cells were also NeuN positive, confirming that this is indeed a neuronal cell population. In conclusion, in the human PFC, supragranular and infragranular somatostatin cells represent two distinct subpopulations of somatostatin neurons.

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.

Schizophrenia is one of the most wide-spread mental brain disorders with complex and largely unknown etiology. To characterize the impact of schizophrenia at a cellular level, we performed single nucleus RNA sequencing of gt;190,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls (7 vs 11, respectively). In addition, to correlate data with cortical anatomy, gt;100,000 neurons were analyzed topographically by immunohistochemistry in an extended cohort of cases with schizophrenia and controls (10 vs 10). Compositional analysis of RNA sequencing data revealed reduction in relative abundance across all families of GABAergic neurons and a concomitant increase in principal neurons, which was most pronounced for supragranular subtypes (layers 2-3). Moreover, supragranular subtypes of GABAergic interneurons showed most dramatic transcriptomic changes. These results were substantiated by histological analysis, which revealed a reduction in the density of calretinin, calbindin and parvalbumin GABAergic interneurons particularly in layer 2. Common effect of schizophrenia on supragranular neuronal networks was underlined by downregulation of protein processing genes and upregulation of neuronal development/plasticity genes across supragranular subtypes of principal neurons and GABAergic interneurons. In situ hybridization and spatial transcriptomics further confirmed supragranular layer neuron vulnerability, revealing complexity of schizophrenia-affected cortical circuits. These point towards general network impairment within supragranular layers being a core substrate associated with schizophrenia symptomatology.Competing Interest StatementThe authors have declared no competing interest.

GABAergic interneurons are a diverse group of cortical neurons commonly identified with the following molecular markers – calretinin, parvalbumin, calbindin and somatostatin. We performed quantitative and qualitative analysis of these interneuron populations in dorsal (Brodmann area 9) and ventral (Brodmann area 14r) regions of the human prefrontal cortex (PFC) on five adult specimens using double labelling immunofluorescence. The interneuron markers were combined with each other as well as with NeuN. The proportion of each interneuron population within the total neuron population in both Brodmann areas (BA) was compared using a paired t-test. The analysis revealed substantial differences in ratios of interneuron populations between supragranular layers but not between infragranular layers. Calretinin neurons were the most numerous interneuron population in supragranular layers and somatostatin neurons were the most numerous in infragranular layers. Furthermore, somatostatin and calbindin neurons had different laminar molecular profiles – in supragranular layers, the level of somatostatin and calbindin co-expression was high, whereas in infragranular layers the level of co- expression was extremely low. The proportion of calretinin neurons was significantly higher in BA14r (average: 10.24%) than in BA9 (average: 8.21%). The proportions of other interneuron populations did not differ significantly between the two cortical regions, though the proportion of somatostatin neurons was on average higher in BA14r than in BA9. In addition, the ratio between calretinin and parvalbumin neurons was significantly higher in BA14r (average: 1.62) than in BA9 (average: 1.15). In conclusion, different proportions of GABAergic interneurons, particularly calretinin neurons, reflect the structural and functional differences between cortical regions.

Arhitektonika neuronske mreže kore velikoga mozga jedinstvena je za svaku osobu i predstavlja biološku osnovu naših kognitivnih i emotivnih kapaciteta. Iako genetska predispozicija određuje okvire, obrazovanje te emocionalno i socijalno okruženje te fizička okolina usmjeravaju razvoj arhitektonike neuralne mreže i determiniraju njenu konačnu strukturu. Djeca i mladi 21. stoljeća predstavljaju generaciju koja je od ranih dana izložena digitalnoj tehnologiji. Brojna istraživanja ukazuju na niz pozitivnih, ali i negativnih posljedica na tjelesni, kognitivni i socio-emocionalni razvoj. Primjerice, plavo svjetlo ekrana utječe na lučenje melatonina i smanjuje kvalitetu spavanja, ali isto tako zajedničko korištenje tehnologije roditelja i djeteta može biti prilika za gradnju skela i pomoć djetetu da bolje razumije online sadržaj i učinke njegovog korištenja. Digitalna tehnologija zasigurno djeluje poticajno na razvoj neuronske mreže koja je bitna za procesuiranje informatičkih, apstraktnih i simboličkih zadataka. No kakav je učinak na razvoj onog dijela neuralne mreže koji procesuira složene komunikacijske i socijalne vještine, kao što je afektivna modulacija emocionalnog izražavanja, konceptualizacija uma, kognitivna fleksibilnost i radna memorija. One su bitne za kvalitetu izravne komunikacije s drugim osobama te snalaženje u situacijama složenih socijalnih odnosa koje su obilježje razvijenog poslovnog i životnog okruženja, jednako kao što su bitne i emocionalne vještine, npr. emocionalna inteligencija. Ona je po svojim karakteristikama slična kristaliziranoj inteligenciji, a razvija se kroz iskustvo i učenje od najranije dobi. Tako su danas diljem svijeta dobro poznati programi za emocionalno opismenjavanje kojim se jačaju socio-emocionalne kompetencije sudionika, a primjena je relativno široka – od poučavanja djece vrtićkog uzrasta do treninga za rukovoditelje i menadžere. Znatan broj roditelja smatra kako je upotreba digitalne tehnologije u ranoj dobi poželjna jer je intuitivna i priprema djecu za kasnije učenje, dok se istovremeno brinu kako to dugoročno utječe na njihov mozak, a samim time na njihov emocionalni razvoj, pažnju i koncentraciju te im je teško pronaći odgovarajuću ravnotežu. Stoga su im nužne smjernice na temelju kojih bi mogli oblikovati obiteljski medijski plan na način koji bi rezultirao zaštitom djece od neželjenih učinaka digitalne tehnologije, jačajući istovremeno njihove prilike za učenje i razvoj, kao i vještine digitalne pismenosti.

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.

Aiming to find molecular, cellular, and structural brain hallmarks and related behavioral outcome after perinatal hypoxia, we designed a non- invasive model of perinatal hypoxia in the newborn rats. In the randomized study, fifty-two Wistar Han rats were subjected to general hypoxia (pO2 73mmHg/2h) or normoxia, 24h after birth. The Western blot method applied 2h, 8h, 12h, and 24h later, showed a gradual increase of the hypoxia- inducible factor 1α, and a decrease of the cytochrome-C- oxygenase (subunit 4, isoform 1) expression, revealing moderate brain lesion in the rats subjected to hypoxia. The behavioral testing at 30th and 70th postnatal days, using open field, hole board, social choice, and T-maze paradigm displayed significant hyperactivity and slower pace of learning in the rats that were exposed to perinatal hypoxia. The closer examination of the cingulate cortex at the age of 4 months, by WFA- and anti-parvalbumin immunohistochemistry, disclosed a significantly increased number of perineuronal nets and parvalbumin-interneurons in the medial and retrosplenial areas. Thus, the moderate hypoxia in the newborn rats causes lifelong changes at the molecular and connectivity level in the cingulate cortex and related behavioral outcome. This new, non- invasive perinatal brain lesion in rats, according to the findings obtained, successfully resembled a frequent but diagnostically neglected moderate perinatal brain injury in humans and may establishes as a complementary model for future search for prevention, diagnostic and therapeutic strategies for perinatal brain injuries in human.

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.