Decoding the development of the human hippocampus.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and the consolidation of information from short-term memory to long-term memory1,2. Here we use single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis to illustrate the cell types, cell linage, molecular features and transcriptional regulation of the developing human hippocampus. Using the transcriptomes of 30,416 cells from the human hippocampus at gestational weeks 16-27, we identify 47 cell subtypes and their developmental trajectories. We also identify the migrating paths and cell lineages of PAX6+ and HOPX+ hippocampal progenitors, and regional markers of CA1, CA3 and dentate gyrus neurons. Multiomic data have uncovered transcriptional regulatory networks of the dentate gyrus marker PROX1. We also illustrate spatially specific gene expression in the developing human prefrontal cortex and hippocampus. The molecular features of the human hippocampus at gestational weeks 16-20 are similar to those of the mouse at postnatal days 0-5 and reveal gene expression differences between the two species. Transient expression of the primate-specific gene NBPF1 leads to a marked increase in PROX1+ cells in the mouse hippocampus. These data provides a blueprint for understanding human hippocampal development and a tool for investigating related diseases.

Aging and neurodegeneration are associated with increased mutations in single human neurons.

It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of 15 normal individuals (aged 4 months to 82 years), as well as 9 individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age-which we term genosenium-shows age-related, region-related, and disease-related molecular signatures and may be important in other human age-associated conditions.

Gladiolus dalenii lyophilisate reverses scopolamine-induced amnesia and reduces oxidative stress in rat brain.

Learning and memory are the most important executive functions performed by the human brain, the loss of which is a prominent feature in dementia. Gladiolus dalenii is traditionally used to treat a number of illnesses such as epilepsy and schizophrenia in Cameroon. This study aims to investigate the anti-amnesia effect of Gladiolus dalenii in scopolamine-induced amnesia in rats and its possible antioxidant properties in this model. Morris water maze, novel object location and recognition tasks were used to assess spatial and working memory. Male rats were treated for 12 days with saline, G. dalenii or Tacrine. Experimental animals were co-treated with scopolamine once daily from day 9 to 12. Acetylcholinesterase activity was measured in the prefrontal cortex and hippocampus. Malondialdehyde and glutathione levels were measured in the hippocampus. G. dalenii reversed memory impairment induced by scopolamine in the Morris water maze, novel object location and recognition tasks. It decreased acetylcholinesterase activity in the hippocampus and prefrontal cortex. It also decreased the level of malondialdehyde and increased the level of glutathione in the hippocampus. The results of this study show that G. dalenii ameliorates the cognitive impairment induced by scopolamine, through inhibition of oxidative stress and enhancement of cholinergic neurotransmission. It can therefore be useful for treatment of conditions associated with memory dysfunction as seen in dementia.

Ablation of ErbB4 from excitatory neurons leads to reduced dendritic spine density in mouse prefrontal cortex.

Dendritic spine loss is observed in many psychiatric disorders, including schizophrenia, and likely contributes to the altered sense of reality, disruption of working memory, and attention deficits that characterize these disorders. ErbB4, a member of the EGF family of receptor tyrosine kinases, is genetically associated with schizophrenia, suggesting that alterations in ErbB4 function contribute to the disease pathology. Additionally, ErbB4 functions in synaptic plasticity, leading us to hypothesize that disruption of ErbB4 signaling may affect dendritic spine development. We show that dendritic spine density is reduced in the dorsomedial prefrontal cortex of ErbB4 conditional whole-brain knockout mice. We find that ErbB4 localizes to dendritic spines of excitatory neurons in cortical neuronal cultures and is present in synaptic plasma membrane preparations. Finally, we demonstrate that selective ablation of ErbB4 from excitatory neurons leads to a decrease in the proportion of mature spines and an overall reduction in dendritic spine density in the prefrontal cortex of weanling (P21) mice that persists at 2 months of age. These results suggest that ErbB4 signaling in excitatory pyramidal cells is critical for the proper formation and maintenance of dendritic spines in excitatory pyramidal cells.

GABAergic precursor transplantation into the prefrontal cortex prevents phencyclidine-induced cognitive deficits.

Phencyclidine (PCP) is a noncompetitive NMDA receptor antagonist, and it induces schizophreniform cognitive deficits in healthy humans and similar cognitive deficits in rodents. Although the PCP-induced cognitive deficits appear to be accompanied and possibly caused by dysfunction of GABAergic inhibitory interneurons in the prefrontal cortex (PFC), the potential benefit(s) of GABAergic interneuron manipulations on PCP-induced cognitive deficits remains unexplored. In this study we show that when embryonic medial ganglionic eminence (MGE) cells, many of which differentiate into cortical GABAergic interneurons in situ, were grafted into the medial PFC (mPFC) of neonatal mice, they differentiated into a specific class of GABAergic interneurons and became functionally integrated into the host neuronal circuitry in adults. Prior MGE cell transplantation into the mPFC significantly prevented the induction of cognitive and sensory-motor gating deficits by PCP. The preventive effects were not reproduced by either transplantation of cortical projection neuron precursors into the mPFC or transplantation of MGE cells into the occipital cortex. The preventive effects of MGE cell transplantation into the mPFC were accompanied by activation of callosal projection neurons in the mPFC. These findings suggest that increasing GABAergic interneuron precursors in the PFC may contribute to the development of a cell-based approach as a novel means of modulating the PFC neuronal circuitry and preventing schizophreniform cognitive deficits.

Alterations induced by chronic lead exposure on the cells of circadian pacemaker of developing rats.

Lead (Pb) exposure alters the temporal organization of several physiological and behavioural processes in which the suprachiasmatic nucleus (SCN) of the hypothalamus plays a fundamental role. In this study, we evaluated the effects of chronic early Pb exposure (CePbe) on the morphology, cellular density and relative optical density (OD) in the cells of the SCN of male rats. Female Wistar rats were exposed during gestation and lactation to a Pb solution containing 320 ppm of Pb acetate through drinking water. After weaning, the pups were maintained with the same drinking water until sacrificed at 90 days of age. Pb levels in the blood, hypothalamus, hippocampus and prefrontal cortex were significantly increased in the experimental group. Chronic early Pb exposure induced a significant increase in the minor and major axes and somatic area of vasoactive intestinal polypeptide (VIP)- and vasopressin (VP)-immunoreactive neurons. The density of VIP-, VP- and glial fibrillary acidic protein (GFAP)-immunoreactive cells showed a significant decrease in the experimental group. OD analysis showed a significant increase in VIP neurons of the experimental group. The results showed that CePbe induced alterations in the cells of the SCN, as evidenced by modifications in soma morphology, cellular density and OD in circadian pacemaker cells. These findings provide a morphological and cellular basis for deficits in circadian rhythms documented in Pb-exposed animals.

Demethylation of specific Wnt/ß-catenin pathway genes and its upregulation in rat brain induced by prenatal valproate exposure.

Valproate (VPA) has been used for decades in the treatment of epilepsy and migraine. However, maternal administration of VPA during pregnancy increases susceptibility to autism spectrum disorders (ASDs) in the offspring. The aim of this study was to investigate the methylation modification and its effects on the activity of Wnt/ß-catenin pathway in the rat brain prenatally exposed to VPA. We exposed the rats in early pregnancy to VPA and found that the prenatal VPA exposure, in comparison with the prenatal vehicle exposure, induced demethylation in the promoter regions of wnt1 and wnt2, but not in those of Wnt inhibitory factor-1 and Dickkopf 1, in the prefrontal cortexes and hippocampi of the offspring. Consequently, both mRNA and protein expression of wnt1 and wnt2 were increased. Furthermore, the activity of Wnt/ß-catenin pathway was upregulated, as indicated by the increased levels of ß-catenin, hence the growing expression of its target genes. This work suggested an epigenetic action via which VPA, when administered in early pregnancy, induced dysregulation of signaling pathway, further facilitating susceptibility to ASDs.

Developmental regulation of neural cell adhesion molecule in human prefrontal cortex.

Neural cell adhesion molecule (NCAM) is a membrane-bound cell recognition molecule that exerts important functions in normal neurodevelopment including cell migration, neurite outgrowth, axon fasciculation, and synaptic plasticity. Alternative splicing of NCAM mRNA generates three main protein isoforms: NCAM-180, -140, and -120. Ectodomain shedding of NCAM isoforms can produce an extracellular 105-115 kilodalton soluble neural cell adhesion molecule fragment (NCAM-EC) and a smaller intracellular cytoplasmic fragment (NCAM-IC). NCAM also undergoes a unique post-translational modification in brain by the addition of polysialic acid (PSA)-NCAM. Interestingly, both PSA-NCAM and NCAM-EC have been implicated in the pathophysiology of schizophrenia. The developmental expression patterns of the main NCAM isoforms and PSA-NCAM have been described in rodent brain, but no studies have examined NCAM expression across human cortical development. Western blotting was used to quantify NCAM in human postmortem prefrontal cortex in 42 individuals ranging in age from mid-gestation to early adulthood. Each NCAM isoform (NCAM-180, -140, and -120), post-translational modification (PSA-NCAM) and cleavage fragment (NCAM-EC and NCAM-IC) demonstrated developmental regulation in frontal cortex. NCAM-180, -140, and -120, as well as PSA-NCAM, and NCAM-IC all showed strong developmental regulation during fetal and early postnatal ages, consistent with their identified roles in axon growth and plasticity. NCAM-EC demonstrated a more gradual increase from the early postnatal period to reach a plateau by early adolescence, potentially implicating involvement in later developmental processes. In summary, this study implicates the major NCAM isoforms, PSA-NCAM and proteolytically cleaved NCAM in pre- and postnatal development of the human prefrontal cortex. These data provide new insights on human cortical development and also provide a basis for how altered NCAM signaling during specific developmental intervals could affect synaptic connectivity and circuit formation, and thereby contribute to neurodevelopmental disorders.

[Effects of heroin exposure on the expression of caspase 3 in prefrontal lobe cortex, hippocampus and nucleus accumbens].

AIM: To investigate the expression of caspase 3 in the brain regions related to addiction, learning and memory in mice prenatally exposed to heroin and to ascertain whether postnatal apoptotic mechanism participates in neurobehavioral teratogenicity induced by maternal heroin abuse. METHODS: A mouse model was established by administration of diacetylmorphine (heroin, purity 98.5%, product ID No.171206-200614) 10 mg/(kg x d) subcutaneously to pregnant BALB/c mice on embryonic day (E)E8-E18. The offspring were divided into heroin(Her) and saline(Sal) groups according to the maternal treatment. The expression of caspase 3 in prefrontal lobe cortex(PFC), hippocampus(HP) and nucleus accumbens(Acb) was detected by RT-PCR and Western blot on mouse postnatal day(P)14. RESULTS: The mRNA and protein expression of caspase 3 were significantly increased in the areas of PFC, HP and Acb in Her group compared with Sal group(P < 0.05). CONCLUSION: E8-E18 prenatal exposure to heroin can induce apoptosis through caspase 3 activation in brain regions related to addiction, learning and memory, which indicates that apoptotic mechanism may be involved in neurobehavioral teratogenicity by heroin exposure in uterus.

Developmental regulation of GABAergic interneuron branching and synaptic development in the prefrontal cortex by soluble neural cell adhesion molecule.

Neural cell adhesion molecule, NCAM, is an important regulator of neuronal process outgrowth and synaptic plasticity. Transgenic mice that overexpress the soluble NCAM extracellular domain (NCAM-EC) have reduced GABAergic inhibitory and excitatory synapses, and altered behavioral phenotypes. Here, we examined the role of dysregulated NCAM shedding, modeled by overexpression of NCAM-EC, on development of GABAergic basket interneurons in the prefrontal cortex. NCAM-EC overexpression disrupted arborization of basket cells during the major period of axon/dendrite growth, resulting in decreased numbers of GAD65- and synaptophysin-positive perisomatic synapses. NCAM-EC transgenic protein interfered with interneuron branching during early postnatal stages when endogenous polysialylated (PSA) NCAM was converted to non-PSA isoforms. In cortical neuron cultures, soluble NCAM-EC acted as a dominant inhibitor of NCAM-dependent neurite branching and outgrowth. These findings suggested that excess soluble NCAM-EC reduces perisomatic innervation of cortical neurons by perturbing axonal/dendritic branching during cortical development.

Fetal alcohol exposure alters the induction of immediate early gene mRNA in the rat prefrontal cortex after an alternation task.

The present study examined fetal alcohol effects (FAE) on the induction of the immediate early genes (IEGs) c-fos, jun B, c-jun, and zif268 mRNAs in the prefrontal cortex, hippocampus, and other brain regions after testing in an alternation task. Subjects were female offspring of Sprague-Dawley rats fed either a 35% ethanol-derived calorie diet, pair-fed with sucrose, or control-fed with laboratory chow during the last week of gestation. At 75-85 days of age, rats were food-deprived and trained in a t-maze for food reward. Then rats were tested at 5-sec, 30-sec, or 60-sec delays on each of 6 days. On the day of killing, a subset of rats was tested at the 60-sec delay for 12 trials and killed 30 min after testing. The remaining animals were killed from their home cage and acted as controls. Expression of the four IEG mRNAs was examined in the brains of these animals using in situ hybridization. FAE rats showed a memory deficit at the 60-sec delay (p < 0.05), but not at the 0-sec or 30-sec delays. Testing in the alternation task induced a significant elevation of c-fos, c-jun, jun B, and zif268 mRNA expression in the prefrontal cortex, hippocampal subfields CA1 and CA3, and several cortical areas. However, FAE rats showed a significantly smaller elevation of both c-fos and jun B mRNA levels in the orbital, prelimbic, and anterior cingulate regions of the prefrontal cortex (p < 0.05). FAE animals also showed a lower expression of jun B mRNA in the caudate nucleus. Significant correlations between the mean performance at the 60-sec delay and mRNA expression of c-fos, jun B, and zif268 in the prefrontal cortical regions (p < 0.05) were observed. These findings suggest that fetal alcohol exposure produces changes in the adult prefrontal cortex that may contribute to the behavioral deficit in the alternation task.

Accelerated resensitization of the D1 dopamine receptor-mediated response in cultured cortical and striatal neurons from the rat: respective role of alpha 1-adrenergic and N-methyl-D-aspartate receptors.

As previously shown in vivo, noradrenergic and glutamatergic neurons can regulate the denervation supersensitivity of D1 dopaminergic (DA) receptors in the rat prefrontal cortex and striatum respectively. Therefore, the effects of methoxamine (an alpha 1-adrenergic agonist) and glutamate on the resensitization of D1 DA receptors were investigated in cultured cortical and striatal neurons from the embryonic rat. In the presence of sulpiride and propranolol, DA stimulated the D1 DA receptor-mediated conversion of 3H-adenine into 3H-cAMP in both intact cortical and striatal cells and these responses were markedly desensitized in cells preexposed for 15 min to DA (50 microM). The complete recovery of the D1 DA response was more rapid in striatal (15 min) than in cortical (80 min) neurons. Methoxamine accelerated the resensitization of the D1 response in cortical but not in striatal neurons. The effect of the alpha 1-adrenergic agonist in cortical neurons was blocked by prazosin and chlorethylclonidine. In contrast, glutamate accelerated the resensitization of the D1 response in striatal but not in cortical neurons and the effect observed in striatal neurons was totally blocked by 2-amino-5-phosphonovaleric acid, an NMDA receptor antagonist. Protein kinase C was shown to be involved in the alpha 1-adrenergic-induced resensitization of the cortical D1 response but not in the glutamate-evoked resensitization of the striatal D1 response. Finally, for comparison, similar experiments were performed on beta-adrenergic receptors using isoproterenol (1 microM) as an agonist. Methoxamine did not modify the resensitization of the beta-adrenergic response in cortical neurons, but glutamate accelerated the resensitization of this response in striatal neurons.