Structural plasticity in the entorhinal and perirhinal cortices following hippocampal lesions in rhesus monkeys.

Immature neurons expressing the Bcl2 protein are present in various regions of the mammalian brain, including the amygdala and the entorhinal and perirhinal cortices. Their functional role is unknown but we have previously shown that neonatal and adult hippocampal lesions increase their differentiation in the monkey amygdala. Here, we assessed whether hippocampal lesions similarly affect immature neurons in the entorhinal and perirhinal cortices. Since Bcl2-positive cells were found mainly in areas Eo, Er, and Elr of the entorhinal cortex and in layer II of the perirhinal cortex, we also used Nissl-stained sections to determine the number and soma size of immature and mature neurons in layer III of area Er and layer II of area 36 of the perirhinal cortex. We found different structural changes in these regions following hippocampal lesions, which were influenced by the time of the lesion. In neonate-lesioned monkeys, the number of immature neurons in the entorhinal and perirhinal cortices was generally higher than in controls. The number of mature neurons was also higher in layer III of area Er of neonate-lesioned monkeys but no differences were found in layer II of area 36. In adult-lesioned monkeys, the number of immature neurons in the entorhinal cortex was lower than in controls but did not differ from controls in the perirhinal cortex. The number of mature neurons in layer III of area Er did not differ from controls, but the number of small, mature neurons in layer II of area 36 was lower than in controls. In sum, hippocampal lesions impacted populations of mature and immature neurons in discrete regions and layers of the entorhinal and perirhinal cortices, which are interconnected with the amygdala and provide major cortical inputs to the hippocampus. These structural changes may contribute to some functional recovery following hippocampal injury in an age-dependent manner.

Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans.

Recently, a population of "immature" neurons generated prenatally, retaining immaturity for long periods and finally integrating in adult circuits has been described in the cerebral cortex. Moreover, comparative studies revealed differences in occurrence/rate of different forms of neurogenic plasticity across mammals, the "immature" neurons prevailing in gyrencephalic species. To extend experimentation from laboratory mice to large-brained mammals, including humans, it is important to detect cell markers of neurogenic plasticity in brain tissues obtained from different procedures (e.g., post-mortem/intraoperative specimens vs. intracardiac perfusion). This variability overlaps with species-specific differences in antigen distribution or antibody species specificity, making it difficult for proper comparison. In this work, we detect the presence of doublecortin and Ki67 antigen, markers for neuronal immaturity and cell division, in six mammals characterized by widely different brain size. We tested seven commercial antibodies in four selected brain regions known to host immature neurons (paleocortex, neocortex) and newly born neurons (hippocampus, subventricular zone). In selected human brains, we confirmed the specificity of DCX antibody by performing co-staining with fluorescent probe for DCX mRNA. Our results indicate that, in spite of various types of fixations, most differences were due to the use of different antibodies and the existence of real interspecies variation.

Brain Plasticity in Humans and Model Systems: Advances, Challenges, and Future Directions.

Plasticity, and in particular, neurogenesis, is a promising target to treat and prevent a wide variety of diseases (e.g., epilepsy, stroke, dementia). There are different types of plasticity, which vary with age, brain region, and species. These observations stress the importance of defining plasticity along temporal and spatial dimensions. We review recent studies focused on brain plasticity across the lifespan and in different species. One main theme to emerge from this work is that plasticity declines with age but that we have yet to map these different forms of plasticity across species. As part of this effort, we discuss our recent progress aimed to identify corresponding ages across species, and how this information can be used to map temporal variation in plasticity from model systems to humans.

CaMKIIα Expressing Neurons to Report Activity-Related Endogenous Hypoxia upon Motor-Cognitive Challenge.

We previously introduced the brain erythropoietin (EPO) circle as a model to explain the adaptive 'brain hardware upgrade' and enhanced performance. In this fundamental circle, brain cells, challenged by motor-cognitive tasks, experience functional hypoxia, triggering the expression of EPO among other genes. We attested hypoxic cells by a transgenic reporter approach under the ubiquitous CAG promoter, with Hif-1α oxygen-dependent degradation-domain (ODD) fused to CreERT2-recombinase. To specifically focus on the functional hypoxia of excitatory pyramidal neurons, here, we generated CaMKIIα-CreERT2-ODD::R26R-tdTomato mice. Behavioral challenges, light-sheet microscopy, immunohistochemistry, single-cell mRNA-seq, and neuronal cultures under normoxia or hypoxia served to portray these mice. Upon complex running wheel performance as the motor-cognitive task, a distinct increase in functional hypoxic neurons was assessed immunohistochemically and confirmed three-dimensionally. In contrast, fear conditioning as hippocampal stimulus was likely too short-lived to provoke neuronal hypoxia. Transcriptome data of hippocampus under normoxia versus inspiratory hypoxia revealed increases in CA1 CaMKIIα-neurons with an immature signature, characterized by the expression of Dcx, Tbr1, CaMKIIα, Tle4, and Zbtb20, and consistent with accelerated differentiation. The hypoxia reporter response was reproduced in vitro upon neuronal maturation. To conclude, task-associated activity triggers neuronal functional hypoxia as a local and brain-wide reaction mediating adaptive neuroplasticity. Hypoxia-induced genes such as EPO drive neuronal differentiation, brain maturation, and improved performance.

Toxic Effect of Streptozotocin on Cultured Mouse Hippocampal Neurons.

In primary dissociated hippocampal cell cultures from 18-day-old mouse embryos, streptozotocin in concentrations of 2-5 mM produced a dose-dependent cytotoxic effect on day 3 in vitro, whereas on day 11 of culturing, the neurons were resistant to streptozotocin. The neurons in the 3-day cultures were functionally immature, which was seen from their weak spontaneous bioelectric activity in the form of rare single action potentials; by day 11 of culturing, the neurons reached a high level of differentiation and their functional properties acquired a character of network burst activity. Thus, streptozotocin had the most pronounced cytotoxic effect on immature hippocampal neurons in vitro.

GABAergic Input Affects Intracellular Calcium Levels in Developing Granule Cells of Adult Rat Hippocampus.

In the dentate gyrus (DG) of the mammalian hippocampus, granule neurons are generated from neural stem cells (NSCs) throughout the life span and are integrated into the hippocampal network. Adult DG neurogenesis is regulated by multiple intrinsic and extrinsic factors that control NSC proliferation, maintenance, and differentiation into mature neurons. γ-Aminobutyric acid (GABA), released by local interneurons, regulates the development of neurons born in adulthood by activating extrasynaptic and synaptic GABAA receptors. In the present work, patch-clamp and calcium imaging techniques were used to record very immature granule cells of adult rat dentate gyrus for investigating the actual role of GABAA receptor activation in intracellular calcium level regulation at an early stage of maturation. Our findings highlight a novel molecular and electrophysiological mechanism, involving calcium-activated potassium channels (BK) and T-type voltage-dependent calcium channels, through which GABA fine-tunes intracellular calcium homeostasis in rat adult-born granule neurons early during their maturation. This mechanism might be instrumental in promoting newborn cell survival.

Non-Newly Generated, "Immature" Neurons in the Sheep Brain Are Not Restricted to Cerebral Cortex.

A newly proposed form of brain structural plasticity consists of non-newly generated, "immature" neurons of the adult cerebral cortex. Similar to newly generated neurons, these cells express the cytoskeletal protein Doublecortin (DCX), yet they are generated prenatally and then remain in a state of immaturity for long periods. In rodents, the immature neurons are restricted to the paleocortex, whereas in other mammals, they are also found in neocortex. Here, we analyzed the DCX-expressing cells in the whole sheep brain of both sexes to search for an indicator of structural plasticity at a cellular level in a relatively large-brained, long-living mammal. Brains from adult and newborn sheep (injected with BrdU and analyzed at different survival times) were processed for DCX, cell proliferation markers (Ki-67, BrdU), pallial/subpallial developmental origin (Tbr1, Sp8), and neuronal/glial antigens for phenotype characterization. We found immature-like neurons in the whole sheep cortex and in large populations of DCX-expressing cells within the external capsule and the surrounding gray matter (claustrum and amygdala). BrdU and Ki-67 detection at neonatal and adult ages showed that all of these DCX+ cells were generated during embryogenesis, not after birth. These results show that the adult sheep, unlike rodents, is largely endowed with non-newly generated neurons retaining immature features, suggesting that such plasticity might be particularly important in large-brained, long-living mammals.SIGNIFICANCE STATEMENT Brain plasticity is important in adaptation and brain repair. Structural changes span from synaptic plasticity to adult neurogenesis, the latter being highly reduced in large-brained, long-living mammals (e.g., humans). The cerebral cortex contains "immature" neurons, which are generated prenatally and then remain in an undifferentiated state for long periods, being detectable with markers of immaturity. We studied the distribution and developmental origin of these cells in the whole brain of sheep, relatively large-brained, long-living mammals. In addition to the expected cortical location, we also found populations of non-newly generated neurons in several subcortical regions (external capsule, claustrum, and amygdala). These results suggests that non-neurogenic, parenchymal structural plasticity might be more important in large mammals with respect to adult neurogenesis.

Activity-Independent Effects of CREB on Neuronal Survival and Differentiation during Mouse Cerebral Cortex Development.

Neuronal survival and morphological maturation depends on the action of the transcription factor calcium responsive element binding protein (CREB), which regulates expression of several target genes in an activity-dependent manner. However, it remains largely unknown whether CREB-mediated transcription could play a role at early stages of neuronal differentiation, prior to the establishment of functional synaptic contacts. Here, we show that CREB is phosphorylated at very early stages of neuronal differentiation in vivo and in vitro, even in the absence of depolarizing agents. Using genetic tools, we also show that inhibition of CREB-signaling affects neuronal growth and survival in vitro without affecting cell proliferation and neurogenesis. Expression of A-CREB or M-CREB, 2 dominant-negative inhibitors of CREB, decreases cell survival and the complexity of neuronal arborization. Similar changes are observed in neurons treated with protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors, which also show decreased levels of pCREBSer133. Notably, expression of CREB-FY, a Tyr134Phe CREB mutant with a lower Km for phosphorylation, partly rescues the effects of PKA and CaMKII inhibition. Our data indicate that CREB-mediated signaling play important roles at early stages of cortical neuron differentiation, prior to the establishment of fully functional synaptic contacts.

Streptozotocin toxicity in vitro depends on maturity of neurons.

Streptozotocin (STZ) is a glucosamine-nitrosourea compound that is particularly toxic to the insulin-producing beta-cells of the pancreas in mammals; it is used for experimental simulation of sporadic Alzheimer's disease by means of intracerebroventricular administration in vivo. Here we show that the application of 3-4 mM STZ to primary culture for 48 h induces neuronal death in immature (2-3 days in vitro) cultures of rat cerebellar granule cells. Mature cultures (7-8 days in vitro) were poorly sensitive to this toxic treatment. Immature cultures demonstrated a high expression of the protein PSA-NCAM, the marker of immature neurons, and they were insensitive to the toxic effect of glutamate. In mature cultures, this protein was poorly expressed, whereas neurons showed a very high sensitivity to the toxic effect of glutamate. Measurements of the concentration of intracellular free calcium ions ([Ca2+]i) showed that the STZ-induced [Ca2+]i increase in young neurons was six times higher than that in mature neurons. Our results show that STZ is very toxic for immature neurons and probably it can significantly impair neurogenesis.

Hippocampal neurogenesis in the C57BL/6J mice at early adulthood following prenatal alcohol exposure.

We examined the effect of chronic prenatal alcohol exposure (PAE) on the process of adult neurogenesis in C57BL/6J mice at early adulthood (PND 56). Pregnant mice, and their in utero litters, were exposed to alcohol, through oral gavage, on gestational days 7-16, with recorded blood alcohol concentrations averaging 184 mg/dL (CA group). Two control groups, sucrose (CAc) and non-treated (NTc) control groups were also examined. The brains of pups at PND 56 from each experimental group were sectioned in a sagittal plane, and stained for Nissl substance with cresyl violet, and immunostained for Ki-67 which labels proliferative cells and doublecortin (DCX) for immature neurons. Morphologically, the neurogenic pattern was identical in all three groups studied. Populations of Ki-67 immunopositive cells in the dentate gyrus were not statistically significantly different between the experimental groups and there were no differences between the sexes. Thus, the PAE in this study does not appear to have a strong effect on the proliferative process in the adult hippocampus. In contrast, the numbers of immature neurons, labeled with DCX, was statistically significantly lower in the prenatal alcohol exposed mice compared with the two control groups. Alcohol significantly lowered the number of DCX hippocampal cells in the male mice, but not in the female mice. This indicates that the PAE appears to lower the rate of conversion of proliferative cells to immature neurons and this effect of alcohol is sexually dimorphic. This lowered number of immature neurons in the hippocampus appears to mirror hippocampal dysfunctions observed in FASD children.