Calretinin-positive Interneurons Research Articles

In utero alcohol exposure impairs retinal angiogenesis and the microvessel-associated positioning of calretinin interneurons.

In addition to brain disorders, which constitute a devastating consequence of prenatal alcohol exposure (PAE), eye development is also significantly affected. Given that the retina is a readily accessible part of the central nervous system, a better understanding of the impact of ethanol on retinal development might provide ophthalmological landmarks helpful for early diagnosis of foetal alcohol syndrome. This study aimed to provide a fine morphometric and cellular characterization of the development of retinal microvasculature and neurovascular interactions in a mouse model of foetal alcohol spectrum disorder. The data revealed that PAE impaired superficial vascular plexus development. In particular, progression of the vascular migration front was significantly decreased in PAE retinas, supporting a delay in plexus progression. Moreover, a significant decrease in the vessel density and number of perforating vessels was quantified in PAE mice, supporting less angiogenesis. The present study provides also the first evidence of a close interaction between migrating calretinin-positive interneurons and perforating microvessels in the inner nuclear layer of the developing retina. This neurovascular association was significantly impaired by PAE. Moreover, projections of amacrine cells were abnormally distributed and densified in stratum S1 and S2. In humans, comparison of a 5-month-old control infant with a 3-month-old alcohol-exposed case revealed a similar mispositioning of calretinin-positive interneurons. This opens new research avenues regarding a neurovascular contribution in the deleterious effects of alcohol in the developing retina and support that ophthalmological examination could become a promising approach for early detection of alcohol-exposed infants presenting with neurovascular brain defects.Significance StatementIn the developing brain, prenatal alcohol exposure (PAE) has been shown to disorganize cortical vasculature leading to defective layering of interneurons which use radial microvessels to enter the neocortex. Using a murine model of FASD, here we show that PAE impairs the retinal microvasculature development and neuronal organization. In particular, migrating calretinin-positive interneurons of the internal nuclear layer are associated with perforating microvessels. PAE reduces the number of vessel-associated calretininergic cells and impairs their positioning. The mispositioning of calretinin-positive interneurons is also observed in a human FAS infant. These findings provide new evidence that PAE-induced cortical impairments are found in retina. They offer promising tools for the early diagnosis of brain impairments in infants in utero exposed to alcohol.

Expression of toll like receptor 8 (TLR8) in specific groups of mouse hippocampal interneurons.

Toll-like receptors (TLR) are one of the main constituents of the innate immune system in mammals. They can detect conserved microbial structures (pathogen-associated molecular patterns) and host-derived ligands that are produced during cellular stress and damage (danger-associated molecular patterns) and may then initiate an intracellular signaling cascade leading to the expression of pro-inflammatory cytokines and immediate immune responses. Some TLR (TLR1, 2, 4, 5, and 6) are expressed on the cell surface while others (TLR3, 7, 8 and 9) are present on the surface of endosomes and their ligands require internalization before recognition is possible. Several TLR have also been detected in neurons where they may serve functions that are not related to immune responses. TLR2, 3, and 4 have been described in cortical neurons and, for TLR4, a seizure-promoting role in epilepsies associated with inflammation has been shown. TLR3, 7, and 8 expressed in neurons seem to influence the growth or withdrawal of neurites and robust activation of TLR8 in neurons may even induce neuronal death. The goal of the current study was to investigate the expression of TLR8 in the hippocampus of mice during postnatal development and in adulthood. We focused on three functionally distinct groups of GABAergic interneurons characterized by the expression of the molecular markers parvalbumin, somatostatin, or calretinin, and we applied double fluorescence immunohistochemistry and cell counts to quantify co-expression of TLR8 in the three groups of GABA-interneurons across hippocampal subregions. We found subregion-specific differences in the expression of TLR8 in these interneurons. During postnatal development, TLR8 was detected only in mice older than P5. While only a small fraction of hippocampal calretinin-positive interneurons expressed TLR8, most parvalbumin-positive interneurons in all hippocampal subregions co-expressed TLR8. Somatostatin-positive interneurons co-expressing TLR8 were mainly present in hippocampal sector CA3 but rare in the dentate gyrus and CA1. High expression of TLR8 in parvalbumin-interneurons may contribute to their high vulnerability in human temporal lobe epilepsy.

The innate immune system stimulating cytokine GM-CSF improves learning/memory and interneuron and astrocyte brain pathology in Dp16 Down syndrome mice and improves learning/memory in wild-type mice

Down syndrome (DS) is characterized by chronic neuroinflammation, peripheral inflammation, astrogliosis, imbalanced excitatory/inhibitory neuronal function, and cognitive deficits in both humans and mouse models. Suppression of inflammation has been proposed as a therapeutic approach to treating DS co-morbidities, including intellectual disability (DS/ID). Conversely, we discovered previously that treatment with the innate immune system stimulating cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which has both pro- and anti-inflammatory activities, improved cognition and reduced brain pathology in a mouse model of Alzheimer's disease (AD), another inflammatory disorder, and improved cognition and reduced biomarkers of brain pathology in a phase II trial of humans with mild-to-moderate AD. To investigate the effects of GM-CSF treatment on DS/ID in the absence of AD, we assessed behavior and brain pathology in 12–14 month-old DS mice (Dp[16]1Yey) and their wild-type (WT) littermates, neither of which develop amyloid, and found that subcutaneous GM-CSF treatment (5 μg/day, five days/week, for five weeks) improved performance in the radial arm water maze in both Dp16 and WT mice compared to placebo. Dp16 mice also showed abnormal astrocyte morphology, increased percent area of GFAP staining in the hippocampus, clustering of astrocytes in the hippocampus, and reduced numbers of calretinin-positive interneurons in the entorhinal cortex and subiculum, and all of these brain pathologies were improved by GM-CSF treatment. These findings suggest that stimulating and/or modulating inflammation and the innate immune system with GM-CSF treatment may enhance cognition in both people with DS/ID and in the typical aging population.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb.

Role of serotonin in modulating the development and function of adult-born neurons in the olfactory bulb.

Reelin restricts dendritic growth of interneurons in the neocortex.

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.

Brain-Derived Neurotrophic Factor Val66Met polymorphism interacts with adolescent stress to alter hippocampal interneuron density and dendritic morphology in mice

Brain-derived neurotrophic factor (BDNF) plays essential roles in GABAergic interneuron development. The common BDNF val66met polymorphism, leads to decreased activity-dependent release of BDNF. The current study used a humanized mouse model of the BDNF val66met polymorphism to determine how reduced activity-dependent release of BDNF, both on its own, and in combination with chronic adolescent stress hormone, impact hippocampal GABAergic interneuron cell density and dendrite morphology.Male and female Val/Val and Met/Met mice were exposed to corticosterone (CORT) or placebo in their drinking water from weeks 6–8, before brains were perfuse-fixed at 15 weeks. Cell density and dendrite morphology of immunofluorescent labelled inhibitory interneurons; somatostatin, parvalbumin and calretinin in the CA1, and 3 and dentate gyrus (DG) across the dorsal (DHP) and ventral hippocampus (VHP) were assessed by confocal z-stack imaging, and IMARIS dendritic mapping software.Mice with the Met/Met genotype showed significantly lower somatostatin cell density compared to Val/Val controls in the DHP, and altered somatostatin interneuron dendrite morphology including branch depth, and spine density. Parvalbumin-positive interneurons were unchanged between genotype groups, however BDNF val66met genotype influenced the dendritic volume, branch level and spine density of parvalbumin interneurons differentially across hippocampal subregions. Contrary to this, no such effects were observed for calretinin-positive interneurons. Adolescent exposure to CORT treatment also significantly altered somatostatin and parvalbumin dendrite branch level and the combined effect of Met/Met genotype and CORT treatment significantly reduced somatostatin and parvalbumin dendrite spine density.In sum, the BDNFVal66Met polymorphism significantly alters somatostatin and parvalbumin-positive interneuron cell development and dendrite morphology. Additionally, we also report a compounding effect of the Met/Met genotype and chronic adolescent CORT treatment on dendrite spine density, indicating that adolescence is a sensitive period of risk for Val66Met polymorphism carriers.

In utero alcohol exposure exacerbates endothelial protease activity from pial microvessels and impairs GABA interneuron positioning

In utero alcohol exposure can induce severe neurodevelopmental disabilities leading to long-term behavioral deficits. Because alcohol induces brain defects, many studies have focused on nervous cells. However, recent reports have shown that alcohol markedly affects cortical angiogenesis in both animal models and infants with fetal alcohol spectrum disorder (FASD). In addition, the vascular system is known to contribute to controlling gamma-aminobutyric acid (GABA)ergic interneuron migration in the developing neocortex. Thus, alcohol-induced vascular dysfunction may contribute to the neurodevelopmental defects in FASD. The present study aimed at investigating the effects of alcohol on endothelial activity of pial microvessels. Ex vivo experiments on cortical slices from mouse neonates revealed that in endothelial cells from pial microvessels acute alcohol exposure inhibits both glutamate-induced calcium mobilization and activities of matrix metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA). The inhibitory effect of alcohol on glutamate-induced MMP-9 activity was abrogated in tPA-knockout and Grin1flox/VeCadcre mice suggesting that alcohol interacts through the endothelial NMDAR/tPA/MMP-9 vascular pathway. Contrasting with the effects from acute alcohol exposure, in mouse neonates exposed to alcohol in utero during the last gestational week, glutamate exacerbated both calcium mobilization and endothelial protease activities from pial microvessels. This alcohol-induced vascular dysfunction was associated with strong overexpression of the N-methyl-d-aspartate receptor subunit GluN1 and mispositioning of the Gad67-GFP interneurons that normally populate the superficial cortical layers. By comparing several human control fetuses with a fetus chronically exposed to alcohol revealed that alcohol exposure led to mispositioning of the calretinin-positive interneurons, whose density was decreased in the superficial cortical layers II-III and increased in deepest layers. This study provides the first mechanistic and functional evidence that alcohol impairs glutamate-regulated activity of pial microvessels. Endothelial dysfunction is characterized by altered metalloproteinase activity and interneuron mispositioning, which was also observed in a fetus with fetal alcohol syndrome. These data suggest that alcohol-induced endothelial dysfunction may contribute in ectopic cortical GABAergic interneurons, that has previously been described in infants with FASD.

Neuroprotective effect of regular swimming exercise on calretinin-positive striatal neurons of Parkinsonian rats.

In Parkinson's disease (PD), physical therapy is one of the mainstays of supportive treatment modalities. This study focused on the neuroprotective effect of regular exercise on striatal calretinin positive interneurons in a rat model of PD. 6-hydroxydopamine (6-OHDA) was injected unilaterally into the medial forebrain bundle of Wistar rats. 6-OHDA lesioned (Parkinsonian) and unlesioned (control) rats were divided into sedentary and exercise groups. Exercise groups had daily swimming sessions for 30min for 6 weeks. After 6-OHDA injections, an apomorphine-induced rotation test was performed (0.05mg/kg, subcutaneous) at the 3rd and 6th weeks. At the end of the 6th week, brains were removed following transcardiac perfusion. The brain sections were stained immunohistochemically for tyrosine hydroxylase and calretinin reactivity. The number of rotations was significantly lower in Parkinsonian exercise group compared to Parkinsonian sedentary group at the 6th week (p = 0.024) and there was significant difference between Parkisonian sedentary groups at the 3rd and 6th weeks (p < 0.002). The calretinin positive interneurons significantly increased in the Parkinsonian exercise group compared to Parkinsonian sedentary group (p = 0.0003) and control exercise group (p < 0.0001). To conclude, the swimming exercise led to a striking increment of calretinin positive interneurons in the striatum of Parkinsonian rat. These findings indicated that the neuroprotective mechanism of exercise increased the number of striatal calretinin positive interneurons that might generate new approaches for the mechanism of neuroprotection. We concluded that striatal calretinin positive interneurons have an important role in the neuroprotective mechanisms of exercise in PD.

Dentate gyrus neurogenesis across different ages in male rats: an immunohistochemical approach

Dentate gyrus is a fundamental sub-region of the hippocampus which is directly engaged in higher memory and cognitive functions. This study was performed to describe the histological and immunohistochemical changes in dentate gyrus in experimental animals during postnatal development. Forty four male albino rats were classified into four equal groups: new born group aged one day, adult group aged 3–6 months, early senile group aged 18–20 months and late senile group aged 30–31 months. Specimens of hippocampus were processed and prepared for routine hematoxylin and eosin stains and immunohistochemical expressions of calretinin, glial fibrillary acidic protein and Ki67 (Kiel 67). Morphometric data were statistically analyzed. The present results showed significant reduction in thickness of granule cell layer of late senile group. Interestingly, the mean number of immature neurons was significantly increased in early senile group, while it was significantly reduced in late senile group. The number of mature granule cells showed marked reduction in both early and late senile groups. Furthermore, the number of astrocytes and optical density of glial fibrillary acidic protein revealed significant age-dependent increase. Measurable difference in number of calretinin positive interneurons was detected between adult and senile groups. However, mean number of Ki67 immune positive nuclei expressed significant age-dependent reduction. This study concluded that interneurons play a substantial role in modulating dentate gyrus neurogenesis which occurs throughout life and steadily decreases during aging. It is recommended to focus on the different stimuli and factors that potentiate neurogenesis to prevent or treat cognitive deficiencies associated with aging.

Age, but Not Amyloidosis, Induced Changes in Global Levels of Histone Modifications in Susceptible and Disease-Resistant Neurons in Alzheimer's Disease Model Mice.

There is increasing interest in the role of epigenetic alterations in Alzheimer’s disease (AD). The epigenome of every cell type is distinct, yet data regarding epigenetic change in specific cell types in aging and AD is limited. We investigated histone tail modifications in neuronal subtypes in wild-type and APP/PS1 mice at 3 (pre-pathology), 6 (pathology-onset) and 12 (pathology-rich) months of age. In neurofilament (NF)-positive pyramidal neurons (vulnerable to AD pathology), and in calretinin-labeled interneurons (resistant to AD pathology) there were no global alterations in histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 27 acetylation (H3K27ac) or histone 3 lysine 27 trimethylation (H3K27me3) in APP/PS1 compared to wild-type mice at any age. Interestingly, age-related changes in the presence of H3K27ac and H3K27me3 were detected in NF-labeled pyramidal neurons and calretinin-positive interneurons, respectively. These data suggest that the global levels of histone modifications change with age, whilst amyloid plaque deposition and its sequelae do not result in global alterations of H3K4me3, H3K27ac and H3K27me3 in NF-positive pyramidal neurons or calretinin-labeled interneurons.

Differential and Overlapping Pattern of Foxp1 and Foxp2 Expression in the Striatum of Adult Mouse Brain

Genetic mutations of FOXP1 and FOXP2 are associated with neurodevelopmental diseases. It is important to characterize the cell types that express Foxp1 and Foxp2 in the brain. Foxp1 and Foxp2 are expressed at high levels in the striatum of mouse brains. There are two populations of striatal projection neurons (SPNs), dopamine D1 receptor (D1R)-expressing striatonigral neurons and D2 receptor (D2R)-expressing striatopallidal neurons. In addition to SPNs, there are different types of striatal interneurons. Here, we quantitatively analyze the expression pattern of Foxp1 and Foxp2 with respect to specific cell types of projection neurons and interneurons in the striatum of adult mouse brains. Double immunostaining and in situ hybridization showed that Foxp1 and Foxp2 were specifically expressed in SPNs, but not in interneurons. For Foxp1, 50–57% of Foxp1-positive neurons co-expressed D1R mRNA, and 45–52% of Foxp1-positive neurons co-expressed D2R mRNA in the striatum at rostrocaudal levels. For Foxp2, 65–77% of Foxp2-positive neurons co-expressed D1R mRNA, and 21–26% of Foxp2-positive neurons co-expressed D2R mRNA in the striatum at rostrocaudal levels. Neither Foxp1 nor Foxp2 was found to co-localize with parvalbumin, somatostatin, nNOS, calretinin and ChAT in interneurons of the striatum. Moreover, none of parvalbumin-, somatostatin-, nNOS-, and calretinin-positive interneurons co-expressed Foxp1 or Foxp2 in the cerebral cortex. As Foxp1 and Foxp2 can form heterodimers for transcriptional regulation, the differential and overlapping expression pattern of Foxp1 and Foxp2 in SPNs implicates coordinate and distinct roles of Foxp1 and Foxp2 in developmental construction and physiologic functions of striatal circuits in the brain.

Two types of somatostatin-expressing GABAergic interneurons in the superficial layers of the mouse cingulate cortex.

Somatostatin-expressing (SOM+), inhibitory interneurons represent a heterogeneous group of cells and given their remarkable diversity, classification of SOM+ interneurons remains a challenging task. Electrophysiological, morphological and neurochemical classes of SOM+ interneurons have been proposed in the past but it remains unclear as to what extent these classes are congruent. We performed whole-cell patch-clamp recordings from 127 GFP-labeled SOM+ interneurons ('GIN') of the superficial cingulate cortex with subsequent biocytin-filling and immunocytochemical labeling. Principal component analysis followed by k-means clustering predicted two putative subtypes of SOM+ interneurons, which we designated as group I and group II GIN. A key finding of our study is the fact that these electrophysiologically and morphologically distinct groups of SOM+ interneurons can be correlated with two neurochemical subtypes of SOM+ interneurons described recently in our laboratory. In particular, all SOM+ interneurons expressing calbindin but no calretinin could be classified as group I GIN, whereas all but one neuropeptide Y- and calretinin-positive interneurons were found in group II.

Mild Traumatic Brain Injury Leads to Decreased Inhibition and a Differential Response of Calretinin Positive Interneurons in the Injured Cortex.

It is clear that even mild forms of traumatic brain injury (TBI) can have lasting cognitive effects; however, the specific cellular changes responsible for the functional deficits remain poorly understood. Previous studies suggest that not all neurons respond in the same way and that changes to neuronal architecture may be subtype specific. The current study aimed to characterize the response of interneurons to TBI. To model TBI in vitro, the neurites of primary cortical neurons were transected at 15 days in vitro. In response, calretinin+ interneurons underwent significant neurite remodeling around the injury site. By examining the response of pyramidal neurons, GAD67-GFP+ interneurons, and calretinin+ interneurons to the injury, we found that this response was specific to the calretinin+ cells. To determine whether calretinin+ interneurons respond in this way to a clinically relevant in vivo model of mild diffuse and focal injury, we subjected mice to the lateral fluid percussion injury model. We found that calretinin+ interneuron density was unaltered by this mild injury, but consistent with our in vitro data, these neurons underwent morphological alterations in their dendrites. These alterations evolved over a 28-day period, and calretinin+ interneurons in the injured mice had a reduction in mean dendrite length and reduced number of secondary dendrites than those in the sham-injured controls by 7 days post-injury. Further, these structural alterations were accompanied by a reduction in the frequency of miniature inhibitory post-synaptic currents in layer V pyramidal neurons. These data suggest that even a mild TBI can lead to an overall change in the excitatory/inhibitory balance of the cortex that may play an important role in the longer-term behavioral pathology associated with mild TBI.

NCAM Regulates Inhibition and Excitability in Layer 2/3 Pyramidal Cells of Anterior Cingulate Cortex.

The neural cell adhesion molecule (NCAM), has been shown to be an obligate regulator of synaptic stability and pruning during critical periods of cortical maturation. However, the functional consequences of NCAM deletion on the organization of inhibitory circuits in cortex are not known. In vesicular gamma-amino butyric acid (GABA) transporter (VGAT)-channelrhodopsin2 (ChR2)-enhanced yellow fluorescent protein (EYFP) transgenic mice, NCAM is expressed postnatally at perisomatic synaptic puncta of EYFP-labeled parvalbumin, somatostatin and calretinin-positive interneurons, and in the neuropil in the anterior cingulate cortex (ACC). To investigate how NCAM deletion affects the spatial organization of inhibitory inputs to pyramidal cells, we used laser scanning photostimulation in brain slices of VGAT-ChR2-EYFP transgenic mice crossed to either NCAM-null or wild type (WT) mice. Laser scanning photostimulation revealed that NCAM deletion increased the strength of close-in inhibitory connections to layer 2/3 pyramidal cells of the ACC. In addition, in NCAM-null mice, the intrinsic excitability of pyramidal cells increased, whereas the intrinsic excitability of GABAergic interneurons did not change. The increase in inhibitory tone onto pyramidal cells, and the increased pyramidal cell excitability in NCAM-null mice will alter the delicate coordination of excitation and inhibition (E/I coordination) in the ACC, and may be a factor contributing to circuit dysfunction in diseases such as schizophrenia and bipolar disorder, in which NCAM has been implicated.

Characterization of focal cortical dysplasia with balloon cells by layer‐specific markers: Evidence for differential vulnerability of interneurons

Focal cortical dysplasia (FCD) is a major cause of pharmacoresistant focal epilepsy. Little is known about the pathomechanisms underlying the characteristic cytoarchitectural abnormalities associated with FCD. In the present study, a broad panel of markers identifying layer-specific neuron subpopulations was applied to characterize dyslamination and structural alterations in FCD with balloon cells (FCD 2b). Pan-neuronal neuronal nuclei (NeuN) and layer-specific protein expression (Reelin, Calbindin, Calretinin, SMI32 (nonphosphorylated neurofilament H), Parvalbumin, transducin-like enhancer protein 4 (TLE4), and Vimentin) was studied by immunohistochemistry on paraffin sections of FCD2b cases (n = 22) and was compared to two control groups with (n = 7) or without epilepsy (n = 4 postmortem cases). Total and layer-specific neuron densities were systematically quantified by cell counting considering age at surgery and brain region. We show that in FCD2b total neuron densities across all six cortical layers were not significantly different from controls. In addition, we present evidence that a basic laminar arrangement of layer-specific neuron subtypes was preserved despite the severe disturbance of cortical structure. SMI32-positive pyramidal neurons showed no significant difference in total numbers, but a reduction in layers III and V. The densities of supragranular Calbindin- and Calretinin-positive interneurons in layers II and III were not different from controls, whereas Parvalbumin-expressing interneurons, primarily located in layer IV, were significantly reduced in numbers when compared to control cases without epilepsy. In layer VI, the density of TLE4-positive projection neurons was significantly increased. Altogether, these data show that changes in cellular composition mainly affect deep cortical layers in FCD2b. The application of a broad panel of markers defining layer-specific neuronal subpopulations revealed that in FCD2b neuronal diversity and a basic laminar arrangement are maintained despite the severe disturbance of cytoarchitecture. Moreover, it showed that Parvalbumin-positive, inhibitory interneurons are highly vulnerable in contrast to other interneuron subtypes, possibly related to the epileptic condition.

Early morphological and functional changes in the GABAergic system of hippocampus in the rat lithium-pilocarpine model of epilepsy.

We studied early alterations in the GABAergic system of the rat hippocampus in the lithium-pilocarpine model of epilepsy. Twenty-four hours after the pilocarpine treatment, a decrease in the number of calretinin-positive interneurons was observed in the CA1 field of the hippocampus, whereas the number of parvalbumin-positive interneurons remained unchanged. The decreased levels of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) and the membrane GABA transporter GAT1 were revealed using Western blot analysis. These data indicate an altered excitation/inhibition balance in the hippocampus with excitation dominance.

GABAρ selective antagonist TPMPA partially inhibits GABA-mediated currents recorded from neurones and astrocytes in mouse striatum

The neostriatum plays a central role in motor coordination where nerve cells operate neuronal inhibition through GABAergic transmission. The neostriatum expresses a wide range of GABA-A subunits, including GABAρ1 and ρ2 which are restricted to a fraction of GABAergic interneurons and astrocytes. Spontaneous postsynaptic currents (sPSCs) evoked by 4-aminopyridine (4-AP) were recorded from neurones of the dorsal neostriatum, and their frequency was reduced > 50% by the selective GABAρ antagonist (1,2,5,6-Tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA). Additionally, we recorded GABA evoked currents from astrocytes in vitro and in situ. Astrocytes in vitro showed modulation by pentobarbital and desensitization upon consecutive applications of GABA. However, modulation by pentobarbital was absent and no significant desensitization was detected from astrocytes in situ. Moreover, TPMPA-sensitive GABA-currents that were insensitive to bicuculline were also recorded from astrocytes in situ, consistent with our previous study where GABAρ expression was demonstrated. Finally, we assessed the mRNA expression of GABAρ3, through different stages of postnatal development; double immunofluorescence disclosed GABAρ3 expression in calretinin-positive interneurons as well as in astrocytes (>70%). These results add new information about the participation of GABAρ subunits in neostriatal interneurons and astrocytes.

NCX1 Exchanger Cooperates with Calretinin to Confer Preconditioning-Induced Tolerance Against Cerebral Ischemia in the Striatum.

Recently, the Na(+)/Ca(+2) exchanger NCX1 and the calcium binding protein calretinin have emerged as new molecular effectors of delayed preconditioning in the brain. In the present study, we investigated whether NCX1 and calretinin cooperate within the preconditioned striatum to confer neurons greater resistance to degeneration. Confocal microscopy analysis revealed that NCX1 expression was upregulated in calretinin-positive interneurons in the rat striatum after tolerance induction. Consistently, coimmunoprecipitation assays performed on human SHSY-5Y cells, a neuronal cell line which constitutively expresses calretinin, revealed a binding between NCX1 and calretinin. Finally, silencing of calretinin expression, both in vitro and in vivo, significantly prevented preconditioning-induced neuroprotection. Interestingly, our biochemical and functional studies showed that the selective silencing of calretinin in brain cells significantly prevented not only the preconditioning-induced upregulation of NCX1 expression and activity but also the activation of the prosurvival protein kinase Akt, which is involved in calretinin and NCX1 protective actions. Collectively, our results indicate that the Na(+)/Ca(+2) exchanger NCX1 and the calcium binding protein calretinin cooperate within the striatum to confer tolerance against cerebral ischemia.

Cortical Abnormalities and Non-Spatial Learning Deficits in a Mouse Model of CranioFrontoNasal Syndrome

Eph receptors and their ephrin ligands play critical roles in the development of the nervous system, however, less is known about their functions in the adult brain. Here, we investigated the function of ephrinB1, an ephrinB family member that is mutated in CranioFrontoNasal Syndrome. We show that ephrinB1 deficient mice (EfnB1Y/−) demonstrate spared spatial learning and memory but exhibit exclusive impairment in non-spatial learning and memory tasks. We established that ephrinB1 does not control learning and memory through direct modulation of synaptic plasticity in adults, since it is not expressed in the adult brain. Rather we show that the cortex of EfnB1Y/− mice displayed supernumerary neurons, with a particular increase in calretinin-positive interneurons. Further, the increased neuron number in EfnB1Y/− mutants correlated with shorter dendritic arborization and decreased spine densities of cortical pyramidal neurons. Our findings indicate that ephrinB1 plays an important role in cortical maturation and that its loss has deleterious consequences on selective cognitive functions in the adult.

Pten Loss in Olig2 Expressing Neural Progenitor Cells and Oligodendrocytes Leads to Interneuron Dysplasia and Leukodystrophy

Therapeutic modulation of phosphatidylinositol 3-kinase (PI3K)/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this, we created Olig2-cre:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy, chronic neurodegeneration, and death by 9 months. In contrast, during immediate postnatal development, oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Pten(fl/fl) mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor, NVP-BKM120, blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary, our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However, such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development.