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Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5) P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5) P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ;1000 nm² rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1a was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons. [ABSTRACT FROM AUTHOR]

Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased upon activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits. [ABSTRACT FROM AUTHOR]

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels. [ABSTRACT FROM AUTHOR]

Neuronal activity has been identified as a key regulator of neuronal network development, but the impact of activity on migration and terminal positioning of interneuron subtypes is poorly understood. The absence of early subpopulation markers and the presence of intermingled migratory and postmigratory neurons make the developing cerebral cortex a difficult model to answer these questions. Postnatal neurogenesis in the subventricular zone (SVZ) offers a more accessible and compartmentalized model. Neural stem cells regionalized along the border of the lateral ventricle produce two main subtypes of neural progenitors, granule cells and periglomerular neurons that migrate tangentially in the rostral migratory stream (RMS) before migrating radially in the olfactory bulb (OB) layers. Here, we used targeted postnatal electroporation to compare the migration of these two populations in male and female mice. We do not observe any obvious differences regarding the mode of tangential or radial migration between these two subtypes. However, we find a striking increase of intrinsic calcium activity in granule cell precursors (GC-Ps) when they switch from tangential to radial migration. By decreasing neuronal excitability in GC-Ps, we find that neuronal activity has little effect on migration but is required for normal positioning and survival of GC-Ps in the OB layers. Strikingly, decreasing activity of periglomerular neuron precursors (PGN-Ps) did not impact their positioning or survival. Altogether these findings suggest that neuronal excitability plays a subtype specific role during the late stage of migration of postnatally born OB interneurons. [ABSTRACT FROM AUTHOR]

Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function. [ABSTRACT FROM AUTHOR]

The olfactory bulb (OB) serves as a relay region for sensory information transduced by receptor neurons in the nose and ultimately routed to a variety of cortical areas. Despite the highly structured organization of the sensory inputs to the OB, even simple monomolecular odors activate large regions of the OB comprising many glomerular modules defined by afferents from different receptor neuron subtypes. OB principal cells receive their primary excitatory input from only one glomerular channel defined by inputs from one class of olfactory receptor neurons. By contrast, interneurons, such as GABAergic granule cells (GCs), integrate across multiple channels through dendodendritic inputs on their distal apical dendrites. Through their inhibitory synaptic actions, GCs appear to modulate principal cell firing to enhance olfactory discrimination, although how GCs contribute to olfactory function is not well understood. In this study, we identify a second synaptic pathway by which principal cells in the rat (both sexes) OB excite GCs by evoking potent nondepressing EPSPs (termed large-amplitude, nondendrodendritic [LANDD] EPSPs). LANDD EPSPs show little depression in response to tetanic stimulation and, therefore, can be distinguished other EPSPs that target GCs. LANDD EPSPs can be evoked by both focal stimulation near GC proximal dendrites and by activating sensory inputs in the glomerular layer in truncated GCs lacking dendrodendritic inputs. Using computational simulations, we show that LANDD EPSPs more reliably encode the duration of principal cell discharges than DD EPSPs, enabling GCs to compare contrasting versions of odor-driven activity patterns. [ABSTRACT FROM AUTHOR]

Delineation of functional synaptic connections is fundamental to understanding sensory processing. Olfactory signals are synaptically processed initially in the olfactory bulb (OB) where neural circuits are formed among inhibitory' interneurons and the output neurons mitral cells (MCs) and tufted cells (TCs). TCs function in parallel with but differently from MCs and are further classified into multiple subpopulations based on their anatomic and functional heterogeneities. Here, we combined optogenetics with electrophysiology' to characterize the synaptic transmission from a subpopulation of TCs, which exclusively express the neuropeptide choleq'stokinin (CCK), to two groups of spatially segregated GABAergic interneurons, granule cells (GCs) and glomerular interneurons in mice of both sexes with four major findings. First, CCKergic TCs receive direct input from the olfactory sensory' neurons (OSNs). This monosynaptic transmission exhibits high fidelity in response to repetitive OSN input. Second, CCKergic TCs drive GCs through two functionally distinct types of monosynaptic connections: (1) dendrodendritic synapses onto GC distal dendrites via their lateral dendrites in the superficial external plexiform layer (EPL); (2) axodendritic synapses onto GC proximal dendrites via their axon collaterals or terminals in the internal plexiform layer (IPL) on both sides of each bulb. Third, CCKergic TCs monosynaptically excite two subpopulations of inhibitory' glomerular interneurons via dendrodendritic synapses. Finally, sniff-like patterned activation of CCKergic TCs induces robust frequency-dependent depression of the dendrodendritic synapses but facilitation of the axodendritic synapses. These results demonstrated important roles of the CCKergic TCs in olfactory processing by orchestrating OB inhibitory' activities. [ABSTRACT FROM AUTHOR]

The endocannabinoid system plays important roles in brain development, but mechanistic studies have focused on neuronal differentiation, migration, and synaptogenesis, with less attention to transcellular interactions that coordinate neurodevelopmental processes across developing neural networks. We determined that, in the developing rodent cerebellar cortex (of both sexes), there is a transient window when the dominant brain cannabinoid receptor, CB1R, is expressed on afferent terminals instead of output neuron Purkinje cell synapses that dominate the adult cerebellum. Activation of these afferent CBIRs suppresses synaptic transmission onto developing granule cells, and consequently also suppresses excitation of downstream neurons in the developing cortical network, including nonsynaptic, migrating neurons. Application of a CB1R antagonist during afferent stimulation trains and depolarizing voltage steps caused a significant, sustained potentiation of synaptic amplitude. Our data demonstrate that transiently expressed afferent CBIRs regulate afferent synaptic strength during synaptogenesis, which enables coordinated dampening of transcortical developmental signals. [ABSTRACT FROM AUTHOR]

In the mammalian olfactory bulb, the inhibitory axonless granule cells (GCs) feature reciprocal synapses that interconnect them with the principal neurons of the bulb, mitral, and tufted cells. These synapses are located within large excitable spines that can generate local action potentials (APs) upon synaptic input ("spine spike"). Moreover, GCs can fire global APs that propagate throughout the dendrite. Strikingly, local postsynaptic Ca2+ entry summates mostly linearly with Ca2+ entry due to coincident global APs generated by glomerular stimulation, although some underlying conductances should be inactivated. We investigated this phenomenon by constructing a compartmental GC model to simulate the pairing of local and global signals as a function of their temporal separation Δt These simulations yield strongly sublinear summation of spine Ca2+ entry for the case of perfect coincidence Δt = 0 ms. Summation efficiency (SE) sharply rises for both positive and negative Δt The SE reduction for coincident signals depends on the presence ofvoltage-gated Na+ channels in the spine head, while NMDARs are not essential. We experimentally validated the simulated SE in slices of juvenile rat brain (both sexes) by pairing two-photon uncaging of glutamate at spines and APs evoked by somatic current injection at various intervals Δt while imaging spine Ca2+ signals. Finally, the latencies of synaptically evoked global APs and EPSPs were found to correspond to Δt ≈10 ms, explaining the observed approximately linear summation of synaptic local and global signals. Our results provide additional evidence for the existence of the GC spine spike. [ABSTRACT FROM AUTHOR]

Aberrant branch formation of granule cell axons (mossy fiber sprouting) is observed in the dentate gyrus of many patients with temporal lobe epilepsy and in animal models of epilepsy. However, the mechanisms underlying mossy fiber sprouting remain elusive. Based on the hypothesis that seizure-mediated gene expression induces abnormal mossy fiber growth, we screened activity-regulated genes in the hippocampus and found that neuritin, an extracellular protein anchored to the cell surface, was rapidly upregulated after electroconvulsive seizures. Overexpression of neuritin in the cultured rat granule cells promoted their axonal branching. Also, kainic acid-dependent axonal branching was abolished in the cultured granule cells from neuritin knock-out mice, suggesting that neuritin may be involved in activity-dependent axonal branching. Moreover, neuritin knock-out mice showed less-severe seizures in chemical kindling probably by reduced mossy fiber sprouting and/or increased seizure resistance. We found that inhibition of the fibroblast growth factor (FGF) receptor attenuated the neuritin-dependent axonal branching. FGF administration also increased branching in granule neurons, whereas neuritin knock-out mice did not show FGF-dependent axonal branching. In addition, FGF and neuritin treatment enhanced the recruitment of FGF receptors to the cell surface. These findings suggest that neuritin and FGF cooperate in inducing mossy fiber sprouting through FGF signaling. Together, these results suggest that FGF and neuritin-mediated axonal branch induction are involved in the aggravation of epilepsy. [ABSTRACT FROM AUTHOR]

Dendrite development of newborn granule cells (GCs) in the dentate gyrus of adult hippocampus is critical for their incorporation into existing hippocampal circuits, but the cellular mechanisms regulating their dendrite development remains largely unclear. In this study, we examined the function of brain-derived neurotrophic factor (BDNF), which is expressed in adult-born GCs, in regulating their dendrite morphogenesis. Using retrovirus-mediated gene transfection, we found that deletion and overexpression of BDNF in adult-born GCs resulted in the reduction and elevation of dendrite growth, respectively. This effect was mainly due to the autocrine rather than paracrine action of BDNF, because deletion of BDNF only in the newborn GCs resulted in dendrite abnormality of these neurons to a similar extent as that observed in conditional knockout (cKO) mice with BDNF deleted in the entire forebrain. Furthermore, selective expression of BDNF in adult-born GCs in BDNF cKO mice fully restored normal dendrite development. The BDNF autocrine action was also required for the development of normal density of spines and normal percentage of spines containing the postsynaptic marker PSD-95, suggesting autocrine BDNF regulation of synaptogenesis. Furthermore, increased dendrite growth of adult-born GCs caused by voluntary exercise was abolished by BDNF deletion specifically in these neurons and elevated dendrite growth due to BDNF overexpression in these neurons was prevented by reducing neuronal activity with coexpression of inward rectifier potassium channels, consistent with activity-dependent autocrine BDNF secretion. Therefore, BDNF expressed in adult-born GCs plays a critical role in dendrite development by acting as an autocrine factor. [ABSTRACT FROM AUTHOR]

Adult neurogenesis is restricted to two distinct areas of the mammalian brain: the olfactory bulb (OB) and the dentate gyrus (DG). Despite its spatial restriction, adult neurogenesis is of crucial importance for sensory processing and learning and memory. Although it has been shown that tens of thousands of new neurons arrive in the OB and DG every day with about half of them surviving after integration, the total contribution of adult neurogenesis to the pre-existing network remains mostly unknown. This is because of previous approaches labeling only a small proportion of adult-generated neurons. Here, we used genetic fate mapping to follow the majority of adult-generated neurons over long periods. Our data demonstrate two distinct modes of neuron addition to the pre-existing network. In the glomerular layer of the OB, there is a constant net addition of adult-generated neurons reaching a third of the total neuronal population within 9 months. In contrast, adult neurogenesis contributes to only a minor fraction of the entire neuronal network in the granular cell layer of the OB and the DG. Although the fraction of adult generated neurons can be further increased by an enriched environment, it still remains a minority of the neuronal network in the DG. Thus, neuron addition is distinct and tightly regulated in the neuronal networks that incorporate new neurons life long. [ABSTRACT FROM AUTHOR]

Single-channel analysis revealed the existence of neuronal L-type Ca2+ channels (LTCCs) with fundamentally different gating properties; in addition to LTCCs resembling cardiac channels, LTCCs with anomalous gating were identified in a variety of neurons, including cerebellar granule cells. Anomalous LTCC gating is mainly characterized by long reopenings after repolarization following strong depolarizations or trains of action potentials. To elucidate the unknown molecular nature of anomalous LTCCs, we performed single-channel patch-clamp recordings from cerebellar granule cells of wild-type, Cav1.3-/- and Cav1.2DHP-/- [containing a mutation in the Cav1.2 α1 subunit that eliminates dihydropyridine (DHP) sensitivity] mice. Quantitative reverse transcription-PCR revealed that Cav1.2 accounts for 89% and Cav1.3 for 11% of the LTCC transcripts in wild-type cerebellar granule cells, whereas Cav1.1 and Cav1.4 are expressed at insignificant levels. Anomalous LTCCs were observed in neurons of Cav1.3-/- mice with a frequency not different from wild type. In the presence of the DHP agonist (+)-(S)-202-791, the typical prepulse-induced reopenings of anomalous LTCCs after repolarization were shorter in Cav1.2DHP-/- neurons than in Cav1.3-/- neurons. Reopenings in Cav1.2DHP-/- neurons in the presence of the DHP agonist were similar to those in wild-type neurons in the absence of the agonist. These data show that Cav1.2α1 subunits are the pore-forming subunits of anomalous LTCCs in mouse cerebellar granule cells. Given the evidence that Cav1.2 channels are specifically involved in sustained Ras-MAPK (mitogen-activated protein kinase)-dependent cAMP response element-binding protein phosphorylation and LTCC-dependent hippocampal long-term potentiation (LTP) (Moosmang et al., 2005), we discuss the hypothesis that anomalous rather than cardiac-type Cav1.2 channels are specifically involved in LTCC-dependent and gene transcription-dependent LTP. [ABSTRACT FROM AUTHOR]

The cerebellum has been a useful model for studying many aspects of neural development because of its relatively simple cytoarchitecture and developmental program. Yet, the genetic mechanisms underlying early differentiation and patterning of the cerebellum are still poorly characterized. Cell expression studies and culture experiments have suggested the importance of bone morphogenetic proteins (BMPs) in development of specific populations of cerebellar neurons. Here, we examined mice with targeted mutations in the BMP type I receptor genes Bmpr1a and Bmpr1b, to genetically test the hypothesis that BMPs play an inductive role in the embryogenesis of cerebellar granule cells. In Bmpr1a;Bmpr1b double knock-out mice, severe cerebellar patterning defects are observed resulting in smaller cerebella that are devoid of foliation. In mutants containing either single BMP receptor gene mutation alone, cerebellar histogenesis appears normal, thereby demonstrating functional redundancy of type I BMP receptors during cerebellar development. Loss of BMP signaling in double mutant animals leads to a dramatic reduction in the number of cerebellar granule cells and ectopic location of many of those that remain. Molecular markers of granule cell specification, including Math1 and Zic1, are drastically downregulated. In addition, Purkinje cells are disorganized and ectopically located, but they appear to be correctly specified. Consistent with the interpretation that granule cells alone are affected, phosphorylated Smad1/5/8 is immunolocalized predominantly to granule cell precursors and not appreciably detected in Purkinje cell precursors. This study demonstrates that BMP signaling plays a crucial role in the specification of granule cells during cerebellar development. [ABSTRACT FROM AUTHOR]

Identifying the brain regions and neuronal cell types that become active at the time of spontaneous seizures remains an important challenge for epilepsy research, and the involvement of dentate granule cells in early seizure events continues to be debated. Although Fos expression is commonly used to evaluate patterns of neuronal activation, there have been few studies of Fos localization after spontaneous seizures. Thus, in a pilocarpine model of recurrent seizures in C57BL/6 mice, Fos expression was examined at multiple time points after spontaneous seizures to follow the temporal and spatial patterns of Fos activation. By 15 min after the beginning of a spontaneous behavioral seizure, Fos labeling was evident in dentate granule cells. This labeling was particularly striking because of its wide extent and relatively uniform appearance in the granule cell layer. At later time points, from 30 min to 4h after a spontaneous seizure, Fos labeling was also detected in interneurons within the dentate gyrus and in widespread regions of the temporal lobe. Interestingly, the timing of Fos activation appeared to differ among different types of GABAergic interneurons in the dentate gyrus, with labeling of parvalbumin neurons along the base of the granule cell layer preceding that of GABA neurons in the molecular layer. The findings in this mouse model are consistent with previous suggestions that spontaneous seizures in temporal lobe epilepsy may result from a periodic breakdown of the normal filter functions of the dentate gyrus and a resulting increase in hypersynchronous activity of dentate granule cells. [ABSTRACT FROM AUTHOR]

Certain neurons choose the neurotransmitter they use in an activity-dependent manner, and trophic factors are involved in this phenotypic differentiation during development. Developing hippocampal granule cells (GCs) constitutively express the markers of the glutamatergic and GABAergic phenotypes, but when development is completed, the GABAergic phenotype shuts off. With electrophysiological, single-cell reverse transcription-PCR and immunohistological techniques, we show here that short-term (24 h) cultures of fully differentiated adult glutamatergic GCs, which express glutamate, VGlut-1 (vesicular glutamate transporter) mRNA, calbindin, and dynorphin mRNA, can be induced to reexpress the GABAergic markers GABA, GAD67 (glutamate decarboxylase 67 kDa isoform), and VGAT (vesicular GABA transporter) mRNA, by sustained synaptic or direct activation of glutamate receptors and by activation of TrkB (tyrosine receptor kinase B) receptors, with brain-derived neurotrophic factor (BDNF) (30 min). The expression of the GABAergic markers was prevented by the blockade of glutamate receptors and sodium or calcium channels, and by inhibitors of protein kinases and protein synthesis. In hippocampal slices of epileptic rats and in BDNF-treated slices from naive rats, we confirmed the appearance of monosynaptic GABAA receptor-mediated responses to GC stimulation, in the presence of glutamate receptors blockers. Accordingly, GC cultures prepared from these slices showed the coexpression of the glutamatergic and GABAergic markers. Our results demonstrate that the neurotransmitter choice of the GCs, which are unique in terms of their continuing birth and death throughout life, depends on programmed and environmental factors, and this process is neither limited by a critical developmental period nor restricted by their insertion in their natural network. [ABSTRACT FROM AUTHOR]

We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with mGlu5 receptors in the corpus striatum. In striatal slices, activation of ephrinB2 by a clustered form of its target receptor, EphB1, amplified the mGlu receptor-mediated stimulation of polyphosphoinositide (PI) hydrolysis. This effect was abolished in slices treated with mGlu1 or NMDA receptor antagonists but was not affected by pharmacological blockade of mGlu5 receptors. An interaction among ephrinB2, mGlu1 receptor, and NMDA was supported by the following observations: (1) the NR1 subunit of NMDA receptors coimmunoprecipitated with mGlu1a receptors and ephrinB2 in striatal lysates; (2) clustered EphB1 amplified excitatory amino acid-stimulated PI hydrolysis in cultured granule cells grown under conditions that favored the expression of mGlu1a receptors; and (3) clustered EphB1 amplified the enhancing effect of mGlu receptor agonists on NMDA toxicity in cortical cultures, and its action was sensitive to mGlu1 receptor antagonists. Finally, fluorescence resonance energy transfer and coclustering analysis in human embryonic kidney 293 cells excluded a physical interaction between ephrinB2 and mGlu1a (or mGlu5 receptors). A functional interaction between ephrinB and mGlu1 receptors, which likely involves adaptor or scaffolding proteins, might have an important role in the regulation of developmental plasticity. [ABSTRACT FROM AUTHOR]