Spine plasticity of dentate gyrus parvalbumin-positive interneurons is regulated by experience.

Experience-driven alterations in neuronal activity are followed by structural-functional modifications allowing cells to adapt to these activity changes. Structural plasticity has been observed for cortical principal cells. However, how GABAergic interneurons respond to experience-dependent network activity changes is not well understood. We show that parvalbumin-expressing interneurons (PVIs) of the dentate gyrus (DG) possess dendritic spines, which undergo behaviorally induced structural dynamics. Glutamatergic inputs at PVI spines evoke signals with high spatial compartmentalization defined by neck length. Mice experiencing novel contexts form more PVI spines with elongated necks and exhibit enhanced network and PVI activity and cFOS expression. Enhanced green fluorescent protein reconstitution across synaptic partner-mediated synapse labeling shows that experience-driven PVI spine growth boosts targeting of PVI spines over shafts by glutamatergic synapses. Our findings propose a role for PVI spine dynamics in regulating PVI excitation by their inputs, which may allow PVIs to dynamically adjust their functional integration in the DG microcircuitry in relation to network computational demands.

Interneuron function and cognitive behavior are preserved upon postnatal removal of Lhx6.

LIM homeobox domain transcription factor 6 (Lhx6) is crucial for the prenatal specification and differentiation of hippocampal GABAergic interneuron precursors. Interestingly, Lhx6 remains to be expressed in parvalbumin-positive hippocampal interneurons (PVIs) long after specification and differentiation have been completed, the functional implications of which remain elusive. We addressed the role of adult-expressed Lhx6 in the hippocampus by knocking down Lhx6 in adult mice (> 8 weeks old) using viral or transgenic expression of Cre-recombinase in Lhx6loxP/loxP mice. Late removal of Lhx6 did not affect the number of PVIs and had no impact on the morphological and physiological properties of PVIs. Furthermore, mice lacking Lhx6 in PVIs displayed normal cognitive behavior. Loss of Lhx6 only partially reduced the expression of Sox6 and Arx, downstream transcription factors that depend on Lhx6 during embryonic development of PVIs. Our data thus suggest that while Lhx6 is vitally important to drive interneuron transcriptional networks during early development, it becomes uncoupled from downstream effectors during postnatal life.

Hippocampal low-frequency stimulation prevents seizure generation in a mouse model of mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common form of focal, pharmacoresistant epilepsy in adults and is often associated with hippocampal sclerosis. Here, we established the efficacy of optogenetic and electrical low-frequency stimulation (LFS) in interfering with seizure generation in a mouse model of MTLE. Specifically, we applied LFS in the sclerotic hippocampus to study the effects on spontaneous subclinical and evoked generalized seizures. We found that stimulation at 1 Hz for 1 hr resulted in an almost complete suppression of spontaneous seizures in both hippocampi. This seizure-suppressive action during daily stimulation remained stable over several weeks. Furthermore, LFS for 30 min before a pro-convulsive stimulus successfully prevented seizure generalization. Finally, acute slice experiments revealed a reduced efficacy of perforant path transmission onto granule cells upon LFS. Taken together, our results suggest that hippocampal LFS constitutes a promising approach for seizure control in MTLE.

Presynaptic GABAB receptors functionally uncouple somatostatin interneurons from the active hippocampal network.

Information processing in cortical neuronal networks relies on properly balanced excitatory and inhibitory neurotransmission. A ubiquitous motif for maintaining this balance is the somatostatin interneuron (SOM-IN) feedback microcircuit. Here, we investigated the modulation of this microcircuit by presynaptic GABAB receptors (GABABRs) in the rodent hippocampus. Whole-cell recordings from SOM-INs revealed that both excitatory and inhibitory synaptic inputs are strongly inhibited by GABABRs, while optogenetic activation of the interneurons shows that their inhibitory output is also strongly suppressed. Electron microscopic analysis of immunogold-labelled freeze-fracture replicas confirms that GABABRs are highly expressed presynaptically at both input and output synapses of SOM-INs. Activation of GABABRs selectively suppresses the recruitment of SOM-INs during gamma oscillations induced in vitro. Thus, axonal GABABRs are positioned to efficiently control the input and output synapses of SOM-INs and can functionally uncouple them from local network with implications for rhythmogenesis and the balance of entorhinal versus intrahippocampal afferents.

Dendritic inhibition differentially regulates excitability of dentate gyrus parvalbumin-expressing interneurons and granule cells.

Fast-spiking parvalbumin-expressing interneurons (PVIs) and granule cells (GCs) of the dentate gyrus receive layer-specific dendritic inhibition. Its impact on PVI and GC excitability is, however, unknown. By applying whole-cell recordings, GABA uncaging and single-cell-modeling, we show that proximal dendritic inhibition in PVIs is less efficient in lowering perforant path-mediated subthreshold depolarization than distal inhibition but both are highly efficient in silencing PVIs. These inhibitory effects can be explained by proximal shunting and distal strong hyperpolarizing inhibition. In contrast, GC proximal but not distal inhibition is the primary regulator of their excitability and recruitment. In GCs inhibition is hyperpolarizing along the entire somato-dendritic axis with similar strength. Thus, dendritic inhibition differentially controls input-output transformations in PVIs and GCs. Dendritic inhibition in PVIs is suited to balance PVI discharges in dependence on global network activity thereby providing strong and tuned perisomatic inhibition that contributes to the sparse representation of information in GC assemblies.

Comprehensive Cell Surface Antigen Analysis Identifies Transferrin Receptor Protein-1 (CD71) as a Negative Selection Marker for Human Neuronal Cells.

Cell state-, developmental stage-, and lineage-specific combinatorial expression of cluster of differentiation (CD) molecules enables the identification of cellular subsets via multicolor flow cytometry. We describe an exhaustive characterization of neural cell types by surface antigens, exploiting human pluripotent stem cell-derived neural cell systems. Using multiwell screening approaches followed by detailed validation of expression patterns and dynamics, we exemplify a strategy for resolving cellular heterogeneity in stem cell paradigms. In addition to providing a catalog of surface antigens expressed in the neural lineage, we identified the transferrin receptor-1 (CD71) to be differentially expressed in neural stem cells and differentiated neurons. In this context, we describe a role for N-Myc proto-oncogene (MYCN) in maintaining CD71 expression in proliferating neural cells. We report that in vitro human stem cell-derived neurons lack CD71 surface expression and that the observed differential expression can be used to identify and enrich CD71- neuronal derivatives from heterogeneous cultures. Stem Cells 2019;37:1293-1306.

Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction.

The induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission entails pre- and postsynaptic Ca2+ signals, which represent transient increments in cytoplasmic free Ca2+ concentration. In diverse synapse types, Ca2+ release from intracellular stores contributes to amplify the Ca2+ signals initially generated by activation of neuronal Ca2+ entry pathways. Here, we used hippocampal slices from young male rats to evaluate whether pharmacological activation or inhibition of Ca2+ release from the endoplasmic reticulum (ER) mediated by ryanodine receptor (RyR) channels modifies LTD induction at Schaffer collateral-CA1 synapses. Pre-incubation of slices with ryanodine (1 µM, 1 h) or caffeine (1 mM, 30 min) to promote RyR-mediated Ca2+ release facilitated LTD induction by low frequency stimulation (LFS), but did not affect the amplitude of synaptic transmission, the profiles of field excitatory postsynaptic potentials (fEPSP) or the paired-pulse (PP) responses. Conversely, treatment with inhibitory ryanodine (20 µM, 1 h) to suppress RyR-mediated Ca2+ release prevented LTD induction, but did not affect baseline synaptic transmission or PP responses. Previous literature reports indicate that LTD induction requires presynaptic CaMKII activity. We found that 1 h after applying the LTD induction protocol, slices displayed a significant increase in CaMKII phosphorylation relative to the levels exhibited by un-stimulated (naïve) slices. In addition, LTD induction (1 h) enhanced the phosphorylation of the presynaptic protein Synapsin I at a CaMKII-dependent phosphorylation site, indicating that LTD induction stimulates presynaptic CaMKII activity. Pre-incubation of slices with 20 µM ryanodine abolished the increased CaMKII and Synapsin I phosphorylation induced by LTD, whereas naïve slices pre-incubated with inhibitory ryanodine displayed similar CaMKII and Synapsin I phosphorylation levels as naïve control slices. We posit that inhibitory ryanodine suppressed LTD-induced presynaptic CaMKII activity, as evidenced by the suppression of Synapsin I phosphorylation induced by LTD. Accordingly, we propose that presynaptic RyR-mediated Ca2+ signals contribute to LTD induction at Schaffer collateral-CA1 synapses.

Publisher Correction: Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Lack of Pannexin 1 Alters Synaptic GluN2 Subunit Composition and Spatial Reversal Learning in Mice.

Long-term potentiation (LTP) and long-term depression (LTD) are two forms of synaptic plasticity that have been considered as the cellular substrate of memory formation. Although LTP has received considerable more attention, recent evidences indicate that LTD plays also important roles in the acquisition and storage of novel information in the brain. Pannexin 1 (Panx1) is a membrane protein that forms non-selective channels which have been shown to modulate the induction of hippocampal synaptic plasticity. Animals lacking Panx1 or blockade of Pannexin 1 channels precludes the induction of LTD and facilitates LTP. To evaluate if the absence of Panx1 also affects the acquisition of rapidly changing information we trained Panx1 knockout (KO) mice and wild type (WT) littermates in a visual and hidden version of the Morris water maze (MWM). We found that KO mice find the hidden platform similarly although slightly quicker than WT animals, nonetheless, when the hidden platform was located in the opposite quadrant (OQ) to the previous learned location, KO mice spent significantly more time in the previous quadrant than in the new location indicating that the absence of Panx1 affects the reversion of a previously acquired spatial memory. Consistently, we observed changes in the content of synaptic proteins critical to LTD, such as GluN2 subunits of N-methyl-D-aspartate receptors (NMDARs), which changed their contribution to synaptic plasticity in conditions of Panx1 ablation. Our findings give further support to the role of Panx1 channels on the modulation of synaptic plasticity induction, learning and memory processes.

Electrical coupling between A17 cells enhances reciprocal inhibitory feedback to rod bipolar cells.

A17 amacrine cells are an important part of the scotopic pathway. Their synaptic varicosities receive glutamatergic inputs from rod bipolar cells (RBC) and release GABA onto the same RBC terminal, forming a reciprocal feedback that shapes RBC depolarization. Here, using patch-clamp recordings, we characterized electrical coupling between A17 cells of the rat retina and report the presence of strongly interconnected and non-coupled A17 cells. In coupled A17 cells, evoked currents preferentially flow out of the cell through GJs and cross-synchronization of presynaptic signals in a pair of A17 cells is correlated to their coupling degree. Moreover, we demonstrate that stimulation of one A17 cell can induce electrical and calcium transients in neighboring A17 cells, thus confirming a functional flow of information through electrical synapses in the A17 coupled network. Finally, blocking GJs caused a strong decrease in the amplitude of the inhibitory feedback onto RBCs. We therefore propose that electrical coupling between A17 cells enhances feedback onto RBCs by synchronizing and facilitating GABA release from inhibitory varicosities surrounding each RBC axon terminal. GJs between A17 cells are therefore critical in shaping the visual flow through the scotopic pathway.

Somatostatin-positive interneurons in the dentate gyrus of mice provide local- and long-range septal synaptic inhibition.

Somatostatin-expressing-interneurons (SOMIs) in the dentate gyrus (DG) control formation of granule cell (GC) assemblies during memory acquisition. Hilar-perforant-path-associated interneurons (HIPP cells) have been considered to be synonymous for DG-SOMIs. Deviating from this assumption, we show two functionally contrasting DG-SOMI-types. The classical feedback-inhibitory HIPPs distribute axon fibers in the molecular layer. They are engaged by converging GC-inputs and provide dendritic inhibition to the DG circuitry. In contrast, SOMIs with axon in the hilus, termed hilar interneurons (HILs), provide perisomatic inhibition onto GABAergic cells in the DG and project to the medial septum. Repetitive activation of glutamatergic inputs onto HIPP cells induces long-lasting-depression (LTD) of synaptic transmission but long-term-potentiation (LTP) of synaptic signals in HIL cells. Thus, LTD in HIPPs may assist flow of spatial information from the entorhinal cortex to the DG, whereas LTP in HILs may facilitate the temporal coordination of GCs with activity patterns governed by the medial septum.

Inhibition of DNA Methylation Impairs Synaptic Plasticity during an Early Time Window in Rats.

Although the importance of DNA methylation-dependent gene expression to neuronal plasticity is well established, the dynamics of methylation and demethylation during the induction and expression of synaptic plasticity have not been explored. Here, we combined electrophysiological, pharmacological, molecular, and immunohistochemical approaches to examine the contribution of DNA methylation and the phosphorylation of Methyl-CpG-binding protein 2 (MeCP2) to synaptic plasticity. We found that, at twenty minutes after theta burst stimulation (TBS), the DNA methylation inhibitor 5-aza-2-deoxycytidine (5AZA) impaired hippocampal long-term potentiation (LTP). Surprisingly, after two hours of TBS, when LTP had become a transcription-dependent process, 5AZA treatment had no effect. By comparing these results to those in naive slices, we found that, at two hours after TBS, an intergenic region of the RLN gene was hypomethylated and that the phosphorylation of residue S80 of MeCP2 was decreased, while the phosphorylation of residue S421 was increased. As expected, 5AZA affected only the methylation of the RLN gene and exerted no effect on MeCP2 phosphorylation patterns. In summary, our data suggest that tetanic stimulation induces critical changes in synaptic plasticity that affects both DNA methylation and the phosphorylation of MeCP2. These data also suggest that early alterations in DNA methylation are sufficient to impair the full expression of LTP.

Persistent discharges in dentate gyrus perisoma-inhibiting interneurons require hyperpolarization-activated cyclic nucleotide-gated channel activation.

Parvalbumin (PV)-expressing perisoma-inhibiting interneurons (PIIs) of the dentate gyrus integrate rapidly correlated synaptic inputs and generate short-duration action potentials that propagate along the axon to their output synapses, supporting fast inhibitory signaling onto their target cells. Here we show that PV-PIIs in rat and mouse dentate gyrus (DG) integrate their intrinsic activity over time and can turn into a persistent firing mode characterized by the ability to generate long-lasting trains of action potentials at ∼50 Hz in the absence of additional inputs. Persistent firing emerges in the axons remote from the axon initial segment and markedly depends on hyperpolarization-activated cyclic nucleotide-gated channel (HCNC) activation. Persistent firing properties are modulated by intracellular Ca(2+) levels and somatic membrane potential. Detailed computational single-cell PIIs models reveal that HCNC-mediated conductances can contribute to persistent firing during conditions of a shift in their voltage activation curve to more depolarized potentials. Paired recordings from PIIs and their target granule cells show that persistent firing supports strong inhibitory output signaling. Thus, persistent firing may emerge during conditions of intense activation of the network, thereby providing silencing to the circuitry and the maintenance of sparse activity in the dentate gyrus.

Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells.

Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhßE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the ß4 subunit. Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

Synaptic properties of SOM- and CCK-expressing cells in dentate gyrus interneuron networks.

Hippocampal GABAergic cells are highly heterogeneous, but the functional significance of this diversity is not fully understood. By using paired recordings of synaptically connected interneurons in slice preparations of the rat and mouse dentate gyrus (DG), we show that morphologically identified interneurons form complex neuronal networks. Synaptic inhibitory interactions exist between cholecystokinin (CCK)-expressing hilar commissural associational path (HICAP) cells and among somatostatin (SOM)-containing hilar perforant path-associated (HIPP) interneurons. Moreover, both interneuron types inhibit parvalbumin (PV)-expressing perisomatic inhibitory basket cells (BCs), whereas BCs and HICAPs rarely target HIPP cells. HICAP and HIPP cells produce slow, weak, and unreliable inhibition onto postsynaptic interneurons. The time course of inhibitory signaling is defined by the identity of the presynaptic and postsynaptic cell. It is the slowest for HIPP-HIPP, intermediately slow for HICAP-HICAP, but fast for BC-BC synapses. GABA release at interneuron-interneuron synapses also shows cell type-specific short-term dynamics, ranging from multiple-pulse facilitation at HICAP-HICAP, biphasic modulation at HIPP-HIPP to depression at BC-BC synapses. Although dendritic inhibition at HICAP-BC and HIPP-BC synapses appears weak and slow, channelrhodopsin 2-mediated excitation of SOM terminals demonstrates that they effectively control the activity of target interneurons. They markedly reduce the discharge probability but sharpen the temporal precision of action potential generation. Thus, dendritic inhibition seems to play an important role in determining the activity pattern of GABAergic interneuron populations and thereby the flow of information through the DG circuitry.

Ablation of Ca(V)2.1 voltage-gated Ca²âº channels in mouse forebrain generates multiple cognitive impairments.

Voltage-gated Ca(V)2.1 (P/Q-type) Ca²âº channels located at the presynaptic membrane are known to control a multitude of Ca²âº-dependent cellular processes such as neurotransmitter release and synaptic plasticity. Our knowledge about their contributions to complex cognitive functions, however, is restricted by the limited adequacy of existing transgenic Ca(V)2.1 mouse models. Global Ca(V)2.1 knock-out mice lacking the α1 subunit Cacna1a gene product exhibit early postnatal lethality which makes them unsuitable to analyse the relevance of Ca(V)2.1 Ca²âº channels for complex behaviour in adult mice. Consequently we established a forebrain specific Ca(V)2.1 knock-out model by crossing mice with a floxed Cacna1a gene with mice expressing Cre-recombinase under the control of the NEX promoter. This novel mouse model enabled us to investigate the contribution of Ca(V)2.1 to complex cognitive functions, particularly learning and memory. Electrophysiological analysis allowed us to test the specificity of our conditional knock-out model and revealed an impaired synaptic transmission at hippocampal glutamatergic synapses. At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity. In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.

Functional characteristics of parvalbumin- and cholecystokinin-expressing basket cells.

Cortical neuronal network operations depend critically on the recruitment of GABAergic interneurons and the properties of their inhibitory output signals. Recent evidence indicates a marked difference in the signalling properties of two major types of perisomatic inhibitory interneurons, the parvalbumin- and the cholecystokinin-containing basket cells. Parvalbumin-expressing basket cells are rapidly recruited by excitatory synaptic inputs, generate high-frequency trains of action potentials, discharge single action potentials phase-locked to fast network oscillations and provide fast, stable and timed inhibitory output onto their target cells. In contrast, cholecystokinin-containing basket cells are recruited in a less reliable manner, discharge at moderate frequencies with single action potentials weakly coupled to the phases of fast network oscillations and generate an asynchronous, fluctuating and less timed inhibitory output. These signalling modes are based on cell type-dependent differences in the functional and plastic properties of excitatory input synapses, integrative qualities and in the kinetics and dynamics of inhibitory output synapses. Thus, the two perisomatic inhibitory interneuron types operate with different speed and precision and may therefore contribute differently to the operations of neuronal networks.

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity.

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.

Nitric oxide amplifies the rat electroretinogram.

It is well established that nitric oxide (NO) participates in retinal signal processing through stimulation of its receptor enzyme, soluble guanylyl cyclase (sGC). However, under pathological conditions such as uveoretinitis, diabetic or ischemic retinopathy, elevated NO concentrations may cause protein S-nitrosation and peroxynitrite formation in the retina, promoting cellular injury and apoptosis. Previous electroretinogram (ERG) studies demonstrated deleterious effects of NO on the retinal light response, but showed no evidence for a role in normal signal processing. To better understand the function of NO in ocular physiology, we investigated the effects of exogenous NO, produced by NO donors with different release kinetics, on the flash ERG of the rat. Within a limited concentration range, NO strongly amplified ERG a- and b-waves, oscillatory potentials, and the scotopic threshold response. Amplification exceeded 100% under dark adaptation, whereas the photopic ERG and the isolated cone response were increased by less than 50%. Blocking photoreceptor-bipolar cell synapses by AP-4 demonstrated a significant increase of the isolated a-wave by NO, and modeling the ERG generator PIII supported photoreceptors as primary NO targets. The sGC inhibitors ODQ and NS2028 did not reduce NO-dependent ERG amplification, ruling out an involvement of the classical NO effector cyclic GMP. Using immunohistochemistry, we show that illumination and exogenous NO altered the S-nitrosation level of the photoreceptor layer, suggesting that direct protein modifications caused by elevated levels of NO may be responsible for the observed phenomenon.