Granule Cell Synapses Research Articles

Low frequency stimulation for seizure suppression: Identification of optimal targets in the entorhinal-hippocampal circuit

BackgroundMesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis (HS) is a common form of drug-resistant focal epilepsy in adults. Treatment for pharmacoresistant patients remains a challenge, with deep brain stimulation (DBS) showing promise for alleviating intractable seizures. This study explores the efficacy of low frequency stimulation (LFS) on specific neuronal targets within the entorhinal-hippocampal circuit in a mouse model of MTLE. ObjectiveOur previous research demonstrated that LFS of the medial perforant path (MPP) fibers in the sclerotic hippocampus reduced seizures in epileptic mice. Here, we aimed to identify the critical neuronal population responsible for this antiepileptic effect by optogenetically stimulating presynaptic and postsynaptic compartments of the MPP-dentate granule cell (DGC) synapse at 1 Hz. We hypothesize that specific targets for LFS can differentially influence seizure activity depending on the cellular identity and location within or outside the seizure focus. MethodsWe utilized the intrahippocampal kainate (ihKA) mouse model of MTLE and targeted specific neural populations using optogenetic stimulation. We recorded intracranial neuronal activity from freely moving chronically epileptic mice with and without optogenetic LFS up to 3 h. ResultsWe found that LFS of MPP fibers in the sclerotic hippocampus effectively suppressed epileptiform activity while stimulating principal cells in the MEC had no impact. Targeting DGCs in the sclerotic septal or non-sclerotic temporal hippocampus with LFS did not reduce seizure numbers but shortened the epileptiform bursts. ConclusionPresynaptic stimulation of the MPP-DGC synapse within the sclerotic hippocampus is critical for seizure suppression via LFS.

Sprague-Dawley rats differ in responses to medial perforant path paired pulse and tetanic activation as a function of sex and age.

Network plasticity in the medial perforant path (MPP) of adult (5-9 mo) and aged (18-20 mo) urethane-anesthetized male and female Sprague-Dawley rats was characterized. Paired pulses probed recurrent networks before and after a moderate tetanic protocol. Adult females exhibited greater EPSP-spike coupling suggesting greater intrinsic excitability than adult males. Aged rats did not differ in EPSP-spike coupling but aged females had larger spikes at high currents than males. Paired pulses suggested lower GABA-B inhibition in females. Absolute population spike measures were larger post-tetani in female rats than male rats. Relative population spike increases were greatest in adult males relative to females and to aged males. EPSP slope potentiation was detected with normalization in some post-tetanic intervals for all groups except aged males. Tetani shortened spike latency across groups. Tetani-associated NMDA-mediated burst depolarizations were larger for the first two trains in each tetanus in adult males than other groups. EPSP slopes over 30 min post-tetani predicted spike size in female rats, but not in males. Replicating newer evidence MPP plasticity in adult males was mediated by increased intrinsic excitability. Female MPP plasticity was related to synaptic drive increases, not excitability increases. Aged male rats were deficient in MPP plasticity.SIGNIFICANCE STATEMENTThe medial perforant path (MPP)-dentate gyrus (DG) granule cell synapse was the site of discovery of long-term potentiation in the mammalian nervous system but considering the current interest in sex and aging, surprisingly few studies have directly examined these variables in relation to tetanus-induced long-term and short-term (paired pulse) plasticity. Using an interleaved current-paired pulse interval protocol and moderate tetanic protocol young (5-9mo) and old (18-20mo) male and female urethane anesthetized rats were found to differ in levels of granule cell intrinsic excitability, E-S coupling, GABA-B inhibition, and tetanic NMDA current contributions to post-tetanic population spike potentiation. This study provides a platform or future examination of sex- and age-related changes in MPP-DG function.

Anatomical and Functional Connectivity at the Dendrodendritic Reciprocal Mitral Cell-Granule Cell Synapse: Impact on Recurrent and Lateral Inhibition.

In the vertebrate olfactory bulb, reciprocal dendrodendritic interactions between its principal neurons, the mitral and tufted cells, and inhibitory interneurons in the external plexiform layer mediate both recurrent and lateral inhibition, with the most numerous of these interneurons being granule cells. Here, we used recently established anatomical parameters and functional data on unitary synaptic transmission to simulate the strength of recurrent inhibition of mitral cells specifically from the reciprocal spines of rat olfactory bulb granule cells in a quantitative manner. Our functional data allowed us to derive a unitary synaptic conductance on the order of 0.2 nS. The simulations predicted that somatic voltage deflections by even proximal individual granule cell inputs are below the detection threshold and that attenuation with distance is roughly linear, with a passive length constant of 650 μm. However, since recurrent inhibition in the wake of a mitral cell action potential will originate from hundreds of reciprocal spines, the summated recurrent IPSP will be much larger, even though there will be substantial mutual shunting across the many inputs. Next, we updated and refined a preexisting model of connectivity within the entire rat olfactory bulb, first between pairs of mitral and granule cells, to estimate the likelihood and impact of recurrent inhibition depending on the distance between cells. Moreover, to characterize the substrate of lateral inhibition, we estimated the connectivity via granule cells between any two mitral cells or all the mitral cells that belong to a functional glomerular ensemble (i.e., which receive their input from the same glomerulus), again as a function of the distance between mitral cells and/or entire glomerular mitral cell ensembles. Our results predict the extent of the three regimes of anatomical connectivity between glomerular ensembles: high connectivity within a glomerular ensemble and across the first four rings of adjacent glomeruli, substantial connectivity to up to eleven glomeruli away, and negligible connectivity beyond. Finally, in a first attempt to estimate the functional strength of granule-cell mediated lateral inhibition, we combined this anatomical estimate with our above simulation results on attenuation with distance, resulting in slightly narrowed regimes of a functional impact compared to the anatomical connectivity.

Disrupted mossy fiber connections from defective embryonic neurogenesis contribute to SOX11-associated schizophrenia.

Abnormal mossy fiber connections in the hippocampus have been implicated in schizophrenia. However, it remains unclear whether this abnormality in the patients is genetically determined and whether it contributes to the onset of schizophrenia. Here, we showed that iPSC-derived hippocampal NPCs from schizophrenia patients with the A/A allele at SNP rs16864067 exhibited abnormal NPC polarity, resulting from the downregulation of SOX11 by this high-risk allele. In the SOX11-deficient mouse brain, abnormal NPC polarity was also observed in the hippocampal dentate gyrus, and this abnormal NPC polarity led to defective hippocampal neurogenesis-specifically, irregular neuroblast distribution and disrupted granule cell morphology. As granule cell synapses, the mossy fiber pathway was disrupted, and this disruption was resistant to activity-induced mossy fiber remodeling in SOX11 mutant mice. Moreover, these mutant mice exhibited diminished PPI and schizophrenia-like behaviors. Activation of hippocampal neurogenesis in the embryonic brain, but not in the adult brain, partially alleviated disrupted mossy fiber connections and improved schizophrenia-related behaviors in mutant mice. We conclude that disrupted mossy fiber connections are genetically determined and strongly correlated with schizophrenia-like behaviors in SOX11-deficient mice. This disruption may reflect the pathological substrate of SOX11-associated schizophrenia.

Carbamazepine Reduces Sharp Wave-Ripple Complexes and Exerts Synapse-Specific Inhibition of Neurotransmission in Ex Vivo Hippocampal Slices

Higher therapeutic concentrations of the antiseizure medication carbamazepine (CBZ) are associated with cognitive side effects. Hippocampal sharp wave-ripple complexes (SPW-Rs) are proposed to participate in memory consolidation during periods of quiet and slow-wave sleep. SPW-Rs are generated in the CA3 region and are regulated by multiple synaptic inputs. Here, we used a multi-electrode array to determine the effects of CBZ on SPW-Rs and synaptic transmission at multiple hippocampal synapses. Our results demonstrate that CBZ reduced SPW-Rs at therapeutically relevant concentrations (IC50 = 37 μM) and altered the core characteristics of ripples, important for information processing and consolidation. Moreover, CBZ inhibited neurotransmission in a synapse-specific manner. CBZ inhibition was most potent at the medial-perforant-path-to-CA3 and mossy-fiber-to-CA3 synapses (IC50s ~ 30 and 60 μM, respectively) and least potent at medial-perforant-path-to-dentate granule cell synapses (IC50 ~ 120 μM). These results suggest that the synapse-specific CBZ inhibition of neurotransmission reduces SPW-Rs and that the CBZ inhibition of SPW-Rs may underlie the cognitive impairments observed with therapeutic doses of CBZ.

Contrasting Functions of Mitogen- and Stress-activated Protein Kinases 1 and 2 in Recognition Memory and In Vivo Hippocampal Synaptic Transmission

The mitogen-activated protein kinases (MAPK) are major signaling components of intracellular pathways required for memory consolidation. Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2) mediate signal transduction downstream of MAPK. MSKs are activated by Extracellular-signal Regulated Kinase 1/2 (ERK1/2) and p38 MAPK. In turn, they can activate cyclic AMP-response-element-binding protein (CREB), thereby modulating the expression of immediate early genes crucial for the formation of long-term memories. While MSK1 has been previously implicated in certain forms of learning and memory, little is known concerning MSK2. Our goal was to explore the respective contribution of MSK1 and MSK2 in hippocampal synaptic transmission and plasticity and hippocampal-dependent recognition memory. In Msk1- and Msk2-knockout mice, we evaluated object and object-place recognition memory, basal synaptic transmission, paired-pulse facilitation (PPF) and inhibition (PPI), and the capacity to induce and sustain long-term potentiation (LTP) in vivo. We also assessed the level of two proteins downstream in the MAPK/ERK1/2 pathway crucial for long-term memory, CREB and the immediate early gene (IEG) Early growth response 1 (EGR1). Loss of Msk1, but not of Msk2, affected excitatory synaptic transmission at perforant path-to-dentate granule cell synapses, altered short-term presynaptic plasticity, impaired selectively long-term spatial recognition memory, and decreased basal levels of CREB and its activated form. LTP in vivo and LTP-induced CREB phosphorylation and EGR1 expression were unchanged after Msk1 or Msk2 deletion. Our findings demonstrate a dissimilar contribution of MSKs proteins in cognitive processes and suggest that Msk1 loss-of-function only has a deleterious impact on neuronal activity and hippocampal-dependent memory consolidation.

Short-Term Plasticity in Cortical GABAergic Synapses on Olfactory Bulb Granule Cells Is Modulated by Endocannabinoids

Olfactory bulb and higher processing areas are synaptically interconnected, providing rapid regulation of olfactory bulb circuit dynamics and sensory processing. Short-term plasticity changes at any of these synapses could modulate sensory processing and potentially short-term sensory memory. A key olfactory bulb circuit for mediating cortical feedback modulation is granule cells, which are targeted by multiple cortical regions including both glutamatergic excitatory inputs and GABAergic inhibitory inputs. There is robust endocannabinoid modulation of excitatory inputs to granule cells and here we explored whether there was also endocannabinoid modulation of the inhibitory cortical inputs to granule cells. We expressed light-gated cation channel channelrhodopsin-2 (ChR2) in GABAergic neurons in the horizontal limb of the diagonal band of Broca (HDB) and their projections to granule cells in olfactory bulb. Selective optical activation of ChR2 positive axons/terminals generated strong, frequency-dependent short-term depression of GABAA-mediated-IPSC in granule cells. As cannabinoid type 1 (CB1) receptor is heavily expressed in olfactory bulb granule cell layer (GCL) and there is endogenous endocannabinoid release in GCL, we investigated whether activation of CB1 receptor modulated the HDB IPSC and short-term depression at the HDB→granule cell synapse. Activation of the CB1 receptor by the exogenous agonist Win 55,212-2 significantly decreased the peak amplitude of individual IPSC and decreased short-term depression, while blockade of the CB1 receptor by AM 251 slightly increased individual IPSCs and increased short-term depression. Thus, we conclude that there is tonic endocannabinoid activation of the GABAergic projections of the HDB to granule cells, similar to the modulation observed with glutamatergic projections to granule cells. Modulation of inhibitory synaptic currents and frequency-dependent short-term depression could regulate the precise balance of cortical feedback excitation and inhibition of granule cells leading to changes in granule cell mediated inhibition of olfactory bulb output to higher processing areas.

The Rac-GEF Tiam1 Promotes Dendrite and Synapse Stabilization of Dentate Granule Cells and Restricts Hippocampal-Dependent Memory Functions.

The dentate gyrus (DG) controls information flow into the hippocampus and is critical for learning, memory, pattern separation, and spatial coding, while DG dysfunction is associated with neuropsychiatric disorders. Despite its importance, the molecular mechanisms regulating DG neural circuit assembly and function remain unclear. Here, we identify the Rac-GEF Tiam1 as an important regulator of DG development and associated memory processes. In the hippocampus, Tiam1 is predominantly expressed in the DG throughout life. Global deletion of Tiam1 in male mice results in DG granule cells with simplified dendritic arbors, reduced dendritic spine density, and diminished excitatory synaptic transmission. Notably, DG granule cell dendrites and synapses develop normally in Tiam1 KO mice, resembling WT mice at postnatal day 21 (P21), but fail to stabilize, leading to dendrite and synapse loss by P42. These results indicate that Tiam1 promotes DG granule cell dendrite and synapse stabilization late in development. Tiam1 loss also increases the survival, but not the production, of adult-born DG granule cells, possibly because of greater circuit integration as a result of decreased competition with mature granule cells for synaptic inputs. Strikingly, both male and female mice lacking Tiam1 exhibit enhanced contextual fear memory and context discrimination. Together, these results suggest that Tiam1 is a key regulator of DG granule cell stabilization and function within hippocampal circuits. Moreover, based on the enhanced memory phenotype of Tiam1 KO mice, Tiam1 may be a potential target for the treatment of disorders involving memory impairments.SIGNIFICANCE STATEMENT The dentate gyrus (DG) is important for learning, memory, pattern separation, and spatial navigation, and its dysfunction is associated with neuropsychiatric disorders. However, the molecular mechanisms controlling DG formation and function remain elusive. By characterizing mice lacking the Rac-GEF Tiam1, we demonstrate that Tiam1 promotes the stabilization of DG granule cell dendritic arbors, spines, and synapses, whereas it restricts the survival of adult-born DG granule cells, which compete with mature granule cells for synaptic integration. Notably, mice lacking Tiam1 also exhibit enhanced contextual fear memory and context discrimination. These findings establish Tiam1 as an essential regulator of DG granule cell development, and identify it as a possible therapeutic target for memory enhancement.

The Role of Astrocytes in the Development of the Cerebellum

Astrocytes, initially described as merely support cells, are now known as a heterogeneous population of cells actively involved in a variety of biological functions such as: neuronal migration and differentiation; regulation of cerebral blood flow; metabolic control of extracellular potassium concentration; and modulation of synapse formation and elimination; among others. Cerebellar glial cells have been shown to play a significant role in proliferation, differentiation, migration, and synaptogenesis. However, less evidence is available about the role of neuron-astrocyte interactions during cerebellar development and their impact on diseases of the cerebellum. In this review, we will focus on the mechanisms underlying cellular interactions, specifically neuron-astrocyte interactions, during cerebellar development, function, and disease. We will discuss how cerebellar glia, astrocytes, and Bergmann glia play a fundamental role in several steps of cerebellar development, such as granule cell migration, axonal growth, neuronal differentiation, and synapse formation, and in diseases associated with the cerebellum. We will focus on how astrocytes and thyroid hormones impact cerebellar development. Furthermore, we will provide evidence of how growth factors secreted by glial cells, such as epidermal growth factor and transforming growth factors, control cerebellar organogenesis. Finally, we will argue that glia are a key mediator of cerebellar development and that identification of molecules and pathways involved in neuron-glia interactions may contribute to a better understanding of cerebellar development and associated disorders.

Interneuronal NMDA receptors regulate long-term depression and motor learning in the cerebellum.

NMDA receptors (NMDARs) are required for long-term depression (LTD) at parallel fibre-Purkinje cell synapses, but their cellular localization and physiological functions invivo are unclear. NMDARs in molecular-layer interneurons (MLIs), but not granule cells or Purkinje cells, are required for LTD, but not long-term potentiation induced by low-frequency stimulation of parallel fibres. Nitric oxide produced by NMDAR activation in MLIs probably mediates LTD induction. NMDARs in granule cells or Purkinje cells are dispensable for motor learning during adaptation of horizontal optokinetic responses. Long-term potentiation (LTP) and depression (LTD), which serve as cellular synaptic plasticity models for learning and memory, are crucially regulated by N-methyl-d-aspartate receptors (NMDARs) in various brain regions. In the cerebellum, LTP and LTD at parallel fibre (PF)-Purkinje cell (PC) synapses are thought to mediate certain forms of motor learning. However, while NMDARs are essential for LTD invitro, their cellular localization remains controversial. In addition, whether and how NMDARs mediate motor learning invivo remains unclear. Here, we examined the contribution of NMDARs expressed in granule cells (GCs), PCs and molecular-layer interneurons (MLIs) to LTD/LTP and motor learning by generating GC-, PC- and MLI/PC-specific knockouts of Grin1, a gene encoding an obligatory GluN1 subunit of NMDARs. While robust LTD and LTP were induced at PF-PC synapses in GC- and PC-specific Grin1 (GC-Grin1 and PC-Grin1, respectively) conditional knockout (cKO) mice, only LTD was impaired in MLI/PC-specific Grin1 (MLI/PC-Grin1) cKO mice. Application of diethylamine nitric oxide (NO) sodium, a potent NO donor, to the cerebellar slices restored LTD in MLI/PC-Grin1 cKO mice, suggesting that NO is probably downstream to NMDARs. Furthermore, the adaptation of horizontal optokinetic responses (hOKR), a cerebellar motor learning task, was normally observed in GC-Grin1 cKO and PC-Grin1 cKO mice, but not in MLI/PC-Grin1 cKO mice. These results indicate that it is the NMDARs expressed in MLIs, but not in PCs or GCs, that play important roles in LTD invitro and motor learning invivo.

Short-Term Plasticity at Olfactory Cortex to Granule Cell Synapses Requires CaV2.1 Activation.

Output projections of the olfactory bulb (OB) to the olfactory cortex (OCX) and reciprocal feedback projections from OCX provide rapid regulation of OB circuit dynamics and odor processing. Short-term synaptic plasticity (STP), a feature of many synaptic connections in the brain, can modulate the strength of feedback based on preceding network activity. We used light-gated cation channel channelrhodopsin-2 (ChR2) to investigate plasticity of excitatory synaptic currents (EPSCs) evoked at the OCX to granule cell (GC) synapse in the OB. Selective activation of OCX glutamatergic axons/terminals in OB generates strong, frequency-dependent STP in GCs. This plasticity was critically dependent on activation of CaV2.1 channels. As acetylcholine (ACh) modulates CaV2.1 channels in other brain regions and as cholinergic projections from the basal forebrain heavily target the GC layer (GCL) in OB, we investigated whether ACh modulates STP at the OCX→GC synapse. ACh decreases OCX→GC evoked EPSCs, it had no effect on STP. Thus, ACh impact on cortical feedback is independent of CaV2.1-mediated STP. Modulation of OCX feedback to the bulb by modulatory transmitters, such as ACh, or by frequency-dependent STP could regulate the precise balance of excitation and inhibition of GCs. As GCs are a major inhibitory source for OB output neurons, plasticity at the cortical feedback synapse can differentially impact OB output to higher-order networks in situations where ACh inputs are activated or by active sniff sampling of odors.

Apparent calcium dependence of vesicle recruitment.

Synaptic transmission relies on the recruitment of neurotransmitter-filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium-dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium-dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium-independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast-recruited vesicles but does not change the vesicle recruitment rates themselves. During sustained synaptic transmission, recruitment of new transmitter-filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca2+ concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca2+ buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity-dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca2+ buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin-Munc13 pathway, which has been proposed to mediate Ca2+ -dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca2+ -independent recruitment rates can explain the slowed recovery from depression upon increased Ca2+ buffering. Our data do not rule out the involvement of the calmodulin-Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca2+ -dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well-established two-pool models predict an apparent Ca2+ -dependence of vesicle recruitment. Thus, previous conclusions of Ca2+ -dependent vesicle recruitment based solely on increased intracellular Ca2+ buffering should be considered with caution.

Synaptotagmin 7 Mediates Both Facilitation and Asynchronous Release at Granule Cell Synapses.

When an action potential invades a presynaptic terminal it evokes large, brief Ca2+ signals that trigger vesicle fusion within milliseconds that is followed by a small residual Ca2+ (Cares) signal. At many synapses Cares produces synaptic facilitation that lasts up to hundreds of milliseconds and, although less common, Cares can also evoke asynchronous release (AR) that persists for tens of milliseconds. The properties of facilitation and AR are very different, which suggests that they are mediated by distinct mechanisms. However, recently it has been shown that the slow calcium sensor synaptotagmin 7 (Syt7) mediates facilitation at many synapses where AR does not occur, and conversely Syt7 can mediate AR without mediating facilitation. Here we study cerebellar granule cell synapses onto stellate cells and Purkinje cells in mice of both sexes to assess the role of Syt7 in these phenomena at the same synapse. This is of particular interest at granule cell synapses where AR is much more calcium dependent and shorter-lived than facilitation. We find that Syt7 can mediate these two processes despite their divergent properties. In Syt7 knock-out animals, facilitation and AR are smaller and shorter lived than in wild-type animals, even though the initial probability of release and Cares signals are unchanged. Although there are short-lived Syt7-independent mechanisms that mediate facilitation and AR in Syt7 KO animals, we find that at granule cell synapses AR and facilitation are both mediated primarily by Syt7.SIGNIFICANCE STATEMENT At synapses made by cerebellar granule cells, presynaptic activity elevates calcium for tens of milliseconds, which in turn evokes both asynchronous release (AR) and synaptic facilitation. AR is more calcium sensitive and shorter-lived than facilitation at these synapses, suggesting that they are mediated by different mechanisms. However, we find that the slow calcium sensor synaptotagmin 7 mediates both of these phenomena. Small, rapidly decaying components of AR and facilitation are present in Syt7 KO animals, indicating that additional mechanisms can contribute to both AR and facilitation at these synapses.

Long-Term Plasticity at the Mitral and Tufted Cell to Granule Cell Synapse of the Olfactory Bulb Investigated with a Custom Multielectrode in Acute Brain Slice Preparations.

Single extracellular stimulation electrodes are a widespread means to locally activate synaptic inputs in acute brain slices. Here we describe the fabrication and application of a multielectrode stimulator that was developed for conditions under which independent stimulation of several nearby sites is desirable. For the construction of the multielectrode we have developed a method by which electrode wires can be spaced at minimal distances of 100μm. This configuration increases the efficiency of stimulation paradigms, such as the comparison of proximal induced and control inputs for studies of synaptic plasticity.In our case the multielectrode was used for acute olfactory bulb slices to independently excite individual nearby glomeruli; the technique allowed us to demonstrate homosynaptic bidirectional long-term plasticity at the mitral/tufted cell to granule cell synapse. We also describe the determinants for successful recordings of long-term plasticity at this synapse, with mechanical and electrophysiological recording stability being tantamount. Finally, we briefly discuss data analysis procedures.

Weakened Intracellular Zn2+-Buffering in the Aged Dentate Gyrus and Its Involvement in Erasure of Maintained LTP.

Memory is lost by the increased influx of extracellular Zn2+ into neurons. It is possible that intracellular Zn2+ dynamics is modified even at non-zincergic medial perforant pathway-dentate granule cell synapses along with aging and that vulnerability to the modification is linked to age-related cognitive decline. To examine these possibilities, vulnerability of long-term potentiation (LTP) maintenance, which underlies memory retention, to modification of synaptic Zn2+ dynamics was compared between young and aged rats. The influx of extracellular Zn2+ into dentate granule cells was increased in aged rats after injection of high K+ into the dentate gyrus, but not in young rats. This increase impaired maintained LTP in aged rats. However, the impairment was rescued by co-injection of CaEDTA, an extracellular Zn2+ chelator, or CNQX, an AMPA receptor antagonist, which suppressed the Zn2+ influx. Maintained LTP was also impaired in aged rats after injection of ZnAF-2DA into the dentate gyrus that chelates intracellular Zn2+, but not in young rats. Interestingly, the capacity of chelating intracellular Zn2+ with intracellular ZnAF-2 was almost lost in the aged dentate gyrus 2h after injection of ZnAF-2DA into the dentate gyrus, suggesting that intracellular Zn2+-buffering is weakened in the aged dentate gyrus, compared to the young dentate gyrus. In the dentate gyrus of aged rats, maintained LTP is more vulnerable to modification of intracellular Zn2+ dynamics than in young rats, probably due to weakened intracellular Zn2+-buffering.

Hebbian Spike-Timing Dependent Plasticity at the Cerebellar Input Stage.

Spike-timing-dependent plasticity (STDP) is a form of long-term synaptic plasticity exploiting the time relationship between postsynaptic action potentials (APs) and EPSPs. Surprisingly enough, very little was known about STDP in the cerebellum, although it is thought to play a critical role for learning appropriate timing of actions. We speculated that low-frequency oscillations observed in the granular layer may provide a reference for repetitive EPSP/AP phase coupling. Here we show that EPSP-spike pairing at 6 Hz can optimally induce STDP at the mossy fiber-granule cell synapse in rats. Spike timing-dependent long-term potentiation and depression (st-LTP and st-LTD) were confined to a ±25 ms time-window. Because EPSPs led APs in st-LTP while APs led EPSPs in st-LTD, STDP was Hebbian in nature. STDP occurred at 6-10 Hz but vanished >50 Hz or <1 Hz (where only LTP or LTD occurred). STDP disappeared with randomized EPSP/AP pairing or high intracellular Ca2+ buffering, and its sign was inverted by GABA-A receptor activation. Both st-LTP and st-LTD required NMDA receptors, but st-LTP also required reinforcing signals mediated by mGluRs and intracellular calcium stores. Importantly, st-LTP and st-LTD were significantly larger than LTP and LTD obtained by modulating the frequency and duration of mossy fiber bursts, probably because STDP expression involved postsynaptic in addition to presynaptic mechanisms. These results thus show that a Hebbian form of STDP occurs at the cerebellum input stage, providing the substrate for phase-dependent binding of mossy fiber spikes to repetitive theta-frequency cycles of granule cell activity.SIGNIFICANCE STATEMENT Long-term synaptic plasticity is a fundamental property of the brain, causing persistent modifications of neuronal communication thought to provide the cellular basis of learning and memory. The cerebellum is critical for learning the appropriate timing of sensorimotor behaviors, but whether and how appropriate spike patterns could drive long-term synaptic plasticity remained unknown. Here, we show that this can actually occur through a form of spike-timing-dependent plasticity (STDP) at the cerebellar inputs stage. Pairing presynaptic and postsynaptic spikes at 6-10 Hz reliably induced STDP at the mossy fiber-granule cell synapse, with potentiation and depression symmetrically distributed within a ±25 ms time window. Thus, STDP can bind plasticity to the mossy fiber burst phase with high temporal precision.

Sniff-Like Patterned Input Results in Long-Term Plasticity at the Rat Olfactory Bulb Mitral and Tufted Cell to Granule Cell Synapse

During odor sensing the activity of principal neurons of the mammalian olfactory bulb, the mitral and tufted cells (MTCs), occurs in repetitive bursts that are synchronized to respiration, reminiscent of hippocampal theta-gamma coupling. Axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between the excitatory MTCs via reciprocal dendrodendritic synapses. We have explored long-term plasticity at this synapse by using a theta burst stimulation (TBS) protocol and variations thereof. GCs were excited via glomerular stimulation in acute brain slices. We find that TBS induces exclusively long-term depression in the majority of experiments, whereas single bursts (“single-sniff paradigm”) can elicit both long-term potentiation and depression. Statistical analysis predicts that the mechanism underlying this bidirectional plasticity involves the proportional addition or removal of presynaptic release sites. Gamma stimulation with the same number of APs as in TBS was less efficient in inducing plasticity. Both TBS- and “single-sniff paradigm”-induced plasticity depend on NMDA receptor activation. Since the onset of plasticity is very rapid and requires little extra activity, we propose that these forms of plasticity might play a role already during an ongoing search for odor sources. Our results imply that components of both short-term and long-term olfactory memory may be encoded at this synapse.

Inhibition of APP gamma-secretase restores Sonic Hedgehog signaling and neurogenesis in the Ts65Dn mouse model of Down syndrome

Neurogenesis impairment starting from early developmental stages is a key determinant of intellectual disability in Down syndrome (DS). Previous evidence provided a causal relationship between neurogenesis impairment and malfunctioning of the mitogenic Sonic Hedgehog (Shh) pathway. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain), a cleavage product of the trisomic gene APP (amyloid precursor protein) up-regulate transcription of Ptch1 (Patched1), the Shh receptor that keeps the pathway repressed. Since AICD results from APP cleavage by γ-secretase, the goal of the current study was to establish whether treatment with a γ-secretase inhibitor normalizes AICD levels and restores neurogenesis in trisomic neural precursor cells. We found that treatment with a selective γ-secretase inhibitor (ELND006; ELN) restores proliferation in neurospheres derived from the subventricular zone (SVZ) of the Ts65Dn mouse model of DS. This effect was accompanied by reduction of AICD and Ptch1 levels and was prevented by inhibition of the Shh pathway with cyclopamine. Treatment of Ts65Dn mice with ELN in the postnatal period P3–P15 restored neurogenesis in the SVZ and hippocampus, hippocampal granule cell number and synapse development, indicating a positive impact of treatment on brain development. In addition, in the hippocampus of treated Ts65Dn mice there was a reduction in the expression levels of various genes that are transcriptionally regulated by AICD, including APP, its origin substrate. Inhibitors of γ-secretase are currently envisaged as tools for the cure of Alzheimer's disease because they lower βamyloid levels. Current results provide novel evidence that γ-secretase inhibitors may represent a strategy for the rescue of neurogenesis defects in DS.

Dendrodendritic synapses in the mouse olfactory bulb external plexiform layer

ABSTRACTOdor information relayed by olfactory bulb projection neurons, mitral and tufted cells (M/T), is modulated by pairs of reciprocal dendrodendritic synaptic circuits in the external plexiform layer (EPL). Interneurons, which are accounted for largely by granule cells, receive depolarizing input from M/T dendrites and in turn inhibit current spread in M/T dendrites via hyperpolarizing reciprocal dendrodendritic synapses. Because the location of dendrodendritic synapses may significantly affect the cascade of odor information, we assessed synaptic properties and density within sublaminae of the EPL and along the length of M/T secondary dendrites. In electron micrographs the M/T to granule cell synapse appeared to predominate and was equivalent in both the outer and inner EPL. However, the dendrodendritic synapses from granule cell spines onto M/T dendrites were more prevalent in the outer EPL. In contrast, individual gephyrin‐immunoreactive (IR) puncta, a postsynaptic scaffolding protein at inhibitory synapses used here as a proxy for the granule to M/T dendritic synapse was equally distributed throughout the EPL. Of significance to the organization of intrabulbar circuits, gephyrin‐IR synapses are not uniformly distributed along M/T secondary dendrites. Synaptic density, expressed as a function of surface area, increases distal to the cell body. Furthermore, the distributions of gephyrin‐IR puncta are heterogeneous and appear as clusters along the length of the M/T dendrites. Consistent with computational models, our data suggest that temporal coding in M/T cells is achieved by precisely located inhibitory input and that distance from the soma is compensated for by an increase in synaptic density. J. Comp. Neurol. 523:1145–1161, 2015. © 2014 Wiley Periodicals, Inc.

Dendrodendritic synapses in the mouse olfactory bulb external plexiform layer.

Odor information relayed by olfactory bulb projection neurons, mitral and tufted cells (M/T), is modulated by pairs of reciprocal dendrodendritic synaptic circuits in the external plexiform layer (EPL). Interneurons, which are accounted for largely by granule cells, receive depolarizing input from M/T dendrites and in turn inhibit current spread in M/T dendrites via hyperpolarizing reciprocal dendrodendritic synapses. Because the location of dendrodendritic synapses may significantly affect the cascade of odor information, we assessed synaptic properties and density within sublaminae of the EPL and along the length of M/T secondary dendrites. In electron micrographs the M/T to granule cell synapse appeared to predominate and was equivalent in both the outer and inner EPL. However, the dendrodendritic synapses from granule cell spines onto M/T dendrites were more prevalent in the outer EPL. In contrast, individual gephyrin-immunoreactive (IR) puncta, a postsynaptic scaffolding protein at inhibitory synapses used here as a proxy for the granule to M/T dendritic synapse was equally distributed throughout the EPL. Of significance to the organization of intrabulbar circuits, gephyrin-IR synapses are not uniformly distributed along M/T secondary dendrites. Synaptic density, expressed as a function of surface area, increases distal to the cell body. Furthermore, the distributions of gephyrin-IR puncta are heterogeneous and appear as clusters along the length of the M/T dendrites. Consistent with computational models, our data suggest that temporal coding in M/T cells is achieved by precisely located inhibitory input and that distance from the soma is compensated for by an increase in synaptic density.