Hierarchical excitatory synaptic connectivity in mouse olfactory cortex

Topological motifs in synaptic connectivity—such as the cortical column—are fundamental to processing of information in cortical structures. However, the mesoscale topology of cortical networks beyond columns remains largely unknown. In the olfactory cortex, which lacks an obvious columnar structure, sensory-evoked patterns of activity have failed to reveal organizational principles of the network and its structure has been considered to be random. We probed the excitatory network in the mouse olfactory cortex using variance analysis of paired whole-cell recording in olfactory cortex slices. On a given trial, triggered network-wide bursts in disinhibited slices had remarkably similar time courses in widely separated and randomly selected cell pairs of pyramidal neurons despite significant trial-to-trial variability within each neuron. Simulated excitatory network models with random topologies only partially reproduced the experimental burst-variance patterns. Network models with local (columnar) or distributed subnetworks, which have been predicted as the basis of encoding odor objects, were also inconsistent with the experimental data, showing greater variability between cells than across trials. Rather, network models with power-law and especially hierarchical connectivity showed the best fit. Our results suggest that distributed subnetworks are weak or absent in the olfactory cortex, whereas a hierarchical excitatory topology may predominate. A hierarchical excitatory network organization likely underlies burst generation in this epileptogenic region, and may also shape processing of sensory information in the olfactory cortex.Structural and functional plasticity at excitatory synapses in cortical networks represents a fundamental mechanism for encoding sensory representations and memory. As a result, neuronal ensembles that are connected with high probability emerge as functional units to produce a population code of the environment. The topology of such excitatory circuits should contain signatures—as global topological motifs—that reflect the encoding strategy. The cortical column is a well-studied example of such a motif (1). Columnar cortices contain substantial distributed connectivity and some brain areas, such as association cortex, high-order cortices, and the piriform cortex, lack a pronounced columnar structure. In the piriform (olfactory) cortex, there exists only a rudimentary understanding of the relationship between network structure and cortical function. The axons of individual piriform pyramidal neurons ramify widely throughout the olfactory cortex, and only show patchiness on a very broad scale (2–4). Consistent with this architecture, neural activity in response to individual odorants is distributed broadly across the olfactory cortex as detected by 2-deoxyglucose, c-fos expression, multiunit recording, and population calcium imaging (5–8). Likewise, the receptive fields of individual neurons in piriform cortex and anterior olfactory cortex are broad (9, 10). Broad receptive fields in piriform cortex reflect convergence of input from many olfactory bulb glomeruli (11) and are strongly influenced by recurrent connectivity (12).These observations support a highly distributed population representation but reveal little about what processing function the piriform cortex performs. Physiological and anatomical studies have provided some clues. For example, neuronal responses in piriform cortex are specific for category of odorant (13), and odor identity and similarity are separately encoded in anterior and posterior piriform cortex, respectively (14), suggesting hierarchical coding. The endopiriform (EN) and preendopiriform nucleus (pEN), immediately subjacent to the piriform cortex, have dense recurrent connectivity and dense connectivity with overlying areas of piriform cortex (15, 16). The pEN, also called area tempestas, is a highly epileptogenic locus (16, 17). However, the physiological role of its dense connectivity is unknown (15, 18).To probe excitatory connectivity in the olfactory cortex, we isolated excitatory synaptic activity in a tailored brain slice containing the ventral anterior piriform cortex (APC_V), the pEN, the anterior olfactory cortex (AOC; also called anterior olfactory nucleus). Using weak stimulation of the lateral olfactory tract (LOT) input while blocking GABAergic inhibition and NMDA receptors, we evoked transient, all-or-none, network-wide bursts of excitation. Network-wide transient bursts are a dynamic circuit property shared by the hippocampus, neocortex, and piriform cortex in disinhibited recording conditions (19–21). We used the pairwise variance patterns detectable in the fine structure of these bursts as a probe of excitatory network topology. We compared whole-cell recordings from randomly selected pairs of principal neurons in olfactory cortex with patterns generated in simulated networks with a range of network topologies. Our findings suggest that excitatory connectivity in olfactory cortex is neither random nor organized into local or distributed subnetworks. Rather, it shows hierarchical connectivity.     

Learning reward timing in cortex through reward dependent expression of synaptic plasticity

The ability to represent time is an essential component of cognition but its neural basis is unknown. Although extensively studied both behaviorally and electrophysiologically, a general theoretical framework describing the elementary neural mechanisms used by the brain to learn temporal representations is lacking. It is commonly believed that the underlying cellular mechanisms reside in high order cortical regions but recent studies show sustained neural activity in primary sensory cortices that can represent the timing of expected reward. Here, we show that local cortical networks can learn temporal representations through a simple framework predicated on reward dependent expression of synaptic plasticity. We assert that temporal representations are stored in the lateral synaptic connections between neurons and demonstrate that reward-modulated plasticity is sufficient to learn these representations. We implement our model numerically to explain reward-time learning in the primary visual cortex (V1), demonstrate experimental support, and suggest additional experimentally verifiable predictions.     

Developmental regulation of protein interacting with C kinase 1 (PICK1) function in hippocampal synaptic plasticity and learning

AMPA-type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the mammalian central nervous system. Modulation of AMPAR trafficking supports several forms of synaptic plasticity thought to underlie learning and memory. Protein interacting with C kinase 1 (PICK1) is an AMPAR-binding protein shown to regulate both AMPAR trafficking and synaptic plasticity at many distinct synapses. However, studies examining the requirement for PICK1 in maintaining basal synaptic transmission and regulating synaptic plasticity at hippocampal Schaffer collateral-cornu ammonis 1 (SC-CA1) synapses have produced conflicting results. In addition, the effect of PICK1 manipulation on learning and memory has not been investigated. In the present study we analyzed the effect of genetic deletion of PICK1 on basal synaptic transmission and synaptic plasticity at hippocampal Schaffer collateral-CA1 synapses in adult and juvenile mice. Surprisingly, we find that loss of PICK1 has no significant effect on synaptic plasticity in juvenile mice but impairs some forms of long-term potentiation and multiple distinct forms of long-term depression in adult mice. Moreover, inhibitory avoidance learning is impaired only in adult KO mice. These results suggest that PICK1 is selectively required for hippocampal synaptic plasticity and learning in adult rodents.     

Synapse elimination accompanies functional plasticity in hippocampal neurons

A critical component of nervous system development is synapse elimination during early postnatal life, a process known to depend on neuronal activity. Changes in synaptic strength in the form of long-term potentiation (LTP) and long-term depression (LTD) correlate with dendritic spine enlargement or shrinkage, respectively, but whether LTD can lead to an actual separation of the synaptic structures when the spine shrinks or is lost remains unknown. Here, we addressed this issue by using concurrent imaging and electrophysiological recording of live synapses. Slices of rat hippocampus were cultured on multielectrode arrays, and the neurons were labeled with genes encoding red or green fluorescent proteins to visualize presynaptic and postsynaptic neuronal processes, respectively. LTD-inducing stimulation led to a reduction in the synaptic green and red colocalization, and, in many cases, it induced a complete separation of the presynaptic bouton from the dendritic spine. This type of synapse loss was associated with smaller initial spine size and greater synaptic depression but not spine shrinkage during LTD. All cases of synapse separation were observed without an accompanying loss of the spine during this period. We suggest that repeated low-frequency stimulation simultaneous with LTD induction is capable of restructuring synaptic contacts. Future work with this model will be able to provide critical insight into the molecular mechanisms of activity- and experience-dependent refinement of brain circuitry during development.     

Olfactory expression of a single and highly variable V1r pheromone receptor-like gene in fish species

Sensory neurons expressing members of the seven-transmembrane V1r receptor superfamily allow mice to perceive pheromones. These receptors, which exhibit no sequence homology to any known protein except a weak similarity to taste receptors, have only been found in mammals. In the mouse, the V1r repertoire contains >150 members, which are expressed by neurons of the vomeronasal organ, a structure present exclusively in some tetrapod species. Here, we report the existence of a single V1r gene in multiple species of a non-terrestrial, vomeronasal organ-lacking taxon, the teleosts. In zebrafish, this V1r gene is expressed in chemosensory neurons of the olfactory rosette with a punctate distribution, strongly suggesting a role in chemodetection. This unique receptor gene exhibits a remarkably high degree of sequence variability between fish species. It likely corresponds to the original V1r present in the common ancestor of vertebrates, which led to the large and very diverse expansion of vertebrate pheromone receptor repertoires, and suggests the presence of V1rs in multiple nonmammalian phyla.     

Essential role for a long-term depression mechanism in ocular dominance plasticity

The classic example of experience-dependent cortical plasticity is the ocular dominance (OD) shift in visual cortex after monocular deprivation (MD). The experimental model of homosynaptic long-term depression (LTD) was originally introduced to study the mechanisms that could account for deprivation-induced loss of visual responsiveness. One established LTD mechanism is a loss of sensitivity to the neurotransmitter glutamate caused by internalization of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Although it has been shown that MD similarly causes a loss of AMPARs from visual cortical synapses, the contribution of this change to the OD shift has not been established. Using an herpes simplex virus (HSV) vector, we expressed in visual cortical neurons a peptide (G2CT) designed to block AMPAR internalization by hindering the association of the C-terminal tail of the AMPAR GluR2 subunit with the AP2 clathrin adaptor complex. We found that G2CT expression interferes with NMDA receptor (NMDAR)-dependent AMPAR endocytosis and LTD, without affecting baseline synaptic transmission. When expressed in vivo, G2CT completely blocked the OD shift and depression of deprived-eye responses after MD without affecting baseline visual responsiveness or experience-dependent response potentiation in layer 4 of visual cortex. These data suggest that AMPAR internalization is essential for the loss of synaptic strength caused by sensory deprivation in visual cortex.     

RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.     

Syngap1 regulates experience-dependent cortical ensemble plasticity by promoting in vivo excitatory synapse strengthening

A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing–dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.

Autism risk genes converge on several neurobiological functions, including the regulation of synapse biology (1–3). Synapse processes directly controlled by autism spectrum disorder (ASD) risk genes include de novo synapse formation, synapse maturation, and activity-driven changes in synapse function (i.e., synapse plasticity). Synapse plasticity, especially in cortical excitatory neurons, is a process enabling neural circuits to store new information, which is essential for experience-dependent modifications of behavior to promote survival (4, 5). Thus, risk genes are thought to contribute to ASD etiology by disrupting how neural circuits change in response to novel experiences, which in turn contributes to maladaptive behaviors. However, the study of risk gene biology and their relationship to neural plasticity is largely restricted to reduced biological preparations that focus on isolated changes to a small subset of synapses. Therefore, it is unclear how risk gene–driven regulation of synapse plasticity contributes to changes in neural dynamics within intact functional networks known to drive adaptive behaviors.Neuronal ensembles, or groups of coactivated neurons, are thought to be the direct neural substrate of cognitive processes and behavior (6). In cortex, ensemble plasticity is a multidimensional process that reflects the distribution of distinct cellular plasticity mechanisms across individual neuronal components within the assembly. For example, neurons within the same sensory-evoked cortical ensemble can undergo either increases or decreases in activity in response to the same sensory experience (7–9). While this general phenomenon has been observed in multiple contexts, it is unclear how neurons within the same functional network can have opposing changes to enduring neuronal activity in response to the same sensory experience. One way that this may occur is through the simultaneous activation of distinct forms of experience-dependent plasticity that are differentially distributed throughout neurons that comprise a functional network. Indeed, sensory experience drives the induction of Hebbian-type synaptic plasticity that can strengthen or weaken excitatory synaptic input onto sensory-responsive neurons (10). Experience-dependent circuit plasticity is not limited to changes in excitatory synaptic strength. Robust changes to the function and connectivity of GABAergic interneurons within cortical microcircuits also occurs in response to novel experience, which in turn regulates the output of pyramidal neurons (11–13). Moreover, intrinsic changes to neuronal excitability have also been observed, and in combination with changes to GABAergic function, these collective processes are thought to maintain a set firing rate within networks even as activity is redistributed among individual neurons (8, 14, 15).We propose that experience induces heterogenous changes in activity within neurons of a cortical assembly through cellular processes controlled, at least in part, by genetic mechanisms linked to ASD risk. This hypothesis originates from the clear overrepresentation of ASD risk genes that regulate the neurobiology of synapses and synapse plasticity (1–3). However, because of the multidimensional nature of cortical network plasticity, one cannot infer how a gene influences experience-dependent changes in distributed network dynamics when the function of the gene has only been studied in isolated subcellular structures, such as synapses. It is therefore important to study major ASD risk genes in the context of intact functional networks. Doing so will help to elucidate how their influence over molecular and cellular functions contribute to intermediate network-level processes more directly linked to behaviors, such as cortical ensemble plasticity.In this study, we investigated how a major ASD risk gene, SYNGAP1/Syngap1 (HUMAN/mouse–mouse only from now on), regulates specific aspects of cellular plasticity in vivo and how this process shapes experience-dependent ensemble plasticity with sensory-responsive cortical networks. The Syngap1 gene, which is a major autism risk factor (16), is a robust regulator of various forms of long-term potentiation (LTP) (17), a cellular model of Hebbian plasticity. It regulates LTP through control of excitatory synapse structure and function by gating NMDA receptor-dependent regulation of AMPA receptor trafficking and dendritic spine size (18–20). The role of Syngap1 in regulating synapse plasticity has been observed by various researchers across distinct neuronal subtypes in a variety of in vitro and ex vivo preparations (21–24). Based on this past work in reduced preparations, we hypothesized that Syngap1 regulates experience-dependent ensemble plasticity by promoting the strengthening of excitatory synapses within functional cortical networks. We found that Syngap1 was required for spike-timing-dependent (STD) synaptic potentiation and experience-mediated synapse bouton formation in layer (L) 2/3 of somatosensory cortex (SSC) but not synaptic depression or synapse bouton elimination. Syngap1 heterozygosity in mice disrupted experience-dependent potentiation of neuronal activity within a subpopulation of L2/3 SSC neurons. Syngap1 loss of function had no effect on plasticity of neurons within the same ensemble that weakens with experience. These findings indicate that disruptions to synapse-level strengthening mechanisms in Syngap1 mice contribute to imbalanced cortical ensemble plasticity driven by novel sensory experience. We propose that a key function of Syngap1 is to promote complex network-level plasticity through the strengthening of excitatory connections within cortical circuits.     

Dendritic coding of multiple sensory inputs in single cortical neurons in vivo

Single cortical neurons in the mammalian brain receive signals arising from multiple sensory input channels. Dendritic integration of these afferent signals is critical in determining the amplitude and time course of the neurons' output signals. As of yet, little is known about the spatial and temporal organization of converging sensory inputs. Here, we combined in vivo two-photon imaging with whole-cell recordings in layer 2 neurons of the mouse vibrissal cortex as a means to analyze the spatial pattern of subthreshold dendritic calcium signals evoked by the stimulation of different whiskers. We show that the principle whisker and the surrounding whiskers can evoke dendritic calcium transients in the same neuron. Distance-dependent attenuation of dendritic calcium transients and the corresponding subthreshold depolarization suggest feed-forward activation. We found that stimulation of different whiskers produced multiple calcium hotspots on the same dendrite. Individual hotspots were activated with low probability in a stochastic manner. We show that these hotspots are generated by calcium signals arising in dendritic spines. Some spines were activated uniquely by single whiskers, but many spines were activated by multiple whiskers. These shared spines indicate the existence of presynaptic feeder neurons that integrate and transmit activity arising from multiple whiskers. Despite the dendritic overlap of whisker-specific and shared inputs, different whiskers are represented by a unique set of activation patterns within the dendritic field of each neuron.     

Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor

Phosphorylation of the GluA1 subunit of AMPA receptors has been proposed to regulate receptor trafficking and synaptic transmission and plasticity. However, it remains unclear whether GluA1 phosphorylation is permissive or sufficient for enacting these functional changes. Here we investigate the role of GluA1 phosphorylation at S831 and S845 residues in the hippocampus through the analyses of GluA1 S831D/S845D phosphomimetic knock-in mice. S831D/S845D mice showed normal total and surface expression and subcellular localization of GluA1 as well as intact basal synaptic transmission. In addition, theta-burst stimulation, a protocol that was sufficient to induce robust long-term potentiation (LTP) in WT mice, resulted in LTP of similar magnitude in S831D/S845D mice. However, S831D/S845D mice showed LTP induced with 10-Hz stimulation, a protocol that is weaker than theta-burst stimulation and was not sufficient to induce LTP in WT mice. Moreover, S831D/S845D mice exhibited LTP induced with spike-timing-dependent plasticity (STDP) protocol at a long pre-post interval that was subthreshold for WT mice, although a suprathreshold STDP protocol at a short pre-post interval resulted in similarly robust LTP for WT and S831D/S845D mice. These results indicate that phosphorylation of GluA1 at S831 and S845 is sufficient to lower the threshold for LTP induction, increasing the probability of synaptic plasticity.     

Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons

l-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons l-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. l-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, l-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of l-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of l-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived l-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of l-lactate as a signaling molecule for neuronal plasticity.The transfer of l-lactate from astrocytes to neurons was recently shown to be necessary for the establishment of long-term memory (LTM) in an inhibitory avoidance (IA) paradigm and for the maintenance of in vivo long-term potentiation (LTP) in the rodent hippocampus (1). This key role of l-lactate in neuronal plasticity mechanisms was demonstrated in experiments in which specific pharmacological and gene expression down-regulation interventions were implemented to prevent the production of l-lactate from glycogen—which is exclusively localized in astrocytes—and its release from these cells in the hippocampus during behavioral training (1). Such interventions completely prevented the establishment of LTM and their effect was fully reversed by the intrahippocampal administration of l-lactate during the training session. The fact that glucose at equicaloric concentrations only marginally mimicked the rescuing effect of l-lactate was taken as an unexpected indication that the primary mechanism of action of l-lactate on plasticity mechanisms was independent of its ability to act as an energy substrate. A role of l-lactate in memory processes was also recently shown in other behavioral paradigms (2, 3). We therefore set out to investigate the molecular mechanisms at the basis of the function of l-lactate on neuronal plasticity.Molecular mechanisms underlying both LTM and long-term plasticity include the induction of expression of a group of immediate early genes (IEGs) such as early growth response 1 (Zif268 or Egr1), CCAAT/enhancer binding protein (C/EBP), and proto-oncogene c-Fos (c-Fos) as well as activity-regulated cytoskeletal-associated protein (Arc or Arg3.1) as a direct effector protein at the synapse, which all participate to different physiological processes associated with neuronal plasticity (4–6). Although stimulation of expression of these IEGs is not restricted to plasticity processes, they are considered as key plasticity-related genes in sustaining such phenomena. In addition, late response genes such as brain-derived neurotrophic factor (BDNF) have also been demonstrated to be major intermediates of plasticity-related processes (7). A role of NMDA receptors (NMDARs) in such plasticity mechanisms is well-established (5, 8).In this article we describe a cascade of molecular events demonstrating that l-lactate stimulates plasticity-related gene expression in neurons through modulation of NMDAR activity associated with changes in redox cellular state. The induction of plasticity gene expression by l-lactate was observed in primary cultures of neurons as well as in vivo in the sensory-motor cortex of mice.     

Histamine in the basolateral amygdala promotes inhibitory avoidance learning independently of hippocampus

Recent discoveries demonstrated that recruitment of alternative brain circuits permits compensation of memory impairments following damage to brain regions specialized in integrating and/or storing specific memories, including both dorsal hippocampus and basolateral amygdala (BLA). Here, we first report that the integrity of the brain histaminergic system is necessary for long-term, but not for short-term memory of step-down inhibitory avoidance (IA). Second, we found that phosphorylation of cyclic adenosine monophosphate (cAMP) responsive-element-binding protein, a crucial mediator in long-term memory formation, correlated anatomically and temporally with histamine-induced memory retrieval, showing the active involvement of histamine function in CA1 and BLA in different phases of memory consolidation. Third, we found that exogenous application of histamine in either hippocampal CA1 or BLA of brain histamine-depleted rats, hence amnesic, restored long-term memory; however, the time frame of memory rescue was different for the two brain structures, short lived (immediately posttraining) for BLA, long lasting (up to 6 h) for the CA1. Moreover, long-term memory was formed immediately after training restoring of histamine transmission only in the BLA. These findings reveal the essential role of histaminergic neurotransmission to provide the brain with the plasticity necessary to ensure memorization of emotionally salient events, through recruitment of alternative circuits. Hence, our findings indicate that the histaminergic system comprises parallel, coordinated pathways that provide compensatory plasticity when one brain structure is compromised.Emotionally arousing experiences create long-term memories that are initially labile, but over time become insensitive to disruption through a process known as consolidation (1). One-trial fear-motivated learning tasks, such as step-down inhibitory avoidance (IA), a hippocampal-dependent associative learning (2), have largely contributed to the knowledge of consolidation process, and convincing evidence indicates that the CA1 region of the hippocampus, the basolateral amygdala (BLA) and the medial prefrontal cortex, are crucially involved in this process (3, 4). However, the actual contribution of each region remains poorly characterized. For instance, it is suggested that they are part of mostly independent circuitries specialized in encoding specific aspects of information, e.g., the emotional component in the BLA and the cognitive aspect in the hippocampus (2, 5). If this relation between structures holds true for an intact brain, however, examples of learning recovery of individuals bearing large brain lesions suggest that the brain can adapt dynamically, and interconnected systems can provide compensation for selective damage (2). Indeed, hippocampal damage resulted in retrograde amnesia for acquired fear memories (6), but did not prevent new learning in rodents (7) as well as in humans (8). Therefore, hippocampal damage before new learning can be overcome, possibly through recruitment of an alternative circuit. The identity of the compensatory structures is still unknown, but the BLA could potentially be one, because it operates to a certain extent in parallel with the CA1 region in memory processing (9).Extensive evidence indicates that emotionally significant experiences activate many hormones and neurotransmitters, including histamine, that regulate the consolidation of newly acquired memories (10, 11). Histamine is synthesized from histidine by histidine-decarboxylase (HDC) (12) and released in the brain from varicosities of axons that ramify extensively throughout the central nervous system. The only source of histaminergic fibers is the hypothalamic tuberomamillary nucleus (TMN) (13). The histaminergic system is crucial in the sleep–wake cycle and is implicated in various brain functions, including the modulation of hippocampal synaptic plasticity (12, 14). Interestingly, when infused into the CA1 region immediately after training of an IA task, histamine induced a dose-dependent promnesic effect through activation of H₂ receptors without altering locomotor activity, exploratory behavior, anxiety state, or retrieval of the avoidance response (15). Consistently, posttraining injections into the dorsal hippocampus of histamine H₂ or H₃ receptor agonists improved memory consolidation after contextual fear conditioning through a mechanism involving extracellular signal-regulated kinase (ERK)₂ phosphorylation (16). Several neurotransmitters, such as dopamine, glutamate, and norepinephrine, activate the ERK cascade in the hippocampus (17), and histamine may interact with these neurotransmitters to orchestrate ERK₂ phosphorylation that appears to play a critical role in consolidating emotional memories (17). Histamine modulates memory of emotionally arousing experiences also in the BLA. Administration of H₃ receptor antagonists into the BLA impaired consolidation of fear memories (18), whereas H₃ receptor agonists ameliorated the expression of adverse memories (19). This effect involved H₂ receptors and was accompanied by a bimodal modulation of the local cholinergic tone (18, 19).Although these findings indicate that administration of histaminergic ligands modulates memory consolidation, studies have not yet investigated the role of endogenous histamine in creating memories for emotionally arousing training. To answer this question, we performed a first set of the experiments to examine how brain histamine depletion obtained by using intralateral ventricle (LV) administration of a-fluoromethylhistidine (a-FMHis), a suicide inhibitor of HDC (20), affected IA memory processes. With the second set of experiments, we investigated IA training-induced CREB phosphorylation in the brain of normal and histamine-depleted rats. The last set of experiments learned whether the local infusion of histamine in the BLA or the CA1 region, respectively, overcame a-FMHis–induced amnesia for IA training.     

Dose-dependent effects of prenatal ethanol exposure on synaptic plasticity and learning in mature offspring

BACKGROUND: We have observed profound deficits in hippocampal synaptic plasticity and one-trial learning in offspring whose mothers drank moderate quantities of ethanol during pregnancy. In the present study, we examined the question of whether lower maternal blood ethanol concentrations (BECs) could produce functional deficits in offspring. METHODS: Rat dams consumed either a 2%, 3%, or 5% ethanol liquid diet throughout gestation. Three other groups of dams were pair-fed a 0% ethanol liquid diet, and a seventh group consumed lab chow ad libitum. Adult offspring from each diet group were assigned either to studies of evoked [3H]-D-aspartate (D-ASP) release from hippocampal slices or spatial learning studies using the Morris Water Task. RESULTS: Consumption of the 2%, 3%, and 5% ethanol liquid diets produced mean peak maternal BECs of 7, 30 and 83 mg/dL, respectively. Consumption of these ethanol diets had no effect on offspring birthweight, litter size or neonatal mortality. Likewise, evoked D-ASP release from hippocampal slices and performance on a standard version of the Morris Water Task were not affected by prenatal ethanol exposure. By contrast, activity-dependent potentiation of evoked D-ASP release from slices and one-trial learning on a "moving platform" version of the Morris Water Task were markedly reduced in offspring whose mothers consumed the 5% ethanol liquid diet. Intermediate deficits in these two parameters were observed in offspring from the 3% ethanol diet group, whereas offspring from the 2% ethanol diet group were not statistically different than controls. CONCLUSIONS: We conclude that the threshold for eliciting subtle, yet significant learning deficits in offspring prenatally exposed to ethanol is less than 30 mg/dL. This BEC is roughly equivalent to drinking 1 to 1.5 ounces of ethanol per day.     

GluN2B subunit-containing NMDA receptor antagonists prevent Aβ-mediated synaptic plasticity disruption in vivo

Currently, treatment with the relatively low-affinity NMDA receptor antagonist memantine provides limited benefit in Alzheimer''s disease (AD). One probable dose-limiting factor in the use of memantine is the inhibition of NMDA receptor-dependent synaptic plasticity mechanisms believed to underlie certain forms of memory. Moreover, amyloid-β protein (Aβ) oligomers that are implicated in causing the cognitive deficits of AD potently inhibit this form of plasticity. Here we examined if subtype-preferring NMDA receptor antagonists could preferentially protect against the inhibition of NMDA receptor-dependent plasticity of excitatory synaptic transmission by Aβ in the hippocampus in vivo. Using doses that did not affect control plasticity, antagonists selective for NMDA receptors containing GluN2B but not other GluN2 subunits prevented Aβ_1–42 -mediated inhibition of plasticity. Evidence that the proinflammatory cytokine TNFα mediates this deleterious action of Aß was provided by the ability of TNFα antagonists to prevent Aβ_1–42 inhibition of plasticity and the abrogation of a similar disruptive effect of TNFα using a GluN2B-selective antagonist. Moreover, at nearby synapses that were resistant to the inhibitory effect of TNFα, Aβ_1–42 did not significantly affect plasticity. These findings suggest that preferentially targeting GluN2B subunit-containing NMDARs may provide an effective means of preventing cognitive deficits in early Alzheimer''s disease.     

Follistatin mediates learning and synaptic plasticity via regulation of Asic4 expression in the hippocampus

The biological mechanisms underpinning learning are unclear. Mounting evidence has suggested that adult hippocampal neurogenesis is involved although a causal relationship has not been well defined. Here, using high-resolution genetic mapping of adult neurogenesis, combined with sequencing information, we identify follistatin (Fst) and demonstrate its involvement in learning and adult neurogenesis. We confirmed that brain-specific Fst knockout (KO) mice exhibited decreased hippocampal neurogenesis and demonstrated that FST is critical for learning. Fst KO mice exhibit deficits in spatial learning, working memory, and long-term potentiation (LTP). In contrast, hippocampal overexpression of Fst in KO mice reversed these impairments. By utilizing RNA sequencing and chromatin immunoprecipitation, we identified Asic4 as a target gene regulated by FST and show that Asic4 plays a critical role in learning deficits caused by Fst deletion. Long-term overexpression of hippocampal Fst in C57BL/6 wild-type mice alleviates age-related decline in cognition, neurogenesis, and LTP. Collectively, our study reveals the functions for FST in adult neurogenesis and learning behaviors.

A wide variety of human disorders such as intellectual disability feature impairment of learning and memory. These conditions have a profound impact on quality of life and social functioning. Despite this, the biological mechanisms underpinning learning are not yet fully understood. However, mounting evidence has suggested that hippocampal neurogenesis is involved (1). Several publications report learning or emotional phenotypes in rodent models, which have little or no neurogenesis in adulthood (2–5), although these, and other findings, have been questioned (6). Despite the lack of consensus on the causal relationship about adult hippocampal neurogenesis on learning, it is possible that the same genes affect both neurogenesis and learning. Indeed, in mouse inbred strains, neurogenesis is genetically correlated with performance in spatial learning and memory tasks (7, 8), and spatial memory in rats is related to the levels of hippocampal neurogenesis (9).We hypothesized that one way to identify genes that influence learning is to identify those that contribute to heritable variation in neurogenesis (10). In this study, we used genetic mapping data from heterogeneous stock (HS) mice to identify loci associated with neurogenesis (11). We increased mapping resolution further by the incorporation of sequence information. This technique has been shown to increase mapping resolution to the point of identifying causal variants (12). One of the target genes, Fst, is predominantly expressed in the cortex, olfactory bulb, and dentate gyrus. Interestingly, two of these regions are where adult neurogenesis occurs. Fst is known to encode the protein follistatin (FST), an activin-binding protein (13, 14), which neutralizes activin bioactivity (15). FST also binds to other members of the transforming growth factor-β superfamily but with a 10-fold lower affinity than for activin A (16). In the brain, activin has been shown to play a role in the maintenance of long-term memory (17). Despite numerous studies about the functions of FST in regulation of muscle growth (18) and energy metabolism (19), its roles in the brain are still unknown.In this study, we used brain-specific Fst knockout (KO) mice to confirm its effect on neurogenesis, and we identified learning deficits in the Fst KO mice as well as deficits in long-term potentiation (LTP) through the regulation of acid-sensing ion channel 4 (ASIC4). Our study demonstrates the power of combining genetic mapping with functional work, and we provide insights into the role of FST in the hippocampus and its influence on learning.     

A role for MHC class I molecules in synaptic plasticity and regeneration of neurons after axotomy

Recently, MHC class I molecules have been shown to be important for the retraction of synaptic connections that normally occurs during development [Huh, G.S., Boulanger, L. M., Du, H., Riquelme, P. A., Brotz, T. M. & Shatz, C. J. (2000) Science 290, 2155-2158]. In the adult CNS, a classical response of neurons to axon lesion is the detachment of synapses from the cell body and dendrites. We have investigated whether MHC I molecules are involved also in this type of synaptic detachment by studying the synaptic input to sciatic motoneurons at 1 week after peripheral nerve transection in beta2-microglobulin or transporter associated with antigen processing 1-null mutant mice, in which cell surface MHC I expression is impaired. Surprisingly, lesioned motoneurons in mutant mice showed more extensive synaptic detachments than those in wild-type animals. This surplus removal of synapses was entirely directed toward inhibitory synapses assembled in clusters. In parallel, a significantly smaller population of motoneurons reinnervated the distal stump of the transected sciatic nerve in mutants. MHC I molecules, which traditionally have been linked with immunological mechanisms, are thus crucial for a selective maintenance of synapses during the synaptic removal process in neurons after lesion, and the lack of MHC I expression may impede the ability of neurons to regenerate axons.     

Experience-dependent functional plasticity and visual response selectivity of surviving subplate neurons in the mouse visual cortex

Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca²⁺ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

The mammalian cerebral cortex consists of six layers, with distinct roles in information processing (1, 2). At the bottom of the neocortex, on the boundary between the gray matter and white matter, there is a thin sheet of neurons called layer 6b (L6b) (3). Layer 6b neurons are thought to be remnants of subplate neurons based on their location and cell-type marker expression (4). During prenatal and early postnatal periods, subplate neurons form transient neuronal circuits that play key roles in cortical maturation (5–7). In the embryonic cortex, subplate neurons form short-lived synapses with early immature neurons to regulate radial migration (8). During perinatal development, subplate neurons transiently receive inputs from ingrowing thalamic axons and innervate layer 4 (L4) to guide thalamic inputs to the eventual target, L4 (5, 6). Thus, the circuits formed by subplate neurons at the perinatal developmental stage are essential to establish basic neuronal circuits before starting experience-dependent refinements (5–7). Subsequently, subplate neurons largely disappear due to programmed cell death, but some survive and reside in L6b (5, 6). In the adult cortex, L6b neurons form neuronal circuits with local and long-distance neurons, which are different from those formed during early development (9–12). Therefore, surviving subplate neurons may acquire a role in information processing after remodeling of neuronal connections. A recent study using three-photon Ca²⁺ imaging demonstrated that L6b neurons show visual responses with broad orientation/direction tuning in the adult mouse primary visual cortex (V1) (13). However, comparable evidence for L6b response properties with other layer neurons in V1 is lacking (14–20). Moreover, L6b neurons have diverse morphology and molecular expression (21–24). Neurons born during subplate neurogenesis show the different expression patterns of subplate markers in postnatal L6b (4). However, the response properties in each subtype of L6b neurons remain unknown.The sensory responsiveness of cortical neurons is considerably refined by sensory experience relatively late in development, referred to as the critical period (25, 26). Previous studies have demonstrated that sensory activities before the onset of the critical period affect the arrangement of subplate neuron neurites in the barrel cortex and local subplate circuits in the auditory cortex (27, 28). However, there is no direct evidence that the sensory responses of surviving subplate neurons are modified by sensory experience during the critical period. If experience-dependent plasticity occurs in subplate neuron responses, they will contribute to the experience-dependent development of sensory functions and possibly to the functions in the mature cortex. Ocular dominance (OD) plasticity in V1 is a canonical model used to examine experience-dependent refinement of sensory responses (25, 26, 29, 30). If one eye is occluded for several days during the critical period, neurons in V1 lose their response to the deprived eye. OD plasticity is robustly preserved across species and cell types. Therefore, OD plasticity is suitable for evaluating experience-dependent plasticity in L6b neurons.This study aimed to characterize the visual responses and OD plasticity of L6b neurons in V1. Toward this goal, two-photon Ca²⁺ imaging was performed in awake juvenile mice, followed by 3D immunohistochemistry with a subplate neuronal marker, connective tissue growth factor (CTGF) (4, 31). L6b neurons showed broader tuning to visual stimuli and lower binocular matching of orientation preference than did layer 2/3 (L2/3) and L6a neurons. Chronic two-photon imaging revealed significant OD plasticity in individual L6b neurons during the critical period. Our results provide strong evidence that L6b neurons, presumed to be subplate neuron remnants, exhibit sensory responses and experience-dependent functional plasticity at a relatively late stage of cortical development.     

Structural plasticity within highly specific neuronal populations identifies a unique parcellation of motor learning in the adult brain

Cortical networks undergo adaptations during learning, including increases in dendritic complexity and spines. We hypothesized that structural elaborations during learning are restricted to discrete subsets of cells preferentially activated by, and relevant to, novel experience. Accordingly, we examined corticospinal motor neurons segregated on the basis of their distinct descending projection patterns, and their contribution to specific aspects of motor control during a forelimb skilled grasping task in adult rats. Learning-mediated structural adaptations, including extensive expansions of spine density and dendritic complexity, were restricted solely to neurons associated with control of distal forelimb musculature required for skilled grasping; neurons associated with control of proximal musculature were unchanged by the experience. We further found that distal forelimb-projecting and proximal forelimb-projecting neurons are intermingled within motor cortex, and that this distribution does not change as a function of skill acquisition. These findings indicate that representations of novel experience in the adult motor cortex are associated with selective structural expansion in networks of functionally related, active neurons that are distributed across a single cortical domain. These results identify a distinct parcellation of cortical resources in support of learning.     

Spine dynamics of PSD-95-deficient neurons in the visual cortex link silent synapses to structural cortical plasticity

Critical periods (CPs) are time windows of heightened brain plasticity during which experience refines synaptic connections to achieve mature functionality. At glutamatergic synapses on dendritic spines of principal cortical neurons, the maturation is largely governed by postsynaptic density protein-95 (PSD-95)-dependent synaptic incorporation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into nascent AMPA-receptor silent synapses. Consequently, in mouse primary visual cortex (V1), impaired silent synapse maturation in PSD-95-deficient neurons prevents the closure of the CP for juvenile ocular dominance plasticity (jODP). A structural hallmark of jODP is increased spine elimination, induced by brief monocular deprivation (MD). However, it is unknown whether impaired silent synapse maturation facilitates spine elimination and also preserves juvenile structural plasticity. Using two-photon microscopy, we assessed spine dynamics in apical dendrites of layer 2/3 pyramidal neurons (PNs) in binocular V1 during ODP in awake adult mice. Under basal conditions, spine formation and elimination ratios were similar between PSD-95 knockout (KO) and wild-type (WT) mice. However, a brief MD affected spine dynamics only in KO mice, where MD doubled spine elimination, primarily affecting newly formed spines, and caused a net reduction in spine density similar to what has been observed during jODP in WT mice. A similar increase in spine elimination after MD occurred if PSD-95 was knocked down in single PNs of layer 2/3. Thus, structural plasticity is dictated cell autonomously by PSD-95 in vivo in awake mice. Loss of PSD-95 preserves hallmark features of spine dynamics in jODP into adulthood, revealing a functional link of PSD-95 for experience-dependent synapse maturation and stabilization during CPs.

Early life of an animal is characterized by time windows of functionally and structurally enhanced brain plasticity known as critical periods (CPs), which have been described initially in the primary visual cortex (V1) of kittens (1). During CPs, experience refines the connectivity of principal excitatory neurons to establish the mature functionality of neural networks. This refinement is governed by the constant generation and elimination of nascent synapses on dendritic spines that sample favorable connections to be consolidated and unfavorable ones to be eliminated (2–5). A fraction of nascent synapses is or becomes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor silent, expressing N-methyl-D-aspartate (NMDA) receptors only (6–8). At eye opening, silent synapses are abundant in the primary visual cortex (V1) (9, 10) and mature during CPs by stable AMPA receptor incorporation (11–14). The pace of silent synapse maturation is governed by the opposing yet cooperative function of postsynaptic density protein of 95 kDa (PSD-95) and its paralog PSD-93, two signaling scaffolds of the postsynaptic density of excitatory synapses (12, 13). However, whether silent synapses are preferential substrates for spine elimination during CPs remains to be investigated.In juvenile mice (postnatal days [P] 20 to 35), a brief monocular deprivation (MD) of the dominant contralateral eye results in a shift of the ocular dominance (OD) of binocular neurons in V1 toward the open eye, mediated by a reduction of responses to visual stimulation of the deprived eye (15–17). Structurally, MD induces an increase in spine elimination in apical dendrites of layer (L) 2/3 and L5 pyramidal neurons (PNs) which is only observed during the CP and constitutes a hallmark of juvenile OD plasticity (jODP) (18–20). After CP closure, cortical plasticity declines progressively, and in standard cage-raised mice beyond P40, a 4-d MD no longer induces the functional nor anatomical changes associated with jODP (21–24).At least three different mechanisms involved in experience-dependent maturation of cortical neural networks have been described, but the molecular and cellular mechanisms that cause CP closure remain highly debated (18, 25, 26). First, plasticity of local inhibitory neurons, such as increased inhibitory tone or a reduction of release probability by experience-dependent endocannabinoid receptor 1 (CB1R) activation was reported to close the critical period in rodent V1 (27–29). Second, the expression of so-called “plasticity brakes,” such as extracellular matrix (ECM), Nogo receptor 1 (NgR1), paired immunoglobulin-like receptor B (PirB), and Lynx1 were correlated with the end of critical periods (30–33). Experimentally decreasing the inhibitory tone or absence of plasticity brakes enhanced ODP expression in various knockout (KO) mouse models (32, 34, 35), among which only Lynx1 KO mice were shown to exhibit functional hallmarks of jODP, such as selective deprived eye depression after a short MD (36). Structurally, Lynx1 KO mice exhibited elevated spine dynamics at baseline; however, MD induced a reduction in spine elimination in apical dendrites of L5 PNs, whereas in L2/3 PNs there was no change (37). Thus, the effects of removing plasticity brakes on structural plasticity are variable, and it remains unclear to what extend manipulating the plasticity brakes can reinstate cellular signatures of CP plasticity in the adult wild-type (WT) brain (38). Third, the progressive maturation of AMPAR-silent synapses was correlated with the closure of the CP for jODP (12, 13). Consequently, in PSD-95 KO mice, the maturation of silent synapses is impaired; their fraction remains at the eye opening level, and jODP is preserved lifelong (13). Furthermore, visual cortex-specific knockdown (KD) of PSD-95 in the adult brain reinstated jODP. In contrast, in PSD-93 KO mice, silent synapses mature precociously and the CP for jODP closes precociously (12), correlating the presence of silent synapses with functional plasticity during CPs.While these three mechanisms of CP closure are not mutually exclusive in regulating cortical plasticity (26), it remains elusive whether CP-like structural plasticity can be expressed in the adult brain and whether silent synapses might be substrates for it. Here, we performed chronic two-photon imaging of dendrites of L2/3 pyramidal neurons in binocular V1 of PSD-95 KO (and KD) and WT mice, tracking the same dendritic spines longitudinally before, during, and after a 4-d period of MD. As previous studies have reported anesthesia effects on spine dynamics (39–41), we performed our experiments in awake mice, thoroughly trained for head fixation under the two-photon microscope. Our chronic spine imaging experiments revealed that in adult PSD-95 KO and KD mice, a brief MD indeed increased spine elimination about twofold, while adult WT mice did not display experience-dependent changes in spine elimination or spine formation. Thus, the loss of PSD-95 led to a high number of AMPAR-silent synapses which were correlated with jODP after MD, and with juvenile-like structural plasticity even in the adult brain, underscoring the importance of silent synapses for CP-timing and network maturation and stabilization.     

NMDARs in granule cells contribute to parallel fiber–Purkinje cell synaptic plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

The ability of an organism to adjust its behavior to environmental demands depends on its capacity to learn and execute coordinated movements. The cerebellum plays a central role in this process by optimizing motor programs through trial-and-error learning (1). Within the cerebellum, the synaptic output from granule cells (GCs) to Purkinje cells (PCs) shapes computational operations during basal motor function and serves as a substrate for motor learning (2). Several forms of motor learning depend on changes in the strength of the parallel fiber (PF), the axon of GCs, to the PC synapse (3, 4).In the mammalian forebrain, synaptic plasticity typically relies on postsynaptic N-methyl-D-aspartate receptor (NMDAR) activation, which alters AMPA receptor (AMPAR) turnover at the postsynaptic site (5). However, this may not extend to the cerebellar synapse between GCs and PCs, since no functional postsynaptic NMDARs have been identified in young or adult rodents (6, 7). Pharmacological approaches, however, have shown that both long-term depression (LTD) and long-term potentiation (LTP) induction depend on NMDAR activation at the PF-PC synapse in young rodents (8–12). Hence, the alternative mechanisms for NMDAR-dependent synaptic modulation may involve presynaptic NMDARs activation [(12–15); for review: refs. 16 and 17]. Indeed, cell-specific deletion of NMDARs in GCs abolishes LTP in young rodents (12). In addition to NMDARs, PF-PC synaptic plasticity also requires nitric-oxide (NO) signaling (18–20). As nitric-oxide synthase (NOS) is expressed in GCs, but not in PCs (21), the activation of presynaptic NMDARs might allow Ca²⁺ influx that activates NO synthesis, which in turn may act upon the PCs. However, in the mature cerebellum, the existence of presynaptic NMDARs on PFs and the role of NO in PF-PC plasticity remains a matter of debate. Previously, we have proposed that the activation of putatively presynaptic NMDARs in young rodents is necessary for inducing PF-PC synaptic plasticity without affecting transmitter release (8, 9, 11, 12). More recently, it has been shown that a subset of PFs express presynaptic NMDARs containing GluN2A subunits and that these receptors are functional (11, 12). Thus, in contrast to their role at other synapses, at least in young rodent, presynaptic NMDARs as part of the PF-PC synapses might act via the production of NO to induce postsynaptic plasticity, without altering neurotransmitter release (9, 11, 12, 18–22). However, a causal link between NMDARs activation in PFs, NO synthesis, and synaptic plasticity induction is still missing.In the cerebral cortex, the expression of presynaptic NMDARs is developmentally regulated (23, 24). However, little is known about the presence and function of presynaptic NMDARs in adult tissue. In the adult cerebellum, PCs only express postsynaptic NMDARs at their synapse with climbing fibers (CFs) (25). It has been proposed that the activation of these receptors could have heterosynaptic effects during PF-PC LTD. This mechanism would explain why LTD in adults depends on NMDARs. According to this model, presynaptic NMDARs would be a transient feature of developing tissue and not necessary for induction of synaptic plasticity and motor learning in adult animals (25).Here, we combine electron microscopy, two-photon calcium imaging, synaptic plasticity experiments, and behavioral measurements to show that presynaptic NMDARs are not developmentally regulated but are required for cerebellar motor learning in adults. We demonstrate that presynaptic NMDARs are present and functional in PFs of mature rodents. By specifically deleting the NMDAR subunit GluN1 either in the post- (PC) or the presynaptic cells (GCs), we demonstrate that NMDAR activation in GCs plays a key role in bidirectional synaptic plasticity and in vestibulo-ocular reflex (VOR) adaptation, an important paradigm for testing cerebellar motor learning (26–28). In contrast, NMDARs in PCs are neither involved in PF-PC synaptic plasticity nor required for cerebellar motor learning.