Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

Nicotinic and muscarinic agonists and acetylcholinesterase inhibitors stimulate a common pathway to enhance GluN2B-NMDAR responses

Nicotinic and muscarinic ACh receptor agonists and acetylcholinesterase inhibitors (AChEIs) can enhance cognitive function. However, it is unknown whether a common signaling pathway is involved in the effect. Here, we show that in vivo administration of nicotine, AChEIs, and an m1 muscarinic (m1) agonist increase glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GluN2B)-containing NMDA receptor (NR2B-NMDAR) responses, a necessary component in memory formation, in hippocampal CA1 pyramidal cells, and that coadministration of the m1 antagonist pirenzepine prevents the effect of cholinergic drugs. These observations suggest that the effect of nicotine is secondary to increased release of ACh via the activation of nicotinic ACh receptors (nAChRs) and involves m1 receptor activation through ACh. In vitro activation of m1 receptors causes the selective enhancement of NR2B-NMDAR responses in CA1 pyramidal cells, and in vivo exposure to cholinergic drugs occludes the in vitro effect. Furthermore, in vivo exposure to cholinergic drugs suppresses the potentiating effect of Src on NMDAR responses in vitro. These results suggest that exposure to cholinergic drugs maximally stimulates the m1/guanine nucleotide-binding protein subunit alpha q/PKC/proline-rich tyrosine kinase 2/Src signaling pathway for the potentiation of NMDAR responses in vivo, occluding the in vitro effects of m1 activation and Src. Thus, our results indicate not only that nAChRs, ACh, and m1 receptors are on the same pathway involving Src signaling but also that NR2B-NMDARs are a point of convergence of cholinergic and glutamatergic pathways involved in learning and memory.Nicotinic and muscarinic agonists can produce cognitive enhancement (1, 2). Acetylcholinesterase inhibitors (AChEIs) also cause cognitive enhancement by increasing ACh levels (3, 4). However, it is largely unknown whether the effect of ACh is mediated by nicotinic ACh receptors (nAChRs), muscarinic receptors, or both. Studies involving cholinergic lesions and local administration of cholinergic antagonists indicate that both nAChRs and muscarinic receptors located in the hippocampus are of particular importance for learning and memory processes (5–8). However, the mechanisms by which these receptors mediate cognitive enhancement largely remain to be elucidated.Synaptic plasticity is thought to be a critical component underlying learning and memory (9, 10), and the NMDA receptor (NMDAR) is a key component of synaptic plasticity (9, 11). Thus, studies of the modulation of NMDAR responses and long-term potentiation (LTP) induction by cholinergic drugs (12–20) help elucidate the mechanisms of cholinergic facilitation of learning and memory. In vitro acute nicotine can potentiate NMDAR-mediated responses in CA1 pyramidal cells in hippocampal slices via at least two different mechanisms (16, 18). One of these mechanisms is absent after a selective cholinergic lesion (21) and is paradoxically blocked by the muscarinic antagonist atropine (18), suggesting not only a critical role of nicotine-induced ACh release but also the involvement of muscarinic receptor activation in the effect of nicotine. This pathway appears to be stimulated by systemic nicotine administration in rats and most likely involves Src signaling (18, 19), which is known to be initiated via acute activation of m1 muscarinic (m1) receptors in CA1 pyramidal cells (22). An implication of these observations is that there is a common signaling pathway stimulated by cognitive-enhancing cholinergic drugs, leading to the enhancement of NMDAR-mediated responses in CA1 pyramidal cells. Thus, in this study, we investigated the link between nicotine and NMDARs in rats by administrating drugs that target different cholinergic proteins.     

Mossy fiber-evoked subthreshold responses induce timing-dependent plasticity at hippocampal CA3 recurrent synapses

Dentate granule cells exhibit exceptionally low levels of activity and rarely elicit action potentials in targeted CA3 pyramidal cells. It is thus unclear how such weak input from the granule cells sustains adequate levels of synaptic plasticity in the targeted CA3 network. We report that subthreshold potentials evoked by mossy fibers are sufficient to induce synaptic plasticity between CA3 pyramidal cells, thereby complementing the sparse action potential discharge. Repetitive pairing of a CA3–CA3 recurrent synaptic response with a subsequent subthreshold mossy fiber response induced long-term potentiation at CA3 recurrent synapses in rat hippocampus in vitro. Reversing the timing of the inputs induced long-term depression. The underlying mechanism depends on a passively conducted giant excitatory postsynaptic potential evoked by a mossy fiber that enhances NMDA receptor-mediated current at active CA3 recurrent synapses by relieving magnesium block. The resulting NMDA spike generates a supralinear depolarization that contributes to synaptic plasticity in hippocampal neuronal ensembles implicated in memory.The CA3 area of the hippocampus exhibits a distinctive, highly recurrent circuitry proposed to support autoassociative memory representation (1, 2). This prediction has been confirmed by experimental work demonstrating the pattern completion capabilities of CA3 networks (3), as well as their roles in the spatial tuning of CA1 pyramidal cells, in one-trial contextual learning (4) and in certain forms of memory consolidation (5). CA3 pyramidal cells receive, via the mossy fibers, information processed by granule cells important for both pattern separation (6, 7) and pattern completion functions (7). The faithful transmission of mossy fiber input appears to be ensured by giant synapses composed of presynaptic boutons with up to 45 release sites (8) that target massive spines, the thorny excrescences, on the apical dendrite of CA3 pyramidal cells. Thus, the mossy fiber synapse is often referred to as a detonator synapse (9). In fact, mossy fiber signaling is more compatible with a gatekeeper function than a high-throughput data relay. Although high-frequency bursts of action potentials in a hippocampal granule cell can discharge a targeted CA3 pyramidal cell, the majority of responses evoked by granule cells in CA3 pyramidal cells do not attain the firing threshold (10). Nevertheless, mossy fibers generate powerful signals evoking subthreshold responses that are much larger than typical synaptic events in the brain, with excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic currents (EPSCs) reaching amplitudes of 10 mV and 1 nA, respectively (11). Here we examined in rat slice cultures how EPSPs generated at mossy fiber synapses are processed in CA3 pyramidal cell dendrites, and evaluated whether subthreshold synaptic responses evoked by mossy fiber stimulation can act as instructive signals to induce plasticity at the pyramidal cell synapses forming the CA3 recurrent network.     

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17β-estradiol or an estrogen receptor (ER)-α (but not ER-β) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently “pruned” by experience.Estradiol rapidly and dramatically increases hippocampal dendritic spine and synapse density within minutes of application (1–4). There is a strong correlative association between estrogen-induced spinogenesis and improvements in cognition (5); however, the relationship of these structural changes to estrogen-induced alterations in hippocampal function is unclear. Our laboratory recently reported that the density of hippocampal CA1 pyramidal dendritic spines increases very rapidly after systemic treatment with 17β-estradiol or estrogen receptor (ER) -selective agonists in ovariectomized female mice, changes that are paralleled by learning enhancements (2, 3). Estrogen-induced rapid structural changes are substantial, increasing spine density by 30–50% within 15–40 min of hormone application (1–3, 6). As a result, adult rodents can experience the addition of thousands of CA1 synapses within a span of minutes after exposure to estradiol. These effects of estrogens reproduce the changes occurring during the 4-d estrous cycle of female rodents, which include the induction of CA1 spines (7).How these processes contribute to the behavioral changes observed after estradiol treatment is not understood. Estradiol enhances excitatory neurotransmission throughout the hippocampus (8–10), and activates BDNF signaling in the mossy fiber system (11). Dendritic spines turn over more rapidly in the hippocampus than in the neocortex (12), particularly in the case of estradiol-induced spines (13). Such rapid, transient, and apparently indiscriminate increases in excitatory synapse formation would seem, at first sight, to be more likely to interfere with preexisting brain circuits and impair normal information processing than to enhance cognitive function.How then, does enhancement of spine formation lead to improved cognitive function? To address this question, we focused specifically on the effects of estradiol in the dorsal CA1 hippocampus, as a site mediating the rapid improving effects of estrogens on learning. Estradiol application, via a mechanism involving ERα, rapidly increases dendritic spine density in the CA1 pyramidal stratum oriens and stratum radiatum subregions. However, contrary to our initial assumptions based on previous work in this field, increased spinogenesis did not result in increased CA1 excitatory input. Rather, estrogen-induced formation of hippocampal spines was associated with a decrease in the ability of CA1 neurons to respond to AMPA receptor (AMPAR) activation, resulting in decreased excitatory input to CA1 neurons. This appears to be the result of AMPAR internalization from the synaptic membrane (6). Taken together, our data suggest that estrogens induce formation of “silent” or immature synapses that act as a substrate for the storage of new memories (14, 15). This finding explains estrogens’ ability to rapidly improve learning without precipitating uncontrolled activation of the hippocampal circuitry. These effects of estradiol on CA1 pyramidal neurons phenotypically resemble the structural and functional properties of neurons during development, when neurons with higher levels of spine density and higher numbers of silent/immature synapses are present before the activity dependent refinement of neural circuitry.     

Glutamatergic regulation prevents hippocampal-dependent age-related cognitive decline through dendritic spine clustering

The dementia of Alzheimer’s disease (AD) results primarily from degeneration of neurons that furnish glutamatergic corticocortical connections that subserve cognition. Although neuron death is minimal in the absence of AD, age-related cognitive decline does occur in animals as well as humans, and it decreases quality of life for elderly people. Age-related cognitive decline has been linked to synapse loss and/or alterations of synaptic proteins that impair function in regions such as the hippocampus and prefrontal cortex. These synaptic alterations are likely reversible, such that maintenance of synaptic health in the face of aging is a critically important therapeutic goal. Here, we show that riluzole can protect against some of the synaptic alterations in hippocampus that are linked to age-related memory loss in rats. Riluzole increases glutamate uptake through glial transporters and is thought to decrease glutamate spillover to extrasynaptic NMDA receptors while increasing synaptic glutamatergic activity. Treated aged rats were protected against age-related cognitive decline displayed in nontreated aged animals. Memory performance correlated with density of thin spines on apical dendrites in CA1, although not with mushroom spines. Furthermore, riluzole-treated rats had an increase in clustering of thin spines that correlated with memory performance and was specific to the apical, but not the basilar, dendrites of CA1. Clustering of synaptic inputs is thought to allow nonlinear summation of synaptic strength. These findings further elucidate neuroplastic changes in glutamatergic circuits with aging and advance therapeutic development to prevent and treat age-related cognitive decline.Cognitive decline often occurs with aging in rodents (1), nonhuman primates (2), and humans (3). Memory loss (4) and executive impairment (5) are of the most functional importance, mediated primarily by the hippocampus and related areas of the medial temporal lobe and the prefrontal cortex (PFC), respectively. The neural circuits vulnerable to aging are composed of glutamatergic pyramidal neurons that furnish corticocortical connections between the association cortices as well as the excitatory hippocampal connections (2, 6). Dendritic spine changes, which appear to be the primary site of structural plasticity in the adult brain (7), occur in the pyramidal neurons of the PFC (5) and in the hippocampus (8, 9) with aging and correlate with behavioral decline. Spines form the postsynaptic component of most excitatory synapses in the cerebral cortex and are capable of rapid formation, expansion, contraction, and elimination (10, 11).Synaptic glutamatergic activity is neuroprotective and critical for long-term potentiation (LTP) and memory formation, whereas extrasynaptic NMDA receptor activity promotes long-term depression and excitotoxicity (12, 13). There is some evidence that astrocytic glutamate transporters decrease with aging (14, 15), and consequently reduce glutamate uptake (14, 16, 17). Reduced glutamate uptake can lead to glutamate spillover to the extrasynaptic space with electrophysiological repercussions (14). The potential use of glutamate modulators as a therapeutic target to regulate the synaptic age-related glutamatergic dysregulation in those vulnerable neural circuits remains to be further investigated.Riluzole is a glutamate modulator that decreases glutamate release (18) and facilitates astrocytic glutamate uptake (19–21). These actions have been suggested to increase glutamate-glutamine cycling, enhancing synaptic glutamatergic activity while preventing excessive glutamate overflow to the extrasynaptic space in rodents (21, 22) and humans (23). Riluzole has also been shown to increase oxidative metabolism with mitochondrial enhancing properties (24) and to increase BDNF expression (25). We hypothesized that improved regulation of the glutamatergic synapse with the glutamate modulator riluzole would promote synaptic NMDA receptor activation while preventing extrasynaptic NMDA activity, thereby protecting against age-related cognitive decline, through induction of neuroplastic changes in the hippocampus and PFC. An important neuroplastic mechanism is clustering of dendritic spines because it significantly empowers neural circuits with nonlinear summation of synaptic inputs (26, 27) and is dependent on neuronal activity (28, 29). For this study, we focused on pyramidal neurons within CA1 and pyramidal neurons in layer 3 of the prelimbic region of medial PFC, an area where we have demonstrated age-related spine loss in middle-aged animals previously (30).     

Left–right dissociation of hippocampal memory processes in mice

Left–right asymmetries have likely evolved to make optimal use of bilaterian nervous systems; however, little is known about the synaptic and circuit mechanisms that support divergence of function between equivalent structures in each hemisphere. Here we examined whether lateralized hippocampal memory processing is present in mice, where hemispheric asymmetry at the CA3–CA1 pyramidal neuron synapse has recently been demonstrated, with different spine morphology, glutamate receptor content, and synaptic plasticity, depending on whether afferents originate in the left or right CA3. To address this question, we used optogenetics to acutely silence CA3 pyramidal neurons in either the left or right dorsal hippocampus while mice performed hippocampus-dependent memory tasks. We found that unilateral silencing of either the left or right CA3 was sufficient to impair short-term memory. However, a striking asymmetry emerged in long-term memory, wherein only left CA3 silencing impaired performance on an associative spatial long-term memory task, whereas right CA3 silencing had no effect. To explore whether synaptic properties intrinsic to the hippocampus might contribute to this left–right behavioral asymmetry, we investigated the expression of hippocampal long-term potentiation. Following the induction of long-term potentiation by high-frequency electrical stimulation, synapses between CA3 and CA1 pyramidal neurons were strengthened only when presynaptic input originated in the left CA3, confirming an asymmetry in synaptic properties. The dissociation of hippocampal long-term memory function between hemispheres suggests that memory is routed via distinct left–right pathways within the mouse hippocampus, and provides a promising approach to help elucidate the synaptic basis of long-term memory.Unilateral specializations may facilitate greater processing power in bilateral brain structures by using the available neuronal circuitry more effectively. Nevertheless, the nature of the mechanisms that can act within the confines of duplicate neural structures to support different cognitive functions in each hemisphere remains elusive.The hippocampus is essential for certain forms of learning and memory, both in humans (1) and in rodents (2, 3), and also plays an important role in navigation (4). The left and right mammalian hippocampi comprise the same anatomical areas and directional connectivity, and yet in the human hippocampus, task-related activity may be localized to only one hemisphere (5). This lateralization may enable the left and right hippocampus to support complementary functions in human episodic memory, with left hippocampal activity associated with an egocentric, sequential representation of space but greater activity in the right hippocampus when an allocentric representation is used (6). It has been suggested that human hippocampal asymmetry is primarily dictated by external asymmetry—namely, the left hemispheric involvement in language processing and the stronger contribution of the right hemisphere to visuospatial attention (7), supported by observations of left hippocampal dominance when semantic information is most task-relevant, compared with right hippocampal dominance when spatial information becomes more pertinent (8). However, a seminal discovery in the mouse brain suggests that left–right asymmetry may actually be a fundamental property of the mammalian hippocampus: it was found that the postsynaptic spine morphology and receptor distribution in CA1 pyramidal neurons is determined by whether the presynaptic input originates in the left or right CA3 (9, 10). Specifically, apical CA1 postsynaptic spines receiving input from the left CA3 are primarily thin and rich in GluN2B subunit-containing NMDA receptors (NMDARs); in contrast, there is a higher proportion of mushroom-shaped spines receiving right CA3 projection, and these larger spines have a lower density of GluN2B subunit-containing NMDARs (9, 10). Interestingly, synaptic plasticity also shows hemispheric asymmetry: irrespective of the hemispheric location of the CA1 neuron, GluN2B NMDAR-requiring spike timing-dependent long-term potentiation (LTP) was induced at synapses where presynaptic input originates in the left CA3, but not in the right CA3 (11).These left–right synaptic differences raise the question as to whether memory processing in mice, as in humans, might differ between the left and right hippocampus. Therefore, in this study, we asked whether acutely inactivating one part of the asymmetric CA3–CA1 network unilaterally would affect learning and memory differentially between hemispheres. To test this, we silenced excitatory cells of CA3 in either the left or the right hippocampus, and consequently also both their ipsilateral and contralateral projections to CA1, using the light-sensitive chloride pump halorhodopsin (eNpHR3.0) coexpressed with enhanced YFP (eYFP) (12).     

The human hippocampus contributes to both the recollection and familiarity components of recognition memory

Despite a substantial body of work comprising theoretical modeling, the effects of medial temporal lobe lesions, and electrophysiological signal analysis, the role of the hippocampus in recognition memory remains controversial. In particular, it is not known whether the hippocampus exclusively supports recollection or both recollection and familiarity—the two latent cognitive processes theorized to underlie recognition memory. We studied recognition memory in a large group of patients undergoing intracranial electroencephalographic (iEEG) monitoring for epilepsy. By measuring high-frequency activity (HFA)—a signal associated with precise spatiotemporal properties—we show that hippocampal activity during recognition predicted recognition memory performance and tracked both recollection and familiarity. Through the lens of dual-process models, these results indicate that the hippocampus supports both the recollection and familiarity processes.Recognition is one’s ability to judge an item as previously encountered. Whereas it is well known that the hippocampus plays a crucial role in human recall memory, the role of the hippocampus in recognition memory remains surprisingly controversial (1–4). A number of studies have reported that bilateral hippocampal injury in humans causes impaired recall, whereas recognition remains intact (5). Others document the preservation of recognition in the setting of hippocampal lesioning in nonhuman primates (6) and rodents (7). On the other hand, a substantial literature describes combined recall and recognition deficits in a similarly injured group of patients (8) and animals (9, 10).Recognition is thought to rely on two processes: familiarity, wherein upon seeing a person’s face, the rememberer has only a vague sense he has met the person before, and recollection, wherein the subject sees the person’s face and vividly remembers details of the encounter (11, 12). What role the hippocampus plays in supporting these processes remains the subject of considerable debate. Many memory researchers have proposed the discrepancy in the lesion data above derives from the fact that the hippocampus, which is well known to play a role in associative and relational memory (13), exclusively subserves recollection, whereas familiarity is supported by the extrahippocampal medial temporal lobe (MTL) (14). By this account, humans and animals are able to compensate for the loss of the hippocampus, and thus recollection, by relying on familiarity (15–18). A contrasting view holds that the hippocampus instead contributes to both recollection and familiarity, thus explaining why hippocampal damage is associated with severe impairment of both processes and consequently the overall recognition performance (4, 8, 19, 20). Whether neural circuitry underlying familiarity and recollection lie within the hippocampus has also been extensively studied using functional MRI (fMRI) (21). Still, that many fMRI experiments show that only recollection signals are found in the hippocampus (22–24) whereas others report blood oxygen level-dependent signal representing both recollection and familiarity in this structure (25), further fuels the debate regarding which aspects of recognition memory are supported by the hippocampus. Although many electrophysiology studies demonstrate neural dissociations in the hippocampus during recognition (24, 26–29), they do not answer the question whether this structure subserves solely recollection or both recollection and familiarity.In this study, we used intracranially recorded high-frequency activity (HFA) to elucidate the role of the human hippocampus in recognition memory. Given that HFA is a spatiotemporally precise marker of neural ensemble activity (30, 31), if hippocampal activity correlated with successful recognition performance at retrieval, then, depending on which theory is correct, it should correlate either with behavioral estimates of recollection only or with behavioral estimates of both recollection and familiarity. Seen through the lens of dual-process theories of recognition (12), hippocampal HFA should identify whether this structure uniquely supports recollection. We tested these hypotheses in a group of 66 epilepsy patients undergoing intracranial monitoring to assess the specific role of the hippocampus in recognition memory.     

HCN channels enhance spike phase coherence and regulate the phase of spikes and LFPs in the theta-frequency range

What are the implications for the existence of subthreshold ion channels, their localization profiles, and plasticity on local field potentials (LFPs)? Here, we assessed the role of hyperpolarization-activated cyclic-nucleotide–gated (HCN) channels in altering hippocampal theta-frequency LFPs and the associated spike phase. We presented spatiotemporally randomized, balanced theta-modulated excitatory and inhibitory inputs to somatically aligned, morphologically realistic pyramidal neuron models spread across a cylindrical neuropil. We computed LFPs from seven electrode sites and found that the insertion of an experimentally constrained HCN-conductance gradient into these neurons introduced a location-dependent lead in the LFP phase without significantly altering its amplitude. Further, neurons fired action potentials at a specific theta phase of the LFP, and the insertion of HCN channels introduced large lags in this spike phase and a striking enhancement in neuronal spike-phase coherence. Importantly, graded changes in either HCN conductance or its half-maximal activation voltage resulted in graded changes in LFP and spike phases. Our conclusions on the impact of HCN channels on LFPs and spike phase were invariant to changes in neuropil size, to morphological heterogeneity, to excitatory or inhibitory synaptic scaling, and to shifts in the onset phase of inhibitory inputs. Finally, we selectively abolished the inductive lead in the impedance phase introduced by HCN channels without altering neuronal excitability and found that this inductive phase lead contributed significantly to changes in LFP and spike phase. Our results uncover specific roles for HCN channels and their plasticity in phase-coding schemas and in the formation and dynamic reconfiguration of neuronal cell assemblies.Local field potentials (LFPs) have been largely believed to be a reflection of the synaptic drive that impinges on a neuron. In recent experimental and modeling studies, there has been a lot of debate on the source and spatial extent of LFPs (1–9). However, most of these studies have used neurons with passive dendrites in their models and/or have largely focused on the contribution of spike-generating conductances to LFPs (7, 8, 10, 11). Despite the widely acknowledged regulatory roles of subthreshold-activated ion channels and their somatodendritic gradients in the physiology and pathophysiology of synapses and neurons (12–17), the implications for their existence on LFPs and neuronal spike phase have surprisingly remained unexplored. This lacuna in LFP analysis is especially striking because local and widespread plasticity of these channels has been observed across several physiological and pathological conditions, translating to putative roles for these channels in neural coding, homeostasis, disease etiology and remedies, learning, and memory (16, 18–23).In this study, we focus on the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that mediate the h current (I_h) in regulating LFPs and theta-frequency spike phase. From a single-neuron perspective, HCN channels in CA1 pyramidal neurons play a critical role in regulating neuronal integration and excitability (14, 24–27) and importantly introduce an inductive phase lead in the voltage response to theta-frequency oscillatory inputs (28), thereby enabling intraneuronal synchrony of incoming theta-frequency inputs (29). Given these and their predominant dendritic expression (25), we hypothesized HCN channels as regulators of LFPs through their ability to alter the amplitude and phase of the intracellular voltage response, thereby altering several somatodendritic transmembrane currents that contribute to LFPs. The CA1 region of the hippocampus offers an ideal setup to test this hypothesis, given the regular, open-field organization (4, 6, 7) of the pyramidal neurons endowed with well-established somatodendritic gradients in ion channel densities (16). As this organization enables us to assess the role of location-dependent channel expression profiles on LFPs across different strata, we tested our hypothesis, using a computational scheme involving morphologically realistic, physiologically constrained conductance-based model neurons. Our results positively test our hypothesis and provide specific evidence for novel roles for HCN channels and their inductive component in regulating LFP and spike phases, apart from enhancing spike-phase coherence. These results identify definite roles for HCN channels in phase-coding schemas and in the formation and dynamic reconfiguration of neuronal cell assemblies and argue for the incorporation of subthreshold-activated ion channels, their gradients, and their plasticity into the computation of LFPs.     

Basolateral amygdala bidirectionally modulates stress-induced hippocampal learning and memory deficits through a p25/Cdk5-dependent pathway

Repeated stress has been suggested to underlie learning and memory deficits via the basolateral amygdala (BLA) and the hippocampus; however, the functional contribution of BLA inputs to the hippocampus and their molecular repercussions are not well understood. Here we show that repeated stress is accompanied by generation of the Cdk5 (cyclin-dependent kinase 5)-activator p25, up-regulation and phosphorylation of glucocorticoid receptors, increased HDAC2 expression, and reduced expression of memory-related genes in the hippocampus. A combination of optogenetic and pharmacosynthetic approaches shows that BLA activation is both necessary and sufficient for stress-associated molecular changes and memory impairments. Furthermore, we show that this effect relies on direct glutamatergic projections from the BLA to the dorsal hippocampus. Finally, we show that p25 generation is necessary for the stress-induced memory dysfunction. Taken together, our data provide a neural circuit model for stress-induced hippocampal memory deficits through BLA activity-dependent p25 generation.Chronic stress can have devastating psychological consequences that include depression and cognitive impairment (1–3). Decades of research suggest that the hippocampus, a structure important for learning and memory and implicated in depression, is particularly sensitive to the effects of chronic stress. In animal models, for example, chronic stress impairs hippocampus-dependent forms of learning and memory (2). This sensitivity is partially conferred by a dense concentration of glucocorticoid receptor (GR) in the hippocampus (4), as well as through hippocampal connectivity to important stress response coordinators, such as the amygdala, from which the hippocampus receives abundant glutamatergic inputs (5–7). Following chronic stress, the hippocampus shows marked reductions in dendritic arborization and neurogenesis, along with impaired plasticity (2). Many of these effects have been attributed to connections between the hippocampus and a specific amygdalar subregion, the basolateral amygdala (BLA) (8–10).Abundant evidence suggests that these BLA inputs have a major impact on hippocampus function; for example, the hippocampus and BLA synchronize their activity during fear memory retrieval and fear extinction (11, 12), whereas electrical stimulation of the BLA disrupts the induction of long-term potentiation (LTP), a measure of synaptic plasticity, in the hippocampal CA1 subregion (13). Lesions of the BLA have been shown to block the detrimental effects of repeated stress, a model of chronic stress in rodents, on LTP and spatial memory (8, 10), as well as the deleterious effect of hippocampal GR activation on hippocampus-dependent memory (9). Although the BLA sends abundant projections to the hippocampus (5–7), this region also projects diffusely throughout the brain and thereby regulates a myriad of behaviors, including valence or social interaction (14), as well as hormonal cascades (15). Because of this complexity, whether BLA activity affects hippocampus-dependent learning and memory directly or indirectly through distinct relay brain regions or other downstream mediators, such as stress hormones, remains unclear.Cdk5 (cyclin-dependent kinase 5) plays a pleiotropic role in the nervous system (16). This enzyme is essential for proper brain development and regulates synaptic plasticity and cognitive function. Activation of Cdk5 requires association with a regulatory subunit known as p35. p35 is subjected to calpain-mediated cleavage into p25 in a process dependent on the activation of glutamate receptors, specifically NR2B-containing NMDA receptors, following neurotoxic stimulation, such as exposure to β-amyloid peptides, oxidative stress, or excitotoxicity, as well as in response to physiological neuronal activity (16).A number of studies have implicated p25 production in Alzheimer’s disease (AD)-like phenotypes, including learning and memory impairments (16), and long-term overexpression of p25 in the forebrain is known to lead to cognitive deficits (16). Furthermore, stress and the heightened sensitivity to stress are known risk factors for the development of AD (1). The role of p25 production after repeated stress remains undetermined, however.One pathway through which p25/Cdk5 might be implicated in stress-induced cognitive dysfunction is stress hormone receptor-mediated epigenetic signaling in the hippocampus. Indeed, it was previously shown that GR is activated by p25/Cdk5-dependent phosphorylation on Ser211 (17, 18), and that increased GR phosphorylation leads to increased expression of histone deacetylase 2 (HDAC2) in a mouse model of AD (18). HDAC2 in turn suppresses the expression of genes important for learning and memory (18, 19), suggesting a mechanism by which elevated p25 generation leads to cognitive impairment. Although GR activation has been shown to be required for stress-induced hippocampal dysfunction and is dependent upon its phosphorylation (20–22), and HDAC2 has been shown to be up-regulated in the ventral striatum of mice following chronic stress (23), the possible up-regulation of HDAC2 in the hippocampus after repeated stress, and the role of p25/Cdk5 signaling in this process, are unknown. We tested the hypothesis that p25 is generated in the hippocampus after repeated stress in an amygdala-dependent manner and contributes to stress-associated learning and memory deficits. Blockade of p25 generation would then protect the hippocampus from the detrimental effects of repeated stress.Here we identify that the activity of a specific BLA to dorsal hippocampus neural circuit mediates the detrimental effects of repeated stress on hippocampal learning and memory via a molecular pathway dependent on p25 generation.     

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome

Haploinsufficiency of the voltage-gated sodium channel Na_V1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of Na_V1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the Na_V1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.Dravet syndrome (DS), also referred to as “severe myoclonic epilepsy in infancy,” is a rare genetic epileptic encephalopathy characterized by frequent intractable seizures, severe cognitive deficits, and premature death (1–3). DS is caused by loss-of-function mutations in SCN1A, the gene encoding type I voltage-gated sodium channel Na_V1.1, which usually arise de novo in the affected individuals (4–7). Like DS patients, mice with heterozygous loss-of-function mutations in Scn1a exhibit ataxia, sleep disorder, cognitive deficit, autistic-like behavior, and premature death (8–14). Like DS patients, DS mice first become susceptible to seizures caused by elevation of body temperature and subsequently experience spontaneous myoclonic and generalized tonic-clonic seizures (11). Global deletion of Na_V1.1 impairs Na⁺ currents and action potential (AP) firing in GABAergic-inhibitory interneurons (8–10), and specific deletion of Na_V1.1 in forebrain interneurons is sufficient to cause DS in mice (13, 15). These data suggest that the loss of interneuron excitability and resulting disinhibition of neural circuits cause DS, but the functional role of different subtypes of interneurons in the cerebral cortex in DS remains unknown.Neocortical GABAergic interneurons shape cortical output and display great diversity in morphology and function (16, 17). The expression of parvalbumin (PV) and somatostatin (SST) defines two large, nonoverlapping groups of interneurons (16, 18, 19). In layer V of the cerebral cortex, PV-expressing fast-spiking interneurons and SST-expressing Martinotti cells each account for ∼40% of interneurons, and these interneurons are the major inhibitory regulators of cortical network activity (17, 20). Layer V PV interneurons make synapses on the soma and proximal dendrites of pyramidal neurons (18, 19), where they mediate fast and powerful inhibition (21, 22). Selective heterozygous deletion of Scn1a in neocortical PV interneurons increases susceptibility to chemically induced seizures (23), spontaneous seizures, and premature death (24), indicating that this cell type may contribute to Scn1a deficits. However, selective deletion of Scn1a in neocortical PV interneurons failed to reproduce the effects of DS fully, suggesting the involvement of other subtypes of interneurons in this disease (23, 24). Layer V Martinotti cells have ascending axons that arborize in layer I and spread horizontally to neighboring cortical columns, making synapses on apical dendrites of pyramidal neurons (17, 25, 26). They generate frequency-dependent disynaptic inhibition (FDDI) that dampens excitability of neighboring layer V pyramidal cells (27–29), contributing to maintenance of the balance of excitation and inhibition in the neocortex. However, the functional roles of Martinotti cells and FDDI in DS are unknown.Because layer V forms the principal output pathway of the neocortex, reduction in inhibitory input to layer V pyramidal cells would have major functional consequences by increasing excitatory output from all cortical circuits. However, the effects of the DS mutation on interneurons and neural circuits that provide inhibitory input to layer V pyramidal cells have not been determined. Here we show that the intrinsic excitability of layer V fast-spiking PV interneurons and SST Martinotti cells and the FDDI mediated by Martinotti cells are reduced dramatically in DS mice, leading to an imbalance in the excitation/inhibition ratio. Our results suggest that loss of Na_V1.1 in these two major types of interneurons may contribute synergistically to increased cortical excitability, epileptogenesis, and cognitive deficits in DS.     

Bidirectional homeostatic plasticity induced by interneuron cell death and transplantation in vivo

Chronic changes in excitability and activity can induce homeostatic plasticity. These perturbations may be associated with neurological disorders, particularly those involving loss or dysfunction of GABA interneurons. In distal-less homeobox 1 (Dlx1^−/−) mice with late-onset interneuron loss and reduced inhibition, we observed both excitatory synaptic silencing and decreased intrinsic neuronal excitability. These homeostatic changes do not fully restore normal circuit function, because synaptic silencing results in enhanced potential for long-term potentiation and abnormal gamma oscillations. Transplanting medial ganglionic eminence interneuron progenitors to introduce new GABAergic interneurons, we demonstrate restoration of hippocampal function. Specifically, miniature excitatory postsynaptic currents, input resistance, hippocampal long-term potentiation, and gamma oscillations are all normalized. Thus, in vivo homeostatic plasticity is a highly dynamic and bidirectional process that responds to changes in inhibition.Prolonged changes in activity levels induce bidirectional changes in neuronal excitability and synaptic activity known as homeostatic plasticity (1, 2). This phenomenon has been described well at excitatory synapses and functions to maintain activity within a preferred dynamic range. Maintaining excitatory/inhibitory synaptic balance is critical for neuronal information processing and a potential problem when confronted with aberrant states of excitability, such as those associated with autism, schizophrenia, Alzheimer’s disease, or epilepsy (3–12).Chronic manipulation of synaptic input and/or action potential (AP) output rates in cortical and hippocampal cell cultures induces homeostatic synaptic scaling, in which the amplitude and then the frequency of pyramidal neuron miniature excitatory postsynaptic currents (mEPSCs) increase when activity is lowered or decrease when activity is raised (13–16). Recent studies have begun to reveal the underlying molecular mechanisms of homeostatic synaptic changes, including the AMPA receptor subunits, synapse-associated calcium-binding proteins, and intracellular signaling cascades involved (14, 17, 18). Changes to activity also trigger homeostatic plasticity of inhibitory synaptic transmission (19–23). Homozygous deletion of glutamate decarboxylase 1 (Gad1), the rate-limiting enzyme in the synthesis of GABA, reduced miniature inhibitory postsynaptic current (mIPSC) amplitudes in cultured hippocampal neurons but also blocked further homeostatic changes to mIPSCs. This suggests a key role for regulation of Gad1 expression in inhibitory homeostatic plasticity (23). Intrinsic excitability is also homeostatically regulated by activity. Changes in input resistance (Rin) and voltage-activated K⁺ and Na⁺ channel number (24–27), and in Na⁺ channel compartmentalization (28, 29), have been described following manipulations that chronically alter neuronal activity. Finally, in vivo manipulation of neuronal activity with TTX results in larger mEPSC amplitudes and reduced Rin of CA1 pyramidal neurons (30), suggesting that multiple mechanisms of homeostatic plasticity can occur simultaneously in the intact nervous system.Loss of GABAergic interneurons is common across different neurological disorders. It is unknown whether homeostatic plasticity can be induced by changes in activity related to interneuronopathy or how the combination of interneuron cell death and compensation alters circuit function. To begin to address these issues, we studied synaptic and intrinsic excitability in a hippocampal circuit in which a subpopulation of interneurons is reduced [i.e., distal-less homeobox 1 (Dlx1^−/−) mice] (31–33). At around 30 d of age, these mice lose a subset of somatostatin (Sst)-, calretinin (CR)-, vasoactive intestinal peptide-, and neuropeptide Y (NPY)-positive interneurons; exhibit decreased inhibitory synaptic activity in some brain regions; and subsequently develop epilepsy (31). Our results show that secondary to the in vivo interneuron loss is a homeostatic reduction in mEPSC frequency, decreased AMPA/NMDA ratio, and decreased intrinsic excitability in CA1 pyramidal neurons (that do not express Dlx1). Transplantation of GABA progenitor cells from the medial ganglionic eminence (MGE) (34) causes a reversal of the homeostatic changes in excitatory synaptic activity and Rin. Additionally, we describe unique changes in Dlx1^−/− circuit function that homeostatic compensation does not correct: enhanced long-term potentiation (LTP) and altered gamma frequency oscillations (GFOs). The severity of these phenotypes is reduced by interneuron transplantation. These studies demonstrate the responsiveness of excitatory circuitry to changes in inhibition, using homeostatic plasticity as a mechanism for maintaining excitatory/inhibitory balance.     

Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus

Ghrelin is a peptide mainly produced by the stomach and released into circulation, affecting energy balance and growth hormone release. These effects are guided largely by the expression of the ghrelin receptor growth hormone secretagogue type 1a (GHS-R1a) in the hypothalamus and pituitary. However, GHS-R1a is expressed in other brain regions, including the hippocampus, where its activation enhances memory retention. Herein we explore the molecular mechanism underlying the action of ghrelin on hippocampal-dependent memory. Our data show that GHS-R1a is localized in the vicinity of hippocampal excitatory synapses, and that its activation increases delivery of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic-type receptors (AMPARs) to synapses, producing functional modifications at excitatory synapses. Moreover, GHS-R1a activation enhances two different paradigms of long-term potentiation in the hippocampus, activates the phosphatidylinositol 3-kinase, and increases GluA1 AMPAR subunit and stargazin phosphorylation. We propose that GHS-R1a activation in the hippocampus enhances excitatory synaptic transmission and synaptic plasticity by regulating AMPAR trafficking. Our study provides insights into mechanisms that may mediate the cognition-enhancing effect of ghrelin, and suggests a possible link between the regulation of energy metabolism and learning.The appetite-stimulating peptide ghrelin is a 28-aa peptide predominantly produced by X/A-like cells in the oxyntic glands of the stomach as well as in the intestine (1), and secreted into the blood stream. This peptide promotes pituitary growth hormone secretion, through activation of the growth hormone secretagogue type 1a receptor (GHS-R1a) or ghrelin receptor (2). Additionally, ghrelin is involved in the regulation of energy balance by increasing food intake and reducing fat utilization (3). Plasma ghrelin levels rise before meals and decrease thereafter (4), a pattern which is consistent with the implication of ghrelin in preprandial hunger and meal initiation. Ghrelin is secreted into the circulation and crosses the blood–brain barrier (5, 6), but there is also evidence for ghrelin synthesis locally in the brain (2, 7, 8). The GHS-R1a receptor mRNA was initially found in the hypothalamus and in the pituitary gland (9), and later detected in the hippocampus (10). GHS-R1a is a G protein-coupled seven-transmembrane domain receptor (3), which can signal through guanine nucleotide-binding protein (G protein) subunit alpha 11 (Gq class) to activate phosphatidylinositol-specific phospholipase C, generating 1,4,5-triphosphate (IP₃) responsible for Ca²⁺ intracellular release from endoplasmic reticulum, and diacylglicerol, which in turn activates protein kinase C (PKC) (11). Ghrelin receptor activation is also coupled to the phosphatidylinositol 3 (PI3)-kinase signaling cascade in different cellular systems through a pertussis toxin-sensitive G protein (G_i/oα) (11), and to protein kinase A (PKA) in isolated hypothalamic neurons, modulating N-type Ca²⁺ channels (12).The finding that GHS-R1a is expressed in the hippocampus raises the possibility that ghrelin, similarly to other appetite-regulating hormones such as leptin (13), may affect brain functions other than those related to endocrine and metabolic regulation (14). Indeed, in the last few years several studies have shown that ghrelin increases memory retention in rodents, and that the hippocampus participates in this effect (6, 15–17). Ghrelin-deficient mice exhibit decreased novel object recognition, a type of memory test dependent on hippocampal function (6), suggesting that endogenous ghrelin has a physiological role in improving learning and memory. Additionally, high-fat and high-glucose diets, which inhibit ghrelin secretion (18, 19), impair hippocampus-dependent synaptic plasticity and spatial memory (20, 21). On the other hand, caloric restriction, which results in an increase in the circulating levels of ghrelin (22), decreases aging-related deficiencies in cognitive processes (23) while increasing learning consolidation and facilitating synaptic plasticity (24). Recent evidence suggests an enhancing effect of ghrelin on long-term potentiation (LTP) in the hippocampus (6, 17), a form of activity-dependent synaptic plasticity which is the cellular correlate for learning and memory (25). However, conclusive evidence is still lacking because one study did not observe effects of ghrelin on LTP induced by theta burst stimulation (6), whereas the other only detected effects of ghrelin on a late phase of LTP [2 h after high-frequency stimulation (17)].Although the function of ghrelin as a cognitive enhancer is well documented, the molecular mechanisms that underlie this function are still poorly understood. Here we have tested whether the activation of GHS-R1a affects the trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), crucial for the expression of changes in synaptic strength in the hippocampus (26). We report that GHS-R1a localizes to excitatory synapses and that its activation induces the synaptic delivery of GluA1-containing AMPAR (GluA1-AMPAR) in rat hippocampal cultures and in CA1 cells in organotypic hippocampal slices. These changes enhance excitatory synaptic transmission. Furthermore, we show that ghrelin receptor activation enhances LTP expression in the CA3–CA1 synapse in organotypic hippocampal slices, and increases the synaptic and cell-surface trafficking of GluA1-AMPAR induced by chemical LTP in hippocampal cultures. Finally, we demonstrate that ghrelin receptor activation in the hippocampus increases the phosphorylation of GluA1 and stargazin. Taken together our data indicate that ghrelin receptor activation regulates AMPARs trafficking underlying synaptic plasticity and learning.     

From the Cover: Place field expansion after focal MEC inactivations is consistent with loss of Fourier components and path integrator gain reduction

Both hippocampal place fields and medial entorhinal cortex (MEC) grid fields increase in scale along the dorsoventral axis. Because the connections from MEC to hippocampus are topographically organized and divergent, it has been hypothesized that place fields are generated by a Fourier-like summation of inputs over a range of spatial scales. This hypothesis predicts that inactivation of dorsal MEC should cause place field expansion, whereas inactivation of ventral MEC should cause field contraction. Inactivation of dorsal MEC caused substantial expansion of place fields; however, as inactivations were made more ventrally, the effect diminished but never switched to contraction. Expansion was accompanied by proportional decreases in theta power, intrinsic oscillation frequencies, phase precession slopes, and firing rates. Our results are most consistent with the predicted loss of specific Fourier components coupled with a path integration gain reduction, which raises the overall place field scale and masks the contraction expected from ventral inactivations.When a rodent navigates through an environment, the principal cells in its hippocampi become tuned to its physical location (1). The continuous updating of positional information through integration of head angular velocity and linear velocity using vestibular, proprioceptive, and visual self-motion signals, termed path integration, is thought to play an important role in this process (2, 3). The grid cell network (4, 5) displays a number of properties, including regularly repeating fields and cells conjunctive for position and direction (6), predicted by a model for path integration in a toroidal attractor map network (7, 8). Given that the connections from the medial entorhinal cortex (MEC) to the hippocampus are topographically organized (9), and both grid and place fields increase in scale along the dorsoventral axes of their respective structures (5, 10–13), it is reasonable to speculate that the features of path integration observed in the hippocampus, including its place fields, may be inherited from the MEC.The periodic nature of grid fields and the range of spatial scales they express suggested they might enable hippocampal place field generation through a Fourier synthesis mechanism (8, 14). In the Fourier model, place field size is proportional to the scales of the input grid fields; a field generated from input grids with a distribution of spatial scales skewed towards smaller scales will be smaller than one whose input spatial scales are skewed in the opposite direction. Therefore, a testable prediction of the model is that lesion of the most dorsal portion of MEC sending inputs to the dorsal hippocampus should increase the scale of place fields recorded there, whereas lesion of the most ventral portion of the MEC sending inputs to the same region should have the opposite effect (14). We performed this experiment by infusing muscimol at multiple sites along the dorsoventral axis of MEC while recording from areas CA3 and CA1 in the dorsal half of the hippocampus, allowing us to track place field properties immediately before and after temporary inactivations. The experiment was conducted on a circular track, as previous studies showed that place fields recorded on narrow tracks are dependent on path integration (15, 16).     

Disruption of hippocampal–prefrontal cortex activity by dopamine D2R-dependent LTD of NMDAR transmission

Functional connectivity between the hippocampus and prefrontal cortex (PFC) is essential for associative recognition memory and working memory. Disruption of hippocampal–PFC synchrony occurs in schizophrenia, which is characterized by hypofunction of NMDA receptor (NMDAR)-mediated transmission. We demonstrate that activity of dopamine D2-like receptors (D2Rs) leads selectively to long-term depression (LTD) of hippocampal–PFC NMDAR-mediated synaptic transmission. We show that dopamine-dependent LTD of NMDAR-mediated transmission profoundly disrupts normal synaptic transmission between hippocampus and PFC. These results show how dopaminergic activation induces long-term hypofunction of NMDARs, which can contribute to disordered functional connectivity, a characteristic that is a hallmark of psychiatric disorders such as schizophrenia.The hippocampus to medial prefrontal cortex (PFC) projection is important for executive function and working and long-term memory (1, 2). Glutamatergic neurons of the ventral hippocampal cornu ammonis 1 (CA1) region project directly to layers 2–6 of ipsilateral PFC, and this connection synchronizes PFC and hippocampal activity during particular behavioral conditions (3–5). Disruption of hippocampal–PFC synchrony is associated with cognitive deficits that occur in disorders such as schizophrenia (6). Hippocampal–PFC uncoupling can be achieved by NMDA receptor (NMDAR) antagonism (7), and NMDAR hypofunction is a recognized feature of schizophrenia (8). However, it is unclear, first, how changes in NMDAR function at this synapse may arise, and second, how NMDAR hypofunction affects hippocampal–PFC synaptic transmission.Canonically, NMDARs are considered to contribute little to single synaptic events, but the slow kinetics of NMDARs contribute to maintaining depolarization, leading to the generation of bursts of action potentials (9–13). Furthermore, NMDARs coordinate spike timing relative to the phase of field potential oscillations (14, 15). NMDAR transmission itself undergoes synaptic plasticity (16, 17), and this can have a profound effect on sustained depolarization, burst firing, synaptic integration, and metaplasticity (9, 11, 18, 19). In PFC, NMDARs are oppositely regulated by dopamine receptors; D1-like receptors (D1Rs) potentiate and D2-like receptors (D2Rs) depress NMDAR currents (20). Interestingly, NMDAR hypofunction (8, 21) and dopamine D2 receptor activity (22) are potentially converging mechanisms contributing to schizophrenia (23).We now examine the contribution of NMDARs to transmission at the hippocampal–PFC synapse. We show that NMDAR activity provides sustained depolarization that can trigger action potentials during bursts of hippocampal input to PFC. We next demonstrate that dopamine D2 receptor-dependent long-term depression (LTD) of NMDAR transmission profoundly attenuates summation of synaptic transmission and neuronal firing at the hippocampal–PFC input. These findings allow for a mechanistic understanding of how alterations in dopamine and NMDAR function can lead to the disruption of hippocampal–PFC functional connectivity, which characterizes certain psychiatric disorders.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

AMPA receptor inhibition by synaptically released zinc

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.The development, function, and experience-dependent plasticity of the mammalian brain depend on the refined neuronal interactions that occur in synapses. In the majority of excitatory synapses, the release of the neurotransmitter glutamate from presynaptic neurons opens transmembrane ion channels in postsynaptic neurons, the ionotropic glutamate receptors, thereby generating the flow of excitatory signaling in the brain. As a result, these receptors play a fundamental role in normal function and development of the brain, and they are also involved in many brain disorders (1).The ionotropic glutamate receptor family consists of AMPA, kainate, and NMDA receptors (NMDARs). Although kainate receptor-mediated excitatory postsynaptic responses occur in a few central synapses (2), AMPA receptors (AMPARs) and NMDARs are localized in the postsynaptic density of the vast majority of glutamatergic synapses in the brain, mediating most of excitatory neurotransmission (1). NMDAR function is regulated by a wide spectrum of endogenous allosteric neuromodulators that fine-tune synaptic responses (3–5); however, much less is known about endogenous AMPAR neuromodulators [(1, 5), but see refs. 6 and 7]. Recent structural studies revealed that the amino terminal domain (ATD) and ligand-binding domain (LBD) are tightly packed in NMDARs but not AMPARs (8–10). These structural differences explain some of the functional differences in allosteric modulation between AMPARs and NMDARs, such as why the ATD of NMDARs, unlike that of AMPARs, modulates function and contains numerous binding sites for allosteric regulators. Nonetheless, given the importance of fine-tuning both synaptic AMPAR and NMDAR responses for brain function, it is puzzling that there is not much evidence for endogenous, extracellular AMPAR modulation. The discovery and establishment of endogenous AMPAR modulators is crucial both for understanding ionotropic glutamate receptor signaling and for developing therapeutic agents for the treatment of AMPAR-related disorders, such as depression, cognitive dysfunctions associated with Alzheimer’s disease, and schizophrenia (1, 11).Free, or readily chelatable, zinc is an endogenous modulator of synaptic and extrasynaptic NMDARs (12–15). Free zinc is stored in glutamatergic vesicles in many excitatory synapses in the cerebral cortex, limbic, and brainstem nuclei (16). In some brain areas, such as in the hippocampus, 50% of boutons synapsing onto CA1 neurons and all mossy fibers synapsing onto CA3 neurons contain synaptic free zinc (17). Whereas earlier studies demonstrated that exogenous zinc inhibits AMPARs (18–21), more recent work suggests that endogenously released synaptic zinc does not modulate AMPARs in central synapses (14, 22). This conclusion was derived from the inability to efficiently chelate and quantify synaptic zinc with the zinc-selective chelators and probes used (15), in apparent support of the hypothesized low levels of released zinc during synaptic stimulation (14).Recent work in our laboratories used new chemical tools that allowed us to intercept and visualize mobile zinc efficiently (15). These studies revealed modulation of extrasynaptic NMDARs by zinc and led us to reinvestigate whether synaptically released zinc might be an endogenous modulator of AMPARs as well. In the present study, we applied these same tools in electrophysiological, laser-based glutamate uncaging and in imaging experiments using wild type and genetically modified mice that lack synaptic zinc.