From the Cover: Joint CP-AMPA and group I mGlu receptor activation is required for synaptic plasticity in dentate gyrus fast-spiking interneurons

Hippocampal principal cell (PC) assemblies provide the brain with a mnemonic representation of space. It is assumed that the formation of cell assemblies is supported by long-lasting modification of glutamatergic synapses onto perisomatic inhibitory interneurons (PIIs), which provide powerful feedback inhibition to neuronal networks. Repetitive activation of dentate gyrus PIIs by excitatory mossy fiber (MF) inputs induces Hebbian long-term potentiation (LTP). In contrast, long-term depression (LTD) emerges in the absence of PII activity. However, little is known about the molecular mechanisms underlying synaptic plasticity in PIIs. Here, we examined the role of group I metabotropic glutamate receptors 1 and 5 (mGluRs1/5) in inducing plastic changes at MF-PII synapses. We found that mGluRs1/5 are located perisynaptically and that pharmacological block of mGluR1 or mGluR5 abolished MF-LTP. In contrast, their exogenous activation was insufficient to induce MF-LTP but cleared MF-LTD. No LTP could be elicited in PIIs loaded with blockers of G protein signaling and Ca²⁺-dependent PKC. Two-photon imaging revealed that the intracellular Ca²⁺ rise necessary for MF-LTP was largely mediated by Ca²⁺-permeable AMPA receptors (CP-AMPARs), but less by NMDA receptors or mGluRs1/5. Thus, our data indicate that fast Ca²⁺ signaling via CP-AMPARs and slow G protein-mediated signaling via mGluRs1/5 converge to a PKC-dependent molecular pathway to induce Hebbian MF-LTP. We further propose that Hebbian activation of mGluRs1/5 gates PIIs into a “readiness mode” to promote MF-LTP, which, in turn, will support timed PII recruitment, thereby assisting in PC assembly formation.Reorganization of hippocampal principal cell (PC) assemblies during spatial learning is supported by the timed recruitment of GABAergic cells, specifically parvalbumin (PV)-expressing perisomatic inhibitory interneurons (PIIs) (1, 2). Dentate gyrus (DG) PIIs are excited by glutamatergic granule cells (GCs) via mossy fiber (MF) synapses, which can undergo activity-dependent synaptic plasticity (3, 4). Indeed, after long-term potentiation (LTP), a single MF input can reliably activate PIIs (4), indicating that MF-LTP may influence spatial representation in the DG (2, 5).Hebbian MF-PII LTP requires precisely timed pre- and postsynaptic activity (4). It is induced in the PII but is expressed presynaptically (3). MF-LTP requires strong intracellular Ca²⁺ elevation and activation of Ca²⁺-permeable AMPA receptors (CP-AMPARs), but, in contrast to Hebbian LTP at CA1 PII inputs (6), it is independent of NMDA receptors (NMDARs) (4), suggesting that CP-AMPARs could provide the required Ca²⁺ rise for plasticity induction (7, 8). However, recent investigations have proposed a role for group I metabotropic glutamate receptors (mGluRs) in the induction of CP-AMPAR–dependent interneuron plasticity (9–13). Indeed, a Hebbian form of CP-AMPAR–dependent LTP at glutamatergic synapses onto somatostatin (SOM)-expressing CA1 stratum oriens/alveus (O/A) interneurons and at MF inputs onto CA3 interneurons requires mGluR1α activation (9, 11). Furthermore, mGluR1α and mGluR5 contribute to LTP at PC inputs on O/A interneurons, including oriens-lacunosum/moleculare (O-LM) cells (10), but induce long-term depression (LTD) in CA1 fast-spiking GABAergic cells (12). Whether mGluRs1/5 are expressed in hippocampal PV-PIIs remained controversial (14, 15), and their contribution to plasticity in PV-PIIs is unknown. Using whole-cell recordings of GCs paired to PV-PIIs and quantitative immunoelectron microscopy, we show that mGluRs1α/5 are expressed in DG PV-PIIs, contribute to Hebbian LTP, and suppress LTD at their MF inputs. Two-photon (2P) imaging further revealed the main Ca²⁺ sources and Ca²⁺-mediated molecular cascades for MF-LTP induction. Finally, we identify one major molecular mechanism underlying the emergence of MF-LTP.     

Thalamic model of awake alpha oscillations and implications for stimulus processing

We describe a unique conductance-based model of awake thalamic alpha and some of its implications for function. The full model includes a model for a specialized class of high-threshold thalamocortical cells (HTC cells), which burst at the alpha frequency at depolarized membrane potentials (∼−56 mV). Our model generates alpha activity when the actions of either muscarinic acetylcholine receptor (mAChR) or metabotropic glutamate receptor 1 (mGluR1) agonists on thalamic reticular (RE), thalamocortical (TC), and HTC cells are mimicked. In our model of mGluR1-induced alpha, TC cells are equally likely to fire during any phase of alpha, consistent with in vitro experiments. By contrast, in our model of mAChR-induced alpha, TC cells tend to fire either at the peak or the trough of alpha, depending on conditions. Our modeling suggests that low levels of mGluR1 activation on a background of mAChR agonists may be able to initiate alpha activity that biases TC cells to fire at certain phases of alpha, offering a pathway for cortical control. If we introduce a strong stimulus by increasing the frequency of excitatory postsynaptic potentials (EPSPs) to TC cells, an increase in alpha power is needed to mimic the level of phasing of TC cells observed in vivo. This increased alpha power reduces the probability that TC cells spike near the trough of alpha. We suggest that mAChR-induced alpha may contribute to grouping TC activity into discrete perceptual units for processing, whereas mGluR1-induced alpha may serve the purpose of blocking unwanted stimuli from reaching the cortex.Alpha rhythms (8–13 Hz) were first observed in humans over the occipital cortex by Berger (1), when subjects were in a relaxed state with their eyes closed. Occipital alpha has been investigated extensively since. However, alpha rhythms are not strictly confined to this area of cortex; alpha activity has also been reported in the somatosensory cortex (2), the auditory cortex (3), and the prefrontal cortex (4).Both the neural substrates responsible for the genesis of alpha and its functional role in cognition remain hotly debated. At the functional level, the point of contention is whether alpha activity serves to process information relevant to the task at hand or serves to filter out irrelevant information. The debate over where alpha activity is generated primarily revolves around whether it is generated by the neocortex, by the thalamus, or by a combination of the two. We make use of recent findings (5–10), as well as prior findings (11–17), to construct a unique conductance-based thalamic model of awake alpha, and use it to address the above controversy.Studies have found that during simultaneous in vivo recordings from the thalamus and neocortex, alpha activity in the neocortex is accompanied by alpha rhythms in the local field potential of the thalamus and in the firing patterns of individual thalamocortical (TC) cells (5, 18). During alpha activity, only 10–30% of TC cells fire in the alpha frequency (5, 6). Their firing pattern consists of high-threshold bursts (HTBs), with the intervals between bursts occurring at the alpha frequency, and gap junctions play a critical role in synchronizing their activity (10). Alpha activity can be induced in thalamic slices in the presence of metabotropic glutamate receptor (mGluR1) agonists (5) or muscarinic acetylcholine receptor (mAChR) agonists (8). As in the in vivo case, only a small fraction of TC cells exhibits HTB at the alpha frequency in the presence of mGluR1 and mAChR agonists. Although the mechanisms by which mGluR1 agonists and mAChR agonists induce HTB may differ, they both seem to do so, in part, by reducing potassium leak conductances and by activating an I_T channel that acts at more depolarized membrane potentials than the standard I_T channel (7).For our model, we developed two submodels: one for a specialized class of high-threshold thalamocortical cells (HTC cells) and one for an I_T-channel variant suggested to play a critical role in the generation of thalamic alpha (5–7, 17). We denote by I_TLT and I_THT the calcium currents associated with the low- and high-threshold variants, respectively. The model generates alpha activity upon choosing parameters to reflect the presence of either mGluR1 or mAChR, consistent with experimental data (5–9). We show that mGluR1- and mAChR-mediated alpha rhythms produce differential effects on the firing of TC cells with respect to the alpha rhythm, and discuss the functional implications.     

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2–related inhibitory peptide; or the blocker of L-voltage–dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by β-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.Frey and Morris (1, 2) and their collaborators (3–7) proposed a mechanism whereby relatively “weak” hippocampal long-term potentiation (LTP) or long-term depression (LTD) lasting only a few minutes can nevertheless “tag” the synapses involved with proteins synthesized ad hoc, so that other plasticity-related proteins (PRPs) produced at other sets of synapses by other LTPs or LTDs can be captured by the tagged synapses and strengthen their activity to “long” LTPs or LTDs lasting hours or days (8). LTDs and LTPs can “cross”-tag each other; that is, LTDs can enhance both LTDs and LTPs, and vice versa (6, 8). Because many learned behaviors rely on hippocampal LTP or LTD (7–9), among them the processing of novelty (9, 10) and the making of extinction (11–13), interactions between consecutive learnings can also be explained by the “tagging-and-capture” hypothesis (9, 10, 13), whose application to behavior became known as “behavioral tagging and capture” (5, 7, 9, 13). Typically, exposure to a novel environment [e.g., a nonanxiogenic 50 × 50 × 40-cm open field (OF) (5, 7, 9, 10, 14)] is interpolated before testing for another task, which becomes enhanced (4–10, 13). The usual reaction to novelty is orienting and exploration (14), followed by habituation of this response (14–16). Habituation is perhaps the simplest form of learning, and it consists of inhibition of the orienting/exploratory response (14, 16).We recently showed that the brief exposure of rats to a novel environment (the OF) within a limited time window enhances the extinction of contextual fear conditioning (CFC) through a mechanism of synaptic tagging and capture (13), which is a previously unidentified example of behavioral tagging of inhibitory learning. Fear extinction is most probably due to LTD in the hippocampus (11, 12), although the possibility that it may also involve LTP is not discarded (13). The enhancement of extinction by novelty probably relies on the habituation to the novel environment, which is also probably due to LTD (15, 16). The enhancement of extinction by the exposure to novelty depends on hippocampal gene expression and ribosomal protein synthesis following extinction training and on both ribosomal and nonribosomal protein synthesis caused by the novel experience (13). Nonribosomal protein synthesis that can be blocked by rapamycin is believed to be dendritic (13, 17), so it would be strategically located for tagging-and-capture processes, but it has not been studied in synaptic tagging to date (3–8) or in other forms of behavioral tagging (7–10). As occurs with the interactions between LTPs and/or LTDs (4), the enhancement of extinction by novelty relies on hippocampal but not amygdalar processes (13).Recent findings indicate that several hippocampal processes related to learning and memory, such as the reconsolidation of spatial learning, are highly dependent on NMDA glutamate receptors, calcium/calmodulin protein kinase II (CaMKII), and long-term voltage channel blockers (L-VDCCs), which, in turn, rely on the proteasomal degradation of proteins (18). Here, we study the effects of an NMDA blocker, 2-amino-5-phosphono pentanoic acid (AP5); the L-VDCC blocker nifedipine (Nife); a CaMKII inhibitor, the autocamtide-2–related inhibitory peptide (AIP); and the irreversible proteasome blocker β-lactacystin (12, 13) on the interaction between novelty and extinction (11). As will be seen, we found that both the setting up of tags by extinction and the presumable production of PRPs by the processing of novelty are dependent on NMDA receptors, CaMKII, and L-VDCCs. This endorses and expands the hypothesis that the novelty–extinction interaction relies on synaptic tagging and capture (13).     

PirB regulates a structural substrate for cortical plasticity

Experience-driven circuit changes underlie learning and memory. Monocular deprivation (MD) engages synaptic mechanisms of ocular dominance (OD) plasticity and generates robust increases in dendritic spine density on L5 pyramidal neurons. Here we show that the paired immunoglobulin-like receptor B (PirB) negatively regulates spine density, as well as the threshold for adult OD plasticity. In PirB^−/− mice, spine density and stability are significantly greater than WT, associated with higher-frequency miniature synaptic currents, larger long-term potentiation, and deficient long-term depression. Although MD generates the expected increase in spine density in WT, in PirB^−/− this increase is occluded. In adult PirB^−/−, OD plasticity is larger and more rapid than in WT, consistent with the maintenance of elevated spine density. Thus, PirB normally regulates spine and excitatory synapse density and consequently the threshold for new learning throughout life.Experience generates both functional and structural changes in neural circuits. The learning process is robust at younger ages during developmental critical periods and continues, albeit at a lower level, into adulthood and old age (1–3). For example, young barn owls exposed to horizontally shifting prismatic spectacles can adapt readily to altered visual input, but adult owls cannot. The experience in the young owls results in a rearranged audiovisual map in tectum that is accompanied by ectopic axonal projections (1). Experience-dependent structural changes have also been observed in the mammalian cerebral cortex. Enriched sensory experience or motor learning are both associated with an increase in dendritic spine density, and a morphological shift from immature thin spines to mushroom spines which harbor larger postsynaptic densities (PSDs) and stronger synapses (4–7). On the flip side, bilateral sensory deprivation induces spine loss (8, 9). Abnormal sensory experience also results in structural modification of inhibitory synapses and circuitry that is temporally and spatially coordinated with changes in excitatory synapses on dendritic spines (10–13).These experience-driven spine changes are thought to involve synaptic mechanisms of long-term potentiation (LTP) and long-term depression (LTD). In hippocampal slices, induction of LTP causes new spines to emerge, as well as spine head enlargement on existing spines (14–16); induction of LTD results in rapid spine regression (14, 17). Importantly, the emergence or regression of spines starts soon after the induction of LTP or LTD, suggesting that these structural changes underlie the persistent expression of long-term plasticity (14, 17).Little is known about molecular mechanisms that restrict experience-dependent plasticity at circuit and synaptic levels and connect it to spine stability. Paired Ig-like receptor B (PirB), a receptor expressed in cortical pyramidal neurons, is known to limit ocular dominance (OD) plasticity both during the critical period and in adulthood (18). PirB binds major histocompatibility class I (MHCI) ligands, whose expression is regulated by visual experience and neural activity (19–21) and thus could act as a key link connecting functional to structural plasticity. If so, mice lacking PirB might be expected to have altered synaptic plasticity rules on the one hand and changes in the density and stability of dendritic spines on the other.     

Long-term depression triggers the selective elimination of weakly integrated synapses

Long-term depression (LTD) weakens synaptic transmission in an activity-dependent manner. It is not clear, however, whether individual synapses are able to maintain a depressed state indefinitely, as intracellular recordings rarely exceed 1 h. Here, we combine optogenetic stimulation of identified Schaffer collateral axons with two-photon imaging of postsynaptic calcium signals and follow the fate of individual synapses for 7 d after LTD induction. Optogenetic stimulation of CA3 pyramidal cells at 1 Hz led to strong and reliable depression of postsynaptic calcium transients in CA1. NMDA receptor activation was necessary for successful induction of LTD. We found that, in the days following LTD, many depressed synapses and their “neighbors” were eliminated from the hippocampal circuit. The average lifetime of synapses on nonstimulated dendritic branches of the same neurons remained unaffected. Persistence of individual depressed synapses was highly correlated with reliability of synaptic transmission, but not with spine size or the amplitude of spine calcium transients. Our data suggest that LTD initially leads to homogeneous depression of synaptic function, followed by selective removal of unreliable synapses and recovery of function in the persistent fraction.Long-lasting modifications of synaptic transmission are thought to underlie learning and information storage in the brain. Intact synaptic plasticity seems to be a precondition for memory formation, and disturbing long-term depression (LTD) or long-term potentiation (LTP) strongly interferes with learning (1–6). Hippocampal field potential recordings have been used to demonstrate that LTD and LTP can be stable for weeks in vivo, at least at the level of large synaptic populations (7–9). It is less clear, however, whether individual synapses can maintain their strength at a specific level over the time scales of memory. Commonly used recording techniques to assess synaptic plasticity (e.g., whole-cell recordings, field recordings, or imaging of Ca²⁺-sensitive dyes) are too short-lived (10–12) or lack single-synapse resolution (7–9). Therefore, it is not known whether the strength of an individual synapse drifts over time, or how specific activity patterns affect the long-term stability of a synapse.In vivo imaging experiments have shown that dendritic spines in mammalian cortex constantly change their morphology and sometimes completely disappear or form de novo (13–15). Life expectancy and turnover of spines is affected by experience and behavioral paradigms, suggesting a regulated, activity-dependent process controlling spine lifetime (16–19). Could LTP and LTD form the missing link between neuronal activity and lasting structural changes? Indeed, induction of LTP at individual excitatory synapses in vitro leads to increased spine head size (12) and insertion of postsynaptic scaffolding proteins (20) and glutamate receptors (21). Potentiation of individual spine synapses selectively increases their stability, pointing to a connection between synaptic plasticity and spine survival (22). Conversely, whether LTD is connected to spine shrinkage or a reduction in spine lifetime is less clear (23–27), as it was not possible to identify individual depressed synapses and to observe them over sufficiently long time periods. Because of these methodological limitations, we know little about LTD at the synaptic level: Are all synapses equally sensitive to depression-inducing activity patterns? For how long do individual synapses stay in the depressed state? Is LTD as synapse-specific as LTP, or does it spread to neighboring synapses?To address these questions, we introduce a noninvasive, optical method based on optogenetic stimulation and two-photon Ca²⁺ imaging to control and measure the activity of individual hippocampal synapses in mature organotypic cultures over a period of 7 d. We found that depressed synapses were frequently eliminated from the circuit in the following days. This delayed elimination was not random: synapses with high release probability were more resistant to elimination than less reliable synapses. Resistant synapses were still connected to the same presynaptic axon after 1 wk and were indistinguishable from nonstimulated synapses, suggesting complete recovery of a subset of initially depressed synapses. Thus, the apparently stable LTD at the level of entire pathways in vivo does not seem to arise from a uniform, long-lasting depression of all stimulated synapses. On the contrary, it might reflect a protracted elimination process that removes a specific subpopulation of all depressed synapses. Our findings suggest that “long-term” depression of Schaffer collateral synapses following optogenetic low-frequency stimulation (oLFS) is, in fact, a transient phenomenon, setting in motion a reorganization of network connectivity.     

Conformational signaling required for synaptic plasticity by the NMDA receptor complex

The NMDA receptor (NMDAR) is known to transmit important information by conducting calcium ions. However, some recent studies suggest that activation of NMDARs can trigger synaptic plasticity in the absence of ion flow. Does ligand binding transmit information to signaling molecules that mediate synaptic plasticity? Using Förster resonance energy transfer (FRET) imaging of fluorescently tagged proteins expressed in neurons, conformational signaling is identified within the NMDAR complex that is essential for downstream actions. Ligand binding transiently reduces FRET between the NMDAR cytoplasmic domain (cd) and the associated protein phosphatase 1 (PP1), requiring NMDARcd movement, and persistently reduces FRET between the NMDARcd and calcium/calmodulin-dependent protein kinase II (CaMKII), a process requiring PP1 activity. These studies directly monitor agonist-driven conformational signaling at the NMDAR complex required for synaptic plasticity.Agonist binding to the NMDAR is required for two major forms of synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD) (1). Surprisingly, activation of NMDARs can produce plasticity in opposite directions, with LTP enhancing transmission and LTD reducing transmission. A model was developed (2, 3) to explain how activation of NMDAR could produce these opposing phenomena: strong stimuli during LTP induction drive a large flux of Ca²⁺ through NMDARs, leading to a large increase in intracellular calcium ion concentration ([Ca²⁺]_i) that activates one series of biochemical steps leading to synaptic potentiation; a weaker stimulus during LTD induction drives a more reduced flux of Ca²⁺ through NMDARs, producing a modest increase in [Ca²⁺]_i that activates a different series of biochemical steps, leading to synaptic depression. However, this model is not consistent with recent studies suggesting that no change in [Ca²⁺]_i is required for LTD, and instead invokes metabotropic signaling by the NMDAR (4). Studies supporting an ion-flow-independent role for NMDARs in LTD (4–7) and other processes (7–13) stand in contrast to studies proposing that flow of Ca²⁺ through NMDAR is required for LTD (14) (see ref. 15 for additional references). An important test of an ion-flow-independent model would be to measure directly signaling actions by NMDARs in the absence of ion flow.     

Epileptic baboons have lower numbers of neurons in specific areas of cortex

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.Epilepsy is associated with structural changes in the cerebral cortex (e.g., refs. 1–6), and partial epilepsies (i.e., seizures originating from a brain region) may lead to loss of neurons (7) and altered connectivity (8). The cerebral cortex is a heterogeneous structure comprised of multiple sensory and motor information-processing systems (e.g., refs. 9 and 10) that vary according to their processing demands, connectivity (e.g., refs. 11 and 12), and intrinsic numbers of cells and neurons (13–16). Chronic seizures have been associated with progressive changes in the region of the epileptic focus and in remote but functionally connected cortical or subcortical structures (3, 17). Because areas of the cortex are functionally and structurally different, they may also differ in susceptibility to pathological changes resulting from epilepsy.The relationship between seizure activity and neuron damage can be difficult to study in humans. Seizure-induced neuronal damage can be convincingly demonstrated in animals using electrically or chemically induced status epilepticus (one continuous seizure episode longer than 5 min) to reveal morphometric (e.g., refs. 18 and 19) or histological changes (e.g., refs. 20 and 21). Subcortical brain regions are often studied for vulnerability to seizure-induced injury (21–27); however, a recent study by Karbowski et al. (28) observed reduction of neurons in cortical layers 5 and 6 in the frontal lobes of rats with seizures. Seizure-induced neuronal damage in the cortex has also been previously demonstrated in baboons with convulsive status epilepticus (29).The goal of the present study was to determine if there is a specific pattern of cell or neuron reduction across the functionally divided areas of the neocortex in baboons with epilepsy. Selected strains of baboons have been studied as a natural primate model of generalized epilepsy (30–36) that is analogous to juvenile myoclonic epilepsy in humans. The baboons demonstrate generalized myoclonic and tonic-clonic seizures, and they have generalized interictal and ictal epileptic discharges on scalp EEG. Because of their phylogenetic proximity to humans, baboons and other Old World monkeys share many cortical areas and other features of cortical organization with humans (e.g., refs. 9 and 10). Cortical cell and neuron numbers were determined using the flow fractionator method (37, 38) in epileptic baboon tissue obtained from the Texas Biomedical Research Institute, where a number of individuals develop generalized epilepsy within a pedigreed baboon colony (31–36). Our results reveal a regionally specific neuron reduction in the cortex of baboons with naturally occurring, generalized seizures.     

Territories of heterologous inputs onto Purkinje cell dendrites are segregated by mGluR1-dependent parallel fiber synapse elimination

In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.Monoinnervation of cerebellar Purkinje cells (PCs) by single climbing fibers (CFs) is established in the early postnatal period (1–3). The soma of a neonatal PC is innervated by more than five CFs with similar synaptic strengths, from which a single CF is functionally strengthened (4, 5). The strengthened (“winner”) CF starts dendritic translocation, whereas the other weaker (“loser”) CFs remaining on the soma are eliminated (6–8). In this process, P/Q-type voltage-dependent Ca²⁺ channels (VDCCs) promote functional differentiation and dendritic translocation of winner CFs, and the early phase of CF synapse elimination (9–11), whereas the late phase of CF synapse elimination is critically dependent on the formation of parallel fiber (PF) synapses and activation of the type 1 metabotropic glutamate receptor (mGluR1)-protein kinase Cγ (PKCγ) pathway (12–17).Segregated dendritic innervation by CFs and PFs is another distinguished feature of the PC synaptic wiring. Although hundreds of thousands of PFs innervate the distal dendritic domain, a single CF monopolizes the proximal dendritic domain, and both innervate a narrow intermediate domain (18). Given that both dendritic translocation of winner CFs and formation of PF synapses proceed upwards from the base of the dendritic tree (6, 19), CFs and PFs must compete with each other to establish their segregated territories. However, the developmental route and the underlying mechanisms of this process are unknown.Our findings indicate that CF and PF territories on PC dendrites are dynamically refined during the early postnatal period, and that the mGluR1-PKCγ signaling pathway regulates segregation by promoting PF synapse elimination. Thus, this signaling cascade plays key roles in sculpting the excitatory synaptic wiring in PCs by eliminating both redundant CF synapses from the soma (3, 20) and competing PF synapses from the proximal dendrites.     

Population receptive field analysis of the primary visual cortex complements perimetry in patients with homonymous visual field defects

Injury to the primary visual cortex (V1) typically leads to loss of conscious vision in the corresponding, homonymous region of the contralateral visual hemifield (scotoma). Several studies suggest that V1 is highly plastic after injury to the visual pathways, whereas others have called this conclusion into question. We used functional magnetic resonance imaging (fMRI) to measure area V1 population receptive field (pRF) properties in five patients with partial or complete quadrantic visual field loss as a result of partial V1+ or optic radiation lesions. Comparisons were made with healthy controls deprived of visual stimulation in one quadrant [“artificial scotoma” (AS)]. We observed no large-scale changes in spared-V1 topography as the V1/V2 border remained stable, and pRF eccentricity versus cortical-distance plots were similar to those of controls. Interestingly, three observations suggest limited reorganization: (i) the distribution of pRF centers in spared-V1 was shifted slightly toward the scotoma border in 2 of 5 patients compared with AS controls; (ii) pRF size in spared-V1 was slightly increased in patients near the scotoma border; and (iii) pRF size in the contralesional hemisphere was slightly increased compared with AS controls. Importantly, pRF measurements yield information about the functional properties of spared-V1 cortex not provided by standard perimetry mapping. In three patients, spared-V1 pRF maps overlapped significantly with dense regions of the perimetric scotoma, suggesting that pRF analysis may help identify visual field locations amenable to rehabilitation. Conversely, in the remaining two patients, spared-V1 pRF maps failed to cover sighted locations in the perimetric map, indicating the existence of V1-bypassing pathways able to mediate useful vision.Cortical damage of the visual pathway often results from posterior or middle cerebral artery infarcts, hemorrhages, and other brain injuries. The most common visual cortex lesions involve the primary visual cortex (V1), the chief relayer of visual information to higher visual areas. Damage to area V1 or its primary inputs leads to the loss of conscious vision in the corresponding region of the contralateral visual hemifield, producing a dense contralateral scotoma that often covers a hemifield (hemianopia) or a single visual field quadrant (quadrantanopia).A much-debated issue is whether the adult V1 is able to reorganize after injury. Reorganization refers to long-term changes in the neuronal circuit (1) and generally requires the growth of new anatomic connections or a permanent change in the strength of existing connections. Several studies report significant remapping in area V1 of patients suffering from macular degeneration and other retinal lesions (2–12). The extent of this remapping has recently been called into question, however (1, 13–19). Less is known about how the visual system remaps to cover the visual field after injury to area V1 or its input projection from the lateral geniculate nucleus (LGN). Enlarged receptive fields have been found in areas surrounding chronic V1 lesions in cats (20–22), and visual point spread functions were seen to enlarge over time in the areas surrounding focal V1 lesions in kittens (23). Smaller, short-term changes (2 d after the lesion) have been reported as well (24). As expected, reorganization is more extensive in young animals (23, 25) compared with adults (26). A change in the balance between excitation and inhibition may underlie this functional reorganization (27–31).In humans, V1 injury is typically followed by a brief period of spontaneous recovery, which rarely lasts beyond 6 mo (32). Whether this recovery is the result of true visual system plasticity or is related to the gradual resolution of perilesional edema and general clinical improvement of the patients is unclear. A recent study in an adult human subject suggested that large-scale reorganization occurs in area V1 after partial deafferentiation by an optic radiation lesion (33); however, quantitative measurements were not performed. To date, there has been no systematic study in humans investigating how spared V1 cortex covers the visual field after chronic V1 injury. The present work is an effort in this direction.We used the population receptive field (pRF) mapping method (34) to study how spared area V1 covers the visual field after chronic injury in five adult human subjects suffering from partial or complete quadrantanopia. Our findings suggest that there is at best a limited degree of reorganization in the spared part of area V1 after partial V1 injury. Interestingly, the pattern of coverage of the visual field measured in spared V1 cortex by functional magnetic resonance imaging (fMRI) typically does not match predictions derived from perimetry maps. Identifying the patterns of mismatch and how they relate to the capacity of early visual areas to reorganize after injury will eventually allow the adoption of more rational strategies for visual rehabilitation.     

Behavioral response inhibition and maturation of goal representation in prefrontal cortex after puberty

Executive functions including behavioral response inhibition mature after puberty, in tandem with structural changes in the prefrontal cortex. Little is known about how activity of prefrontal neurons relates to this profound cognitive development. To examine this, we tracked neuronal responses of the prefrontal cortex in monkeys as they transitioned from puberty into adulthood and compared activity at different developmental stages. Performance of the antisaccade task greatly improved in this period. Among neural mechanisms that could facilitate it, reduction of stimulus-driven activity, increased saccadic activity, or enhanced representation of the opposing goal location, only the latter was evident in adulthood. Greatly accentuated in adults, this neural correlate of vector inversion may be a prerequisite to the formation of a motor plan to look away from the stimulus. Our results suggest that the prefrontal mechanisms that underlie mature performance on the antisaccade task are more strongly associated with forming an alternative plan of action than with suppressing the neural impact of the prepotent stimulus.Behavioral response inhibition, and cognitive task performance more generally, improves substantially between the time of puberty and adulthood (1–4). Risky decision-making peaks in adolescence, the time period between puberty and adulthood that is most closely linked to delinquent behavior in humans (5–7). Performance in tasks that assay response inhibition, such as the antisaccade task, improves into adulthood, reflecting the progressive development of behavioral control (3). This period of cognitive enhancement parallels the maturation of the prefrontal cortex (8–11). Anatomical changes in the prefrontal cortex continue during adolescence, involving gray and white matter volumes and myelination of axon fibers within the prefrontal cortex and between the prefrontal cortex and other areas (8–15). Changes in prefrontal activation, including increases (12, 16–20) and decreases (21, 22), have been documented in imaging studies for tasks that require inhibition of prepotent behavioral responses and filtering of distractors.Much less is known about how the physiological properties of prefrontal neurons develop after puberty. Similar to the human pattern of development, the monkey prefrontal cortex undergoes anatomical maturation in adolescence and early adulthood (23, 24). Male monkeys enter puberty at ∼3.5 y of age and reach full sexual maturity at 5 y, approximately equivalent to the human ages of 11 y and 16 y, respectively (25, 26). By some accounts, biochemical and anatomical changes characteristic of adolescence in humans occur at an earlier, prepubertal age in the monkey prefrontal cortex (27, 28), so it is not known if cognitive maturation or neurophysiological changes occur in monkeys after puberty. The contribution of prefrontal cortex to antisaccade performance has also been a matter of debate, with contrasting views favoring mechanisms of inhibiting movement toward the visual stimulus or enhancing movement away from it (29–31). Potential maturation of behavioral response inhibition may therefore be associated with a more efficient suppression of the stimulus representation in neural activity (weaker visual responses to stimuli inside the receptive field), stronger motor responses (higher activity to saccades), or enhancement of the appropriate goal representation (stronger activity for planning a saccade away from the stimulus). To examine the mechanisms that facilitate the mature ability to resist generating a response toward a salient stimulus, we used developmental markers to track transition from puberty to adulthood in monkeys and sought to identify neural correlates of changes in antisaccade performance within the visual and saccade-related activations of prefrontal neurons.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

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.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

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.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.