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While the mechanisms responsible for LTP and LTD of excitatory synaptic responses mediated by AMPA receptors (AMPARs) have been extensively characterized, much less is known about the regulation of NMDA receptors (NMDARs) by synaptic activity. In hippocampal CA1 cells, prolonged low frequency afferent stimulation depresses synaptic responses mediated by either NMDARs or AMPARs. However, this apparently similar LTD is accompanied by a change in the coefficient of variation (CV) of only the AMPAR-mediated synaptic responses; the CV of the NMDAR-mediated synaptic responses is unaffected. Moreover, by varying the pattern of synaptic stimulation, the responses mediated by one receptor subtype can be modified without affecting the responses mediated by the other. These results indicate that the mechanisms underlying activity-dependent plasticity of NMDAR-mediated synaptic responses are different from those responsible for plasticity of AMPAR-mediated synaptic responses.

Summary Recent work has suggested that some proportion of excitatory synapses on hippocampal CA1 pyramidal cells that express NMOA receptors (NMDARa) may not express functional AMPA receptors (AMPARs), thus making these synapses silent at the resting membrane potential. In agreement with this hypothesis, we demonstrete here that it is pQssible to stimulate synapses that yield no detectable excitatory postsynaptic currents (EPSCs) when the cell is held at -60 mV; yet at positive holding potentials (+30 to +60 mV), EPSCs can be elicited that are completely blocked by the NMOAR antagonist, D-APV. When these functionally silent synapses are subjected to an LTP Induction protocol, EPSCs mediated by AMPARs appear and remain for the duration of the experiment. This conversion of silent synapses to functional synapses is blocked by D-APV. These results suggest that LTP may involve modification of AMPARs that, prior to LTP, were either not present in the postsynaptic membrane or electrophysiologically silent. This mechanism may account for several experimental results previously attributed to presynaptic changes in quantal content.

The use of human embryonic stem cells (hESCs) to repair diseased or injured brain is promising technology with significant humanitarian, societal and economic impact. Parkinson's disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. The generation of this cell type will fulfill a currently unmet therapeutic need. We report on the isolation and perpetuation of a midbrain-specified self-renewable human neural stem cell line (hNSCs) from hESCs. These hNSCs grew as a monolayer and uniformly expressed the neural precursor markers nestin, vimentin and a radial glial phenotype. We describe a process to direct the differentiation of these hNSCs towards the DA lineage. Glial conditioned media acted synergistically with fibroblastic growth factor and leukemia inhibitory factor to induce the expression of the DA marker, tyrosine hydroxylase (TH), in the hNSC progeny. The glial-derived neurotrophic factor did not fully mimic the effects of conditioned media. The hNSCs expressed the midbrain-specific transcription factors Nurr1 and Pitx3. The inductive effects did not modify the level of the glutamic acid decarboxylase (GAD) transcript, a marker for GABAergic neurons, while the TH transcript increased 10-fold. Immunocytochemical analysis demonstrated that the TH-expressing cells did not co-localize with GAD. The transplantation of these DA-induced hNSCs into the non-human primate MPTP model of PD demonstrated that the cells maintain their DA-induced phenotype, extend neurite outgrowths and express synaptic markers.

The role of protein phosphatases in regulating synaptic transmission in the CA1 region of the hippocampus was examined using slices pretreated with calyculin A, a specific inhibitor of protein phosphatases 1 and 2A. Stimulation of afferents at 1 Hz (but not 0.1 Hz) for periods of 5– 10 min caused a long-lasting enhancement of synaptic transmission. The increase in synaptic responses was not due to a change in fiber excitability, as there was a shift to the left in the input-output curve following the synaptic enhancement. The enhancement was observed only in the input that received the 1 Hz stimulation and not in an independent control pathway, indicating that the increase in synaptic strength is input specific and limited to repetitively activated synapses. Applying 1 Hz stimulation when synaptic transmission was blocked by replacing extracellular Ca2+ with Mg2+ prevented or significantly reduced any change in synaptic efficacy after reperfusion with normal Ca(2+)-containing medium. In contrast, 1 Hz stimulation given when synaptic transmission was blocked by non-NMDA and NMDA glutamate receptor antagonists still caused a synaptic enhancement following washout of the antagonists. The enhancement of synaptic transmission also was not blocked by loading CA1 cells with the calcium chelator BAPTA. Thus, influx of Ca2+ into presynaptic elements is required for the synaptic enhancement elicited by 1 Hz stimulation in calyculin A-treated hippocampal slices. Consistent with the activation of processes that cause an increase in transmitter release, the magnitude of paired-pulse facilitation decreased following the synaptic enhancement, and the NMDA receptor-mediated component of the synaptic response was increased by 1 Hz stimulation. These results suggest that when protein phosphatases are inhibited by calyculin A, prolonged periods of 1 Hz stimulation lead to activation of presynaptic Ca(2+)- dependent protein kinases, resulting in a persistent increase in evoked transmitter release. They also indicate that the activity of presynaptic protein phosphatases is critically important for limiting increases in synaptic strength following repetitive afferent activity.

Several recent studies have suggested a critical role for nitric oxide (NO) production in hippocampal LTP and LTD. In this study we show that normal LTP and LTD can be induced in rat hippocampal slices incubated in the NO synthase inhibitor L-NG-nitroarginine (NOArg) (100 microM). A test of NMDA-stimulated cGMP production demonstrated that incubation of slices in 100 microM NOArg effectively inhibited NO synthase. Our results suggest that NO synthase activity may not be required for the generation of LTP or LTD in CA1 of rat hippocampus.

Long-term potentiation (LTP) is a synaptic mechanism thought to be involved in learning and memory. Long-term depression (LTD), an activity-dependent decrease in synaptic efficacy, may be an equally important mechanism which permits neural networks to store information more effectively. One form of LTD that has been observed in the hippocampus requires activation of postsynaptic NMDA (N-methyl-D-aspartate) receptors, a change in postsynaptic calcium concentration, and activation of postsynaptic serine/threonine protein phosphatase 1 (PP1) or 2A (PP2A). The mechanism by which PP1 or PP2A is regulated by synaptic activity is unclear because these protein phosphatases are not directly influenced by calcium concentration. LTD induction may require activation of a more complex protein phosphatase cascade consisting of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, its phosphoprotein substrate, inhibitor-1, and PP1. We tested this hypothesis using calcineurin inhibitors as well as different forms of inhibitor-1 loaded into postsynaptic cells. Our results suggest a signalling pathway in which calcineurin dephosphorylates and inactivates inhibitor-1. This in turn increases PP1 activity and contributes to the generation of LTD.

Long-term potentiation in the CA1 region of the hippocampus is the most extensively studied model of activity-dependent synaptic plasticity in the mammalian brain. Its induction normally involves activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors, which are thought to control the occurrence of long-term potentiation at individual synapses. Recent work in the hippocampus indicates that NMDA receptor activation does not necessarily lead to induction of long-term potentiation but instead may elicit a repertoire of distinct forms of synaptic plasticity including short-term potentiation or long-term depression. Furthermore, mechanisms exist such that the induction of long-term potentiation can be inhibited by modest activation of NMDA receptors. Experimental results are beginning to clarify the mechanistic relationships between these different phenomena, although much remains unknown. Whatever their underlying mechanisms, these additional forms of NMDA-receptor-dependent synaptic plasticity confer increased flexibility to neural circuits involved in information processing and storage.

Synapsin I, the major phosphoprotein of synaptic vesicles, is thought to play a central role in neurotransmitter release. Here we introduce a null mutation into the murine synapsin I gene by homologous recombination. Mice with no detectable synapsin I manifest no apparent changes in well-being or gross nervous system function. Thus, synapsin I is not essential for neurotransmitter release. Electrophysiology reveals that mice lacking synapsin I exhibit a selective increase in paired pulse facilitation, with no major alterations in other synaptic parameters such as long-term potentiation. In addition to potential redundant functions shared with other proteins, synapsin I in normal mice may function to limit increases in neurotransmitter release elicited by residual Ca2+ after an initial stimulus.

The effectiveness of long-term potentiation (LTP) as a mechanism for information storage would be severely limited if processes that decrease synaptic strength did not also exist. In area CA1 of the rat hippocampus, prolonged periods of low-frequency afferent stimulation elicit a long-term depression (LTD) that is specific to the stimulated input. The induction of LTD was blocked by the extracellular application of okadaic acid or calyculin A, two inhibitors of protein phosphatases 1 and 2A. The loading of CA1 cells with microcystin LR, a membrane-impermeable protein phosphatase inhibitor, or calmodulin antagonists also blocked or attenuated LTD. The application of calyculin A after the induction of LTD reversed the synaptic depression, suggesting that phosphatase activity is required for the maintenance of LTD. These findings indicate that the synaptic activation of protein phosphatases plays an important role in the regulation of synaptic transmission.

Long-lasting activity-dependent changes in the efficacy of synaptic transmission in the mammalian brain are considered to be of fundamental importance for the development of neural circuitry and for the storage of information. The most compelling and reliable model for such changes has been long-term potentiation (LTP) in the hippocampus, a long-lasting increase in synaptic strength normally induced by repetitive high-frequency activation of presynaptic afferents (1). With the advent of in vitro brain slice preparations and their inherent experimental advantages, an enormous effort by a cadre of researchers over the last decade has resulted in detailed (and sometimes controversial) hypotheses concerning the cellular and biochemical mechanisms responsible for LTP (2). A nagging problem concerning the utility of LTP as either an important developmental or memory-storage mechanism has been the inability to define and characterize experimental paradigms that reliably decrease synaptic strength. Although a long-term depression (LTD) of synaptic transmission is not absolutely necessary to constrain LTP, which does exhibit a finite decay, neural networks with the ability to modify synaptic strength in both directions have enormous flexibility and power. Recently, over the last year or so, significant progress has been made in the experimental analysis of LTD in the mammalian brain. It appears that LTD, like LTP, is not a single uniform phenomenon but rather must be considered a generic term that is used to describe any long-lasting decrease in synaptic strength. Nevertheless, some common themes concerning the mechanisms of LTD in different brain regions are beginning to emerge. Because the induction of most forms of LTP requires synaptic activation of N-methyl-D-aspartate (NMDA) receptors (one of the several subtypes of receptor activated by the excitatory neurotransmitter glutamate) during strong postsynaptic depolarization (2), LTP is considered a biological correlate of the Hebb postulate (3), which in principle predicts an increase in synaptic efficacy when presynaptic and postsynaptic activity are strongly correlated. The requirement for NMDA receptor activation also explains the important property of LTP referred to as input or synapse specificity; only those synapses activated by the LTP-inducing stimulus exhibit LTP. In contrast, early work in the hippocampus both in vivo and in vitro demonstrated that when LTP is induced in one population of synapses, a modest depression may occur at some of the inactive synapses on the same population of postsynaptic cells (4-6). In a classic paper that anticipated this finding (and that was published in this journal almost exactly 20 years ago) Stent (7) proposed that such "heterosynaptic" decreases in synaptic efficacy could be explained by an extension of the Hebb rule; synaptic strength will decrease at inputs that are quiescent or weakly active when the postsynaptic cell is very active. While theoreticians over the ensuing two decades convincingly demonstrated the importance of algorithms that could decrease synaptic strength (8-10), experimental work on LTD lagged far behind, especially when compared to the enormous effort aimed at understanding the mechanisms of LTP. Thus, several years ago, considerable excitement was generated by the report that LTD could be generated in the CAl region of hippocampal slices when synaptic inputs were stimulated either outof-phase with short bursts of stimuli given to an independent conditioning input or during direct hyperpolarization of the postsynaptic cell (11). This LTD was of particular interest because it was "homosynaptic," occurring only at those synapses activated by the stimulation, and because its induction obeyed the converse of the Hebb rule (i.e., synapses were weakened when their activity did not correlate with significant postsynaptic activity). Unfortunately, disappointment soon followed as many laboratories had difficulty replicating this phenomenon (12). However, optimism and enthusiasm are on the rise recently as it appears that there are indeed forms of LTD that are amenable to rigorous experimental attack (not only in the hippocampus but also in the cerebellum and cerebral cortex). A common theme that emerges from the results to date is that, like LTP, changes in postsynaptic Ca2+ levels are required for the generation of LTD, although the specific characteristics of this Ca2+ signal may differ. In the CAl region of the hippocampus, prolonged, low-frequency (1 Hz) afferent stimulation produces a saturable and stable homosynaptic (i.e., synapse specific) LTD (13, 14). Surprisingly, the induction of this form of LTD is quite similar to LTP in that it requires activation of NMDA receptors (13, 14) and is blocked by strong hyperpolarization or buffering postsynaptic Ca2+ (14). Homosynaptic LTD has much in common with previous reports of "depotentiation" following LTP (15, 16); in fact, recent evidence suggests that homosynaptic LTD and LTP are reversal modifications of some common expression mechanism (17). However, a significant difference between homosynaptic LTD and LTP may be their developmental profile, since LTD appears to be more robust in slices prepared from young animals (14, 17). The mechanisms of heterosynaptic LTD have also been examined recently in the CAl region of hippocampal slices. This form of LTD is often quite small in magnitude (18) or requires unusual induction conditions (i.e., complete blockade of synaptic transmission during induction) (19). Nonetheless, changes in postsynaptic Ca2+ again appear to be required, although in this case Ca2+ is supplied via activation of voltage-dependent Ca2+ channels (18, 19). In the cerebellum, an absolute requirement for the LTD of the parallel fiber (PF)-Purkinje cell (PC) synapse induced by simultaneous climbing-fiber (CF) activation is a rise in postsynaptic Ca2+. The evidence includes the following observations: (i) CF activation can be replaced by direct depolarization of PCs (20, 21), (ii) both manipulations result in large increases in dendritic Ca2+ level (21), and (iii) LTD is blocked by loading PCs with Ca2+ chelators (21, 22) or by removing external Ca2+ (20). Activation of a metabotropic glutamate receptor (20, 23) possibly coupled to activation of protein kinase C (24) also appears to be necessary for cerebellar LTD. Questions that remain about this form of LTD include whether activation of the synaptic non-NMDA receptor is necessary for

Because LTP and LTD may contribute to experience-dependent plasticity, a prominent hypothesis is that developmental changes in the biophysical and molecular properties of NMDA receptors (NMDARs) may regulate the duration of critical periods1,2,3,4,5,6,7. Here we report that susceptibility to LTP at thalamocortical synapses in early postnatal mouse slices is lost at a time point when the duration of NMDAR-mediated excitatory post-synaptic currents (NMDAR EPSCs) is not significantly altered. However, changes in the subunit composition of NMDARs, as defined pharmacologically, correlate strongly with the loss of the ability to generate LTP.

The mechanisms responsible for long-lasting, activity-dependent decreases in synaptic efficacy are not well understood. We have examined the initial steps required for the induction of long-term depression (LTD) in CA1 pyramidal cells by repetitive low frequency (1 Hz) synaptic stimulation. This form of LTD was synapse specific, was saturable, and required activation of post-synaptic NMDA receptors. Loading CA1 cells with the Ca2+ chelator BAPTA prevented LTD, whereas lowering extracellular Ca2+ resulted in the induction of LTD by stimulation that previously elicited long-term potentiation. Following LTD, synaptic strength could be increased to its original maximal level, indicating that LTD is reversible and not due to deterioration of individual synapses. Induction of homosynaptic LTD therefore requires an NMDA receptor-dependent change in postsynaptic Ca2+ which may be distinct from that required for long-term potentiation.

In area CA1 of the hippocampus, synaptic activation of NMDA receptors during postsynaptic depolarization can generate either a decremental synaptic potentiation termed short-term potentiation (STP) or stable, long-term potentiation (LTP). Examining the relationship between these two forms of synaptic enhancement should provide information about the intracellular processes responsible for the stabilization of LTP. Using the hippocampal slice preparation, initial experiments confirmed that STP can be generated either by a weak tetanus or by pairing a single EPSP with postsynaptic depolarization. Following the generation of submaximal LTP, application of a weak, STP-inducing tetanus resulted in STP (not LTP), suggesting that the processes responsible for stabilizing LTP must be activated during induction and cannot be accessed at later times. To determine the interval over which processes activated during STP can be integrated and result in stable LTP (the “integration time” for the stabilization of LTP), a fixed number of afferent stimuli were given at varying intervals (5–60 sec) during postsynaptic depolarization. Using either extracellular or whole-cell recording, LTP was rarely (11% of experiments) elicited at 1 min intervals and frequently (76% of experiments) elicited at 10 sec intervals. These results indicate that following a single EPSP during postsynaptic depolarization, the processes responsible for the stabilization of LTP decay significantly within approximately 1 min, although this value may depend on the level of activation of the requisite intracellular processes.

A curious property of excitatory synapses in the hippocampus and some other neural tissues is that when they are heavily used, they undergo a long­ lasting increase in their efficacy. Brief repetitive activation of hippocampal excitatory synapses results in a substantial increase in synaptic strength that can last for several hours and has been detected even weeks after induction. This use-dependent strengthening of a synapse is known as long-term potentiation, or more commonly, LTP (Bliss & Lorna 1973, Lomo 1966, Bliss & Lynch 1988). LTP occurs most prominently in the hippocampus, where consolidation of experience into long-term memory is thought to occur. LTP is the most compelling and widely studied model

Long-term potentiation (LTP) is a persistent enhancement of synaptic transmission observed at excitatory synapses in the mammalian hippocampus (Bliss and Lomo, 1973). This phenomenon is one of the most striking examples of synaptic plasticity in the vertebrate brain, and has been intensively studied as a model for learning and memory. LTP can be divided into two parts, the triggering or initiation events, and the long-lasting alteration in synaptic strength. In the past six years a considerable body of work has clarified some of the processes involved in triggering LTP, and it has become widely accepted that these processes are localized to the postsynaptic neuron. In contrast, the processes responsible for maintaining the potentiation over time are not as clearly understood, and the synaptic site of these processes remains controversial. This chapter will focus on work from our laboratory studying the cellular mechanisms involved in LTP at the Schaffer collateral-pyramidal cell synapse in the CA1 region of the hippocampal slice preparation.

There is plenty of indirect evidence that the postsynaptic density (PSD)-95 protein is involved in the clustering and, perhaps, targeting of postsynaptic proteins. Direct evidence was lacking, but PSD-95 knockout mice have now arrived, and their properties support a clustering function. However, knocking out PSD-95 also disrupts synaptic plasticity, and this could mean that PSD-95 acts as a kind of scaffold protein.

Dale's principle states that each neuron uses one and only one neurotransmitter at all of its synapses. In their commentary, Nicoll and Malenka recount how the truth of this axiom was seriously undermined when some neurons were discovered to use two neurotransmitters. They go on to describe a report by page [ Jonas et al .][1] in this issue, which deals a further blow to Dale's principle by showing that two small molecule transmitters, glycine and GABA, can be released from the very same vesicle. [][2] [1]: http://www.sciencemag.org/cgi/content/short/281/5375/419 [2]: http://

Hippocampal long-term potentiation (LTP) has long been suspected to be involved in learning and memory, but definitive proof has been hard to come by. Two groups now report, however, that fear conditioning (which is a form of pavlovian conditioning) causes an increase in the strength of synapses in a region of the brain that is associated with learning and memory. Animals were trained to associate a tone with a foot shock, eliciting a response of ‘fear’. After training, this response occurred when the tone was given but the foot shock was not.

Long-lasting changes in the strength of synapses in the brain underlie many forms of learning and memory. Depending on the pattern of activation, synapses can show either long-term potentiation (LTP) or long-term depression (LTD). A provocative study now shows that a form of LTD may spread over considerable distances, and that this spread may even involve signals that travel backwards.

Heterosynaptic long-term depression (hetLTD) at one input can be induced by applying a conditioning stimulus to an adjacent set of synapses. In hippocampal CA1 pyramidal cells, our results suggest that hetLTD is triggered by an extracellular diffusible factor that is released following tetanic activation of NMDA receptors. This hetLTD occludes with homosynaptic LTD suggesting common underlying mechanisms.

THE phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate1 with concomitant postsynaptic membrane depolarization2-5. This relieves the voltage-dependent magnesium block of the NMD A-receptor ion channel6,7, allowing calcium to flow into the dendritic spine8-10. Although calcium has been shown to be a necessary trigger for LTP (refs 11,12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/cal-modulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain20. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Further-more, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.

Both serotonin and the selective gamma-aminobutyric acidB (GABAB) agonist, baclofen, increase potassium (K+) conductance in hippocampal pyramidal cells. Although these agonists act on separate receptors, the potassium currents evoked by the agonists are not additive, indicating that the two receptors share the same potassium channels. Experiments with hydrolysis-resistant guanosine triphosphate (GTP) and guanosine diphosphate analogs and pertussis toxin indicate that the opening of the potassium channels by serotonin and GABAB receptors involves a pertussis toxin-sensitive GTP-binding (G) protein, which may directly couple the two receptors to the potassium channel.

Brief repetitive activation of excitatory synapses in the hippocampus leads to an increase in synaptic strength that lasts for many hours. This long-term potentiation (LTP) of synaptic transmission is the most compelling cellular model in the vertebrate brain for learning and memory. The critical role of postsynaptic calcium in triggering LTP has been directly examined using three types of experiment. First, nitr-5, a photolabile nitrobenzhydrol tetracarboxylate calcium chelator, which releases calcium in response to ultraviolet light, was used. Photolysis of nitr-5 injected into hippocampal CA1 pyramidal cells resulted in a large enhancement of synaptic transmission. Second, in agreement with previous results, buffering intracellular calcium at low concentrations blocked LTP. Third, depolarization of the postsynaptic membrane so that calcium entry is suppressed prevented LTP. Taken together, these results demonstrate that an increase in postsynaptic calcium is necessary to induce LTP and sufficient to potentiate synaptic transmission.

A previous study suggested that schizophrenic subjects exhibit an impaired ability to correct their own errors of movement without using exteroceptive signals. However, the performance of schizophrenic subjects was compared to that of only one other psychiatric group (alcoholic subjects), and a relatively small number of subjects was studied. To investigate the specificity of the postulated impairment, 9 schizophrenic, 11 depressed, and 8 normal subjects performed a tracking task designed to prevent the use of exteroceptive cues in correcting errors of movement. The depressed and normal groups did not differ significantly on any performance measure, but the schizophrenic subjects again demonstrated a gross impairment in correcting errors, yet no impairment in initiating correct responses. These findings suggest that the impaired ability to monitor ongoing motor behavior on the basis of internal, self-generated cues may be specific to schizophrenia among major psychiatric disorders.

The interspike interval for a pair of propagating impulses was studied from a simulation model of axons with a triphasic recovery cycle (relative refractory, supernormal, and subnormal periods) and from electrophysiologic results on the parallel fibers of the cerebellar cortex. Impulse entrainment, whereby convergence of the interspike interval to a fixed value occurs, was predicted from the model for impulses initiated at any point during the recovery cycle. Convergence of the interspike interval toward a fixed value was also observed for the parallel fibers when impulses were initiated during the relative refractory or the early supernormal periods. We suggest that convergence of the interspike interval contributes to regularization of constitutent impulses within a spike train.

Muscarinic receptor stimulation in the hippocampus has been associated with inositol phospholipid breakdown. In other systems this leads to the formation of inositol trisphosphate and diacylglycerol, which promotes the activation of protein kinase C. Phorbol esters, which directly activate protein kinase C, exhibit high and specific binding in the hippocampus. This, along with the advantages of the hippocampal slice preparation, including direct pharmacological access to a cell population (CA1 pyramidal cells) having clearly defined muscarinic responses, makes this an ideal preparation to examine whether protein kinase C serves as the intracellular signal for muscarinic receptor occupation. Like muscarinic agonists, phorbol esters abolish the slow calcium-activated potassium afterhyperpolarizing potential (AHP) and its underlying current without reducing calcium action potentials. Those phorbol analogs that do not activate kinase C have no effect, suggesting that activation of this enzyme is required to reduce the AHP. The accommodation of spike discharge normally seen during a long depolarizing stimulus is also markedly reduced by phorbol esters as well as by muscarinic receptor activation. However, unlike muscarinic agonists, phorbol esters have no effect on the muscarine-sensitive, voltage-dependent, potassium current termed IM, nor do they consistently cause an increase in input resistance. Moreover, unlike ACh, they do not appear to have a presynaptic inhibitory action on the fast EPSP elicited by orthodromic stimulation. The slow cholinergic EPSP was blocked by phorbol esters, but this could be accounted for by a postsynaptic action. Thus, if inositol phospholipid turnover is involved in mediating muscarinic responses in the hippocampus, the activation of protein kinase C can account for only part of the electrophysiological response.

Recent studies have gone a long way to explain the steps involved in generating long-term potentiation (LTP). This review focuses on the triggering role of postsynaptic calcium, the sequence of events which might be initiated by calcium, and where the persistent change may ultimately occur during LTP.

The effect of dopamine (DA) on the calcium-activated potassium conductance underlying the slow afterhyperpolarization (AHP) which follows a train of action potentials in hippocampal pyramidal cells was studied utilizing the in vitro hippocampal slice preparation. Bath-applied DA (1-100 microM) significantly reduced the AHP in a reversible, dose-dependent manner. Neither the amount of current injected to elicit the AHP nor its initial amplitude had an effect on the reduction of the AHP by DA. DA did not depress calcium spikes, suggesting that the blockade of the AHP likely occurs at a step subsequent to the entry of calcium. Since DA's actions on the AHP closely mimicked those of norepinephrine, we examined the effect of beta-adrenergic antagonists on DA's actions. At concentrations which in other systems have been shown not to block DA stimulated adenylate cyclase, beta-adrenergic antagonists completely inhibited the reduction of the AHP by DA. In some cells DA also elicited small hyperpolarizations which were not blocked by application of dopamine receptor antagonists. These findings strongly suggest that a major electrophysiological action of DA in the hippocampus (i.e. blockade of the AHP) is due to its cross reactivity with beta-adrenergic receptors and that rigid pharmacologic criteria must be used before attributing an action of DA unambiguously to its interaction with DA receptors.

Despite extensive research, much controversy remains regarding the locus of expression of long-term potentiation (LTP) in area CA1 of the hippocampus, specifically, whether LTP is accompanied by an increase in the probability of release (p(r)) of synaptic vesicles. We have developed a novel method for assaying p(r), which utilizes the synaptic refractory period--a brief 5-6 ms period following release during which the synapse is incapable of transmission (Stevens and Wang, 1995). We show that this assay is sensitive to a battery of manipulations that affect p(r) but find no change following either NMDA receptor-dependent LTP or long-term depression (LTD).

SummaryVentral tegmental area (VTA) dopamine (DA) neurons have been implicated in reward, aversion, salience, cognition, and several neuropsychiatric disorders. Optogenetic approaches involving transgenic Cre-driver mouse lines provide powerful tools for dissecting DA-specific functions. However, the emerging complexity of VTA circuits requires Cre-driver mouse lines that restrict transgene expression to a precisely defined cell population. Because of recent work reporting that VTA DA neurons projecting to the lateral habenula release GABA, but not DA, we performed an extensive anatomical, molecular, and functional characterization of prominent DA transgenic mouse driver lines. We find that transgenes under control of the tyrosine hydroxylase, but not the dopamine transporter, promoter exhibit dramatic non-DA cell-specific expression patterns within and around VTA nuclei. Our results demonstrate how Cre expression in unintentionally targeted cells in transgenic mouse lines can confound the interpretation of supposedly cell-type-specific experiments. This Matters Arising paper is in response to Stamatakis et al. (2013), published in Neuron. See also the Matters Arising Response paper by Stuber et al. (2015), published concurrently with this Matters Arising in Neuron.

SummaryActive neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). By using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma xenograft model, we demonstrate that active neurons similarly promote HGG proliferation and growth in vivo. Conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures, indicating secretion of activity-regulated mitogen(s). The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen, and soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feedforward expression of NLGN3 in glioma cells. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth.

The N-methyl-D-aspartate receptor–independent form of long-term potentiation (LTP) at hippocampal mossy fiber synapses requires presynaptic Ca 2+ –dependent activation of adenylyl cyclase. To determine whether this form of LTP might occur at other synapses, we examined cerebellar parallel fibers that, like hippocampal mossy fiber synapses, express high levels of the Ca 2+ /calmodulin-sensitive adenylyl cyclase I. Repetitive stimulation of parallel fibers caused a long-lasting increase in synaptic strength that was associated with a decrease in paired-pulse facilitation. Blockade of glutamate receptors did not prevent LTP induction, nor did loading of Purkinje cells with a Ca 2+ chelator. LTP was occluded by forskolin-induced potentiation and blocked by the protein kinase A inhibitor Rp-8-CPT-cAMPS. These findings suggest that parallel fiber synapses express a form of LTP that is dependent on the activation of a presynaptic adenylyl cyclase and is indistinguishable from LTP at hippocampal mossy fiber synapses.

Neural circuit development requires concurrent morphological and functional changes. Here, we identify coordinated and inversely correlated changes in dendritic morphology and mEPSC amplitude following increased neural activity. We show that overexpression of beta-catenin, a molecule that increases total dendritic length, mimics the effects of increased neuronal activity by scaling down mEPSC amplitudes, while postsynaptic expression of a protein that sequesters beta-catenin reverses the effects of activity on reducing mEPSC amplitudes. These results were confirmed immunocytochemically as changes in the size and density of surface synaptic AMPA receptor clusters. In individual neurons there was an inverse linear relationship between total dendritic length and average mEPSC amplitude. Importantly, beta-catenin overexpression in vivo promoted dendritic growth and reduced mEPSC amplitudes. Together, these results demonstrate that coordinated changes in dendritic morphology and unitary excitatory synaptic strength may serve as an important intrinsic mechanism that helps prevent neurons from overexcitation during neural circuit development.

SummaryThe transcription factor CREB is critical for several forms of experience-dependent plasticity in a range of species and is commonly activated in neurons by calcium/calmodulin-dependent protein kinase IV (CaMKIV). Surprisingly, little is known about the neural circuit adaptations caused by activation of CaMKIV and CREB. Here, we use viral-mediated gene transfer in vivo to examine the consequences of acute expression of constitutively active forms of CaMKIV and CREB on synaptic function in the rodent hippocampus. Acute expression of active CaMKIV or CREB caused an enhancement of both NMDA receptor-mediated synaptic responses and long-term potentiation (LTP). This was accompanied by electrophysiological and morphological changes consistent with the generation of “silent synapses,” which provide an ideal substrate for further experience-dependent modifications of neural circuitry and which may also be important for the consolidation of long-term synaptic plasticity and memories.

SummaryNeurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell-type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell-type-specific expression patterns of multiple neurexins at the single-cell level and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity.

Recently, leucine-rich repeat transmembrane proteins (LRRTMs) were found to be synaptic cell-adhesion molecules that, when expressed in nonneuronal cells, induce presynaptic differentiation in contacting axons. We now demonstrate that LRRTM2 induces only excitatory synapses, and that it also acts to induce synapses in transfected neurons similarly to neuroligin-1. Using affinity chromatography, we identified alpha- and beta-neurexins as LRRTM2 ligands, again rendering LRRTM2 similar to neuroligin-1. However, whereas neuroligins bind neurexins containing or lacking an insert in splice site #4, LRRTM2 only binds neurexins lacking an insert in splice site #4. Binding of neurexins to LRRTM2 can produce cell-adhesion junctions, consistent with a trans-interaction regulated by neurexin alternative splicing, and recombinant neurexin-1beta blocks LRRTM2's ability to promote presynaptic differentiation. Thus, our data suggest that two unrelated postsynaptic cell-adhesion molecules, LRRTMs and neuroligins, unexpectedly bind to neurexins as the same presynaptic receptor, but that their binding is subject to distinct regulatory mechanisms.

Although much has been learned about the mechanisms underlying NMDA receptor–dependent long-term potentiation (LTP), considerable debate remains as to whether LTP is expressed as an increase in the synaptic release of glutamate or as an increase in the sensitivity of the postsynaptic glutamate receptors. We have directly measured changes in the synaptic release of glutamate by recording synaptically evoked glial glutamate transporter currents with whole-cell recording. Glial cell responses were very sensitive to manipulations known to change the release of glutamate yet remained constant during LTP. These results argue strongly for a postsynaptic expression mechanism for LTP.

SummaryLeucine-rich repeat transmembrane proteins (LRRTMs) are synaptic cell adhesion molecules that trigger excitatory synapse assembly in cultured neurons and influence synaptic function in vivo, but their role in synaptic plasticity is unknown. shRNA-mediated knockdown (KD) of LRRTM1 and LRRTM2 in vivo in CA1 pyramidal neurons of newborn mice blocked long-term potentiation (LTP) in acute hippocampal slices. Molecular replacement experiments revealed that the LRRTM2 extracellular domain is sufficient for LTP, probably because it mediates binding to neurexins (Nrxs). Examination of surface expression of endogenous AMPA receptors (AMPARs) in cultured neurons suggests that LRRTMs maintain newly delivered AMPARs at synapses after LTP induction. LRRTMs are also required for LTP of mature synapses on adult CA1 pyramidal neurons, indicating that the block of LTP in neonatal synapses by LRRTM1 and LRRTM2 KD is not due to impairment of synapse maturation.