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We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

Spinal muscular atrophy (SMA) is a monogenic disease that clinically manifests as severe muscle weakness owing to neurotransmission defects and motoneuron degeneration. Individuals affected by SMA experience neuromuscular weakness that impacts functional activities of daily living. We have used a mouse model of severe SMA (SMNΔ7) to test whether a calcium channel gating modifier (GV-58), alone or in combination with a potassium channel antagonist (3,4-diaminopyridine; 3,4-DAP), can improve neuromuscular function in this mouse model. Bath application of GV-58 alone or in combination with 3,4-DAP significantly restored neuromuscular transmission to control levels in both a mildly vulnerable forearm muscle and a strongly vulnerable trunk muscle in SMNΔ7 mice at postnatal days 10–12. Similarly, acute subcutaneous administration of GV-58 to postnatal day 10 SMNΔ7 mice, alone or in combination with 3,4-DAP, significantly increased a behavioral measure of muscle strength. These data suggest that GV-58 may be a promising treatment candidate that could address deficits in neuromuscular function and strength and that the addition of 3,4-DAP to GV-58 treatment could aid in restoring function in SMA.

PDF file - 170K, Representative LEC microingjections showing dye transfer. Pre-images obtained using DIC and gfp (a,c,e) showing dsred (pink) for reference cell and lucifer yellow dye transfer in blue. LECs electroporated with empty EGFP vector (a,b); WT- hCx47-EGFP (c,d); H409Y-EGFP (e,f). 40x oil, 1.3 n.a.

Purpose: Secondary lymphedema is a frequent complication of breast cancer associated with surgery, chemotherapy, or radiation following breast cancer treatment. The potential contribution of genetic susceptibility to risk of developing secondary lymphedema following surgical trauma, radiation, and other tissue insults has not been studied.Experimental Design: To determine whether women with breast cancer and secondary lymphedema had mutations in candidate lymphedema genes, we undertook a case–control study of 188 women diagnosed with breast cancer recruited from the University of Pittsburgh Breast Cancer Program (http://www.upmccancercenter.com/breast/index.cfm) between 2000 and 2010.Candidate lymphedema genes, GJC2 (encoding connexin 47 [Cx47]), FOXC2, HGF, MET, and FLT4 (encoding VEGFR3), were sequenced for mutation. Bioinformatics analysis and in vitro functional assays were used to confirm significance of novel mutations.Results: Cx47 mutations were identified in individuals having secondary lymphedema following breast cancer treatment but not in breast cancer controls or normal women without breast cancer. These novel mutations are dysfunctional as assessed through in vitro assays and bioinformatics analysis and provide evidence that altered gap junction function leads to lymphedema.Conclusions: Our findings challenge the view that secondary lymphedema is solely due to mechanical trauma and support the hypothesis that genetic susceptibility is an important risk factor for secondary lymphedema. A priori recognition of genetic risk (i) raises the potential for early detection and intervention for a high-risk group and (ii) allows the possibility of altering surgical approach and/or chemo- and radiation therapy, or direct medical treatment of secondary lymphedema with novel connexin-modifying drugs. Clin Cancer Res; 18(8); 2382–90. ©2012 AACR.

PDF file - 154K, Mutation chromatograms.

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The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.SIGNIFICANCE STATEMENTThe AP waveform plays an essential role in determining the behavior of neurotransmission at the presynaptic terminal. Although the frog NMJ is a model synapse for the study of synaptic transmission, there are many unknowns centered around the shape and propagation of its presynaptic AP waveform. Here, we demonstrate that the presynaptic terminal of the frog NMJ has a very brief AP waveform and that the motor nerve terminal contains three distinct electrical regions. We propose that the changes in the AP waveform as it propagates along the terminal can explain the proximal-distal gradient in transmitter release seen in electrophysiological studies.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 μM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 μM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-μM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-μM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-μM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

Calcium-triggered neurotransmission underlies most communication in the nervous system. Yet, despite the conserved and essential nature of this process, the molecular underpinnings of calcium-triggered neurotransmission have been difficult to study directly and our understanding to this date remains incomplete. Here we frame more recent efforts to understand this process with a historical perspective of the study of neurotransmitter release at the neuromuscular junction. We focus on the role of calcium channel distribution and organization relative to synaptic vesicles, as well as the nature of the calcium sensors that trigger release. Importantly, we provide a framework for understanding how the function of neurotransmitter release sites, or active zones, contributes to the function of the synapse as a whole.

The spatiotemporal calcium dynamics within presynaptic neurotransmitter release sites (active zones, AZs) at the time of synaptic vesicle fusion is critical for shaping the dynamics of neurotransmitter release. Specifically, the relative arrangement and density of voltage-gated calcium channels (VGCCs) as well as the concentration of calcium buffering proteins can play a large role in the timing, magnitude, and plasticity of release by shaping the AZ calcium profile. However, a high-resolution understanding of the role of AZ structure in spatiotemporal calcium dynamics and how it may contribute to functional heterogeneity at an adult synapse is currently lacking. We demonstrate that synaptic delay varies considerably across, but not within, individual synapses at the frog neuromuscular junction (NMJ). To determine how elements of the AZ could contribute to this variability, we performed a parameter search using a spatially realistic diffusion reaction-based computational model of a frog NMJ AZ (Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD. Biophys J 104: 2751–2763, 2013; Ma J, Kelly L, Ingram J, Price TJ, Meriney SD, Dittrich M. J Neurophysiol 113: 71–87, 2015). We demonstrate with our model that synaptic delay is sensitive to significant alterations in the spatiotemporal calcium dynamics within an AZ at the time of release caused by manipulations of the density and organization of VGCCs or by the concentration of calcium buffering proteins. Furthermore, our data provide a framework for understanding how AZ organization and structure are important for understanding presynaptic function and plasticity. NEW & NOTEWORTHY The structure of presynaptic active zones (AZs) can play a large role in determining the dynamics of neurotransmitter release across many model preparations by influencing the spatiotemporal calcium dynamics within the AZ at the time of vesicle fusion. However, less is known about how different AZ structural schemes may influence the timing of neurotransmitter release. We demonstrate that variations in AZ structure create different spatiotemporal calcium profiles that, in turn, lead to differences in synaptic delay.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder caused by antibodies directed against the voltage-gated calcium channels that provide the calcium ion flux that triggers acetylcholine release at the neuromuscular junction. To study the pathophysiology of LEMS and test candidate therapeutic strategies, a passive-transfer animal model has been developed in mice, which can be created by daily intraperitoneal injections of LEMS patient serum or IgG into mice for 2-4 weeks. Results from studies of the mouse neuromuscular junction have revealed that each synapse has hundreds of transmitter release sites but that the probability for release at each one is likely to be low. LEMS further reduces this low probability such that transmission is no longer effective at triggering a muscle contraction. The LEMS-mediated attack reduces the number of presynaptic calcium channels, disorganizes transmitter release sites, and results in the homeostatic upregulation of other calcium channel types. Symptomatic treatment is focused on increasing the probability of release from dysfunctional release sites. Current treatment uses the potassium channel blocker 3,4-diaminopyridine (DAP) to broaden the presynaptic action potential, providing more time for calcium channels to open. Current research is focused on testing new calcium channel gating modifiers that work synergistically with DAP.

Until the recent development of disease-modifying therapeutics, spinal muscular atrophy (SMA) was considered a devastating neuromuscular disease with a poor prognosis for most affected individuals. Symptoms generally present during early childhood and manifest as muscle weakness and progressive paralysis, severely compromising the affected individual’s quality of life, independence, and lifespan. SMA is most commonly caused by the inheritance of homozygously deleted SMN1 alleles with retention of one or more copies of a paralog gene, SMN2, which inversely correlates with disease severity. The recent advent and use of genetically targeted therapies have transformed SMA into a prototype for monogenic disease treatment in the era of genetic medicine. Many SMA-affected individuals receiving these therapies achieve traditionally unobtainable motor milestones and survival rates as medicines drastically alter the natural progression of this disease. This review discusses historical SMA progression and underlying disease mechanisms, highlights advances made in therapeutic research, clinical trials, and FDA-approved medicines, and discusses possible second-generation and complementary medicines as well as optimal temporal intervention windows in order to optimize motor function and improve quality of life for all SMA-affected individuals.

The nervous system utilizes about 100 molecules to signal between neurons ( intercellular signaling ), including neurotransmitters, which can either directly mediate or modulate fast synaptic transmission. However, not all molecules that affect the nervous system are considered neurotransmitters. As such, researchers have developed a number of criteria that are used to determine whether a molecule can be classified as a neurotransmitter. Neurotransmitters have proven difficult to study, given that they are released in small amounts and are often found in a milieu of other neurotransmitters released by neighboring neurons. To aid in understanding how neurotransmitters are generated and used, we categorize them into four groups based on certain characteristics. Our hope is that this grouping will make it easier to grasp the general concepts that govern neurotransmitter synthesis and use. It is important to remember that the actions of neurotransmitters are determined not by the molecules themselves, but rather by the downstream effects of their receptors.

Glial cells were originally thought to be fairly passive players in the nervous system, mainly involved in supporting neurons by regulating ion concentrations and other substances in the extracellular fluid. However, it is now clear that glial cells also have a much more direct, integral role in regulating synaptic transmission. In particular, a subset of glial cells called astrocytes often surround synapses and are able to affect synaptic transmission by removing released neurotransmitters from the synaptic cleft, maintaining appropriate ion concentrations in the extracellular fluid, and releasing neurotransmitters themselves in a manner similar to synaptic vesicular release from neurons. Additionally, astrocytes are connected to one another by gap junctions, which allows coordinated modulation of neighboring astrocytes by waves of calcium that propagate via these gap junctions, and, by extension, coordinate the activity of local neurons.

Neurotransmitters affect cells by binding to receptor proteins, inducing conformational changes in the receptors that affect neuronal function either directly, by opening ion channels (ionotropic receptors), or indirectly, by initiating intracellular cascades that can affect neurons in many different ways (metabotropic receptors).

The fusion of synaptic vesicles and release of chemical transmitter from the axon terminal occurs very rapidly (in less than 1 ms), and is dependent upon an influx of calcium ions. What are the biochemical processes that trigger vesicle fusion, and how can they occur so quickly? First, vesicles are moved to the active zone and tethered to the plasma membrane to prepare them for release in advance of action potential activity. Second, a calcium sensor protein on synaptic vesicles holds the vesicle on the verge of fusion, waiting for the calcium ion trigger. Finally, when calcium ions enter the nerve terminal, they bind to the calcium sensor protein, releasing the hold on the synaptic vesicle, which facilitates very rapid fusion with the plasma membrane.

Synaptic integration is the postsynaptic process of collecting information from many presynaptic inputs and combining them to determine the output of the postsynaptic neuron. In its most basic form, this concept can be reduced to a single question: how much synaptic input, and at what location on a neuron, is required to generate an action potential in a postsynaptic neuron?

In order for chemical neurotransmission to occur, transmitter molecules must escape from the presynaptic axon and diffuse to their site of action, and this must happen rapidly in order for the nervous system to function in the way it does. Based on clues from investigations of both evoked and spontaneous neurotransmission, largely at the neuromuscular junction, scientists discovered that neurotransmitter molecules are released from neurons via synaptic vesicles. They determined that these vesicles each contain approximately the same amount of neurotransmitter, termed a “quantum,” and that the probability of vesicle release can be altered by a number of factors, including the concentration of intracellular calcium. The behavior of these vesicles during presynaptic action potential activity can be described by the quantal theory of transmitter release, a conceptual and computational framework that predicts normal neurotransmitter release from synapses and has also allowed researchers to study how synapses change in response to things such as drugs and disease.

Ion channels mediate many aspects of neuronal signaling, from the responses of neurons to neurotransmitters to the generation of action potentials that allow signals to travel along axons. By understanding the relationship between ion flow (or flux) through channels and membrane potential, it is possible to determine how action of a particular type of ion channel will affect a neuron. Voltage-gated ion channels, which respond to electrical activity and are essential for the generation of action potentials, are among the most common types of ion channels in neurons. Specifically, action potentials are generated by current moving through voltage-gated sodium and potassium ion channels. In this chapter, we will cover roles voltage-gated ion channels play in the generation and conduction of action potentials and how this underlies communication between neurons.

Amino acids are ubiquitous in the body, so it was not initially intuitive that they would be likely to act as neurotransmitters. Nonetheless, the three neurotransmitters with an amino acid structure—glutamate, GABA, and glycine—underlie the vast majority of fast synaptic transmission, and can also have neuromodulatory effects. Neurons that release glutamate and glycine as neurotransmitters must be identified indirectly (e.g., by specific plasma membrane or vesicular transporters) because these amino acids are present in every cell and also serve roles outside of neurotransmission. Given the prevalence of amino acid neurotransmitters in the central nervous system, it is not surprising that they are implicated in many neurological disorders.

During the exocytosis of small clear vesicles, vesicle membranes fuse with the plasma membrane of the axon terminal. Many lines of experimental evidence show that following exocytosis, neurons selectively retrieve vesicular membrane from the plasma membrane and reuse it to create new synaptic vesicles that are subsequently used to release more neurotransmitter. This process of bringing vesicular membrane back into the presynaptic terminal is called endocytosis. We discuss three forms of endocytosis used by neurons.

The ability of neurons to send signals to one another is crucial for the function of the nervous system, but early scientists had no way of knowing how communication from one neuron to another occurred. We now take for granted that synapses exist between neurons, but before this could be verified using the advanced experimental techniques available relatively recently, there was debate among early neuroscientists about whether this was true. Some scientists proposed that neurons were contiguous with one another, much like capillaries in the body, while others concluded that neurons were in fact separate cells. This latter concept was called the Neuron Doctrine, which we now know to be correct. The junctions between neurons were given the name “synapse,” and the study of synapses has revealed ultrastructural details of synaptic structure as well as an understanding of how the nervous system assembles synapses during nervous system development.

The impact of presynaptic transmitter release site organization on synaptic function has been a vibrant area of research for synaptic physiologists. Because there is a highly nonlinear relationship between presynaptic calcium influx and subsequent neurotransmitter release at synapses, the organization and density of calcium sources (voltage-gated calcium channels [VGCCs]) relative to calcium sensors located on synaptic vesicles is predicted to play a major role in shaping the dynamics of neurotransmitter release at a synapse. Here we review the history of structure-function studies within transmitter release sites at the neuromuscular junction across three model preparations in an effort to discern the relationship between VGCC organization and synaptic function, and whether that organizational structure imparts evolutionary advantages for each species.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

We have investigated the impact of transmitter release site (active zone; AZ) structure on synaptic function by physically rearranging the individual AZ elements in a previously published frog neuromuscular junction (NMJ) AZ model into the organization observed in a mouse NMJ AZ. We have used this strategy, purposefully without changing the properties of AZ elements between frog and mouse models (even though there are undoubtedly differences between frog and mouse AZ elements in vivo), to directly test how structure influences function at the level of an AZ. Despite a similarly ordered ion channel array substructure within both frog and mouse AZs, frog AZs are much longer and position docked vesicles in a different location relative to AZ ion channels. Physiologically, frog AZs have a lower probability of transmitter release compared with mouse AZs, and frog NMJs facilitate strongly during short stimulus trains in contrast with mouse NMJs that depress slightly. Using our computer modeling approach, we found that a simple rearrangement of the AZ building blocks of the frog model into a mouse AZ organization could recapitulate the physiological differences between these two synapses. These results highlight the importance of simple AZ protein organization to synaptic function. NEW & NOTEWORTHY A simple rearrangement of the basic building blocks in the frog neuromuscular junction model into a mouse transmitter release site configuration predicted the major physiological differences between these two synapses, suggesting that transmitter release site structure and organization is a strong predictor of function.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca(2+) channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are thought to be removed. There is no cure for LEMS, and the current most commonly used symptomatic treatment option is a potassium channel blocker [3,4-diaminopyridine (3,4-DAP)] that does not completely reverse symptoms and can have dose-limiting side-effects. We previously reported the development of a novel Ca(2+) channel agonist, GV-58, as a possible alternative treatment strategy for LEMS. In this study, we tested the hypothesis that the combination of GV-58 and 3,4-DAP will elicit a supra-additive increase in neurotransmitter release at LEMS model NMJs. First, we tested GV-58 in a cell survival assay to assess potential effects on cyclin-dependent kinases (Cdks) and showed that GV-58 did not affect cell survival at the relevant concentrations for Ca(2+) channel effects. Then, we examined the voltage dependence of GV-58 effects on Ca(2+) channels using patch clamp techniques; this showed the effects of GV-58 to be dependent upon Ca(2+) channel opening. Based on this mechanism, we predicted an interaction between 3,4-DAP and GV-58. We tested this hypothesis using a mouse passive transfer model of LEMS. Using intracellular electrophysiological ex vivo recordings, we demonstrated that a combined application of 3,4-DAP plus GV-58 had a supra-additive effect that completely reversed the deficit in neurotransmitter release magnitude at LEMS model NMJs. This reversal contrasts with the less significant improvement observed with either compound alone. Our data indicate that a combination of 3,4-DAP and GV-58 represents a promising treatment option for LEMS and potentially for other disorders of the NMJ.

The neuromuscular junction is known as a strong and reliable synapse. It is strong because it releases an excess of chemical transmitter, beyond what is required to bring the postsynaptic muscle cell to threshold. Because the synapse can sustain suprathreshold muscle activation during short trains of action potentials, it is also reliable. The presynaptic mechanisms that lead to reliability during short trains of activity have only recently been elucidated. It appears that there are relatively few calcium channels in individual active zones, that channels open with a low probability during action potential stimulation and that even if channels open the resulting calcium flux only rarely triggers vesicle fusion. Thus, each synaptic vesicle may only associate with a small number of calcium channels, forming an unreliable single vesicle release site. Strength and reliability of the neuromuscular junction emerge as a result of its assembly from thousands of these unreliable single vesicle release sites. Hence, these synapses are strong while at the same time only releasing a small subset of available docked vesicles during each action potential, thus conserving transmitter release resources. This prevents significant depression during short trains of action potential activity and confers reliability.

Voltage-gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity-dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter-containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2-selective VGCC gating modifiers (notably GV-58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV-58 structure that extend our understanding of the structure-activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF-06) was more efficacious than GV-58, another (KK-75) acts more quickly on VGCCs than GV-58, and a third (KK-20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function.

The frog neuromuscular junction (NMJ) is a strong and reliable synapse because, during activation, sufficient neurotransmitter is released to trigger a postsynaptic action potential (AP). Recent evidence supports the hypothesis that this reliability emerges from the assembly of thousands of unreliable single vesicle release sites. The mechanisms that govern this unreliability include a paucity of voltage-gated calcium channels, a low probability of calcium channel opening during an AP, and the rare triggering of synaptic vesicle fusion even when a calcium channel does open and allows calcium flux. Here, we discuss the evidence that these unreliable single vesicle release sites may be the fundamental building blocks of many types of synapses in both the peripheral and central nervous system (PNS and CNS, respectively).

Lambert-Eaton myasthenic syndrome (LEMS) causes neuromuscular weakness as a result of an autoimmune attack on the calcium channels that normally regulate chemical transmitter release at the neuromuscular junction. Currently there are limited treatment options for patients with this and other forms of neuromuscular weakness. A novel, first-in-class calcium channel agonist that is selective for the types of voltage-gated calcium channels that regulate transmitter release at neuromuscular synapses has recently been developed. This compound (GV-58) slows deactivation (closing) of the channel, resulting in a large increase in total calcium entry during motor nerve action potential activity. This new calcium channel agonist is currently being evaluated for the treatment of neuromuscular weakness. Potential applications include development as single therapeutics, or for combination treatments.

Purpose: Secondary lymphedema is a frequent complication of breast cancer associated with surgery, chemotherapy, or radiation following breast cancer treatment. The potential contribution of genetic susceptibility to risk of developing secondary lymphedema following surgical trauma, radiation, and other tissue insults has not been studied. Experimental Design: To determine whether women with breast cancer and secondary lymphedema had mutations in candidate lymphedema genes, we undertook a case–control study of 188 women diagnosed with breast cancer recruited from the University of Pittsburgh Breast Cancer Program (http://www.upmccancercenter.com/breast/index.cfm) between 2000 and 2010. Candidate lymphedema genes, GJC2 (encoding connexin 47 [Cx47]), FOXC2, HGF, MET, and FLT4 (encoding VEGFR3), were sequenced for mutation. Bioinformatics analysis and in vitro functional assays were used to confirm significance of novel mutations. Results: Cx47 mutations were identified in individuals having secondary lymphedema following breast cancer treatment but not in breast cancer controls or normal women without breast cancer. These novel mutations are dysfunctional as assessed through in vitro assays and bioinformatics analysis and provide evidence that altered gap junction function leads to lymphedema. Conclusions: Our findings challenge the view that secondary lymphedema is solely due to mechanical trauma and support the hypothesis that genetic susceptibility is an important risk factor for secondary lymphedema. A priori recognition of genetic risk (i) raises the potential for early detection and intervention for a high-risk group and (ii) allows the possibility of altering surgical approach and/or chemo- and radiation therapy, or direct medical treatment of secondary lymphedema with novel connexin-modifying drugs. Clin Cancer Res; 18(8); 2382–90. ©2012 AACR.

We used high-resolution fluorescence imaging and single-pixel optical fluctuation analysis to estimate the opening probability of individual voltage-gated calcium (Ca2+) channels during an action potential and the number of such Ca2+channels within active zones of frog neuromuscular junctions. Analysis revealed ∼36 Ca2+channels within each active zone, similar to the number of docked synaptic vesicles but far less than the total number of transmembrane particles reported based on freeze-fracture analysis (∼200–250). The probability that each channel opened during an action potential was only ∼0.2. These results suggest why each active zone averages only one quantal release event during every other action potential, despite a substantial number of docked vesicles. With sparse Ca2+channels and low opening probability, triggering of fusion for each vesicle is primarily controlled by Ca2+influx through individual Ca2+channels. In contrast, the entire synapse is highly reliable because it contains hundreds of active zones.

Excitatory transmission within hippocampal area CA3 stems from three major glutamatergic pathways: the perforant path formed by axons of layer II stellate cells in the entorhinal cortex, the mossy fiber axons originating from the dentate gyrus granule cells, and the recurrent axon collaterals of CA3 pyramidal cells. The synaptic communication of each of these pathways is modulated by metabotropic glutamate receptors that fine-tune the signal by affecting both the timing and strength of the connection. Within area CA3 of the hippocampus, group I mGluRs (mGluR1 and mGluR5) are expressed postsynaptically, whereas group II (mGluR2 and mGluR3) and III mGluRs (mGluR4, mGluR7, and mGluR8) are expressed presynaptically. Receptors from each group have been demonstrated to be required for different forms of pre- and postsynaptic long-term plasticity and also have been implicated in regulating short-term plasticity. A recent observation has demonstrated that a presynaptically expressed mGluR can affect the timing of action potentials elicited in the postsynaptic target. Interestingly, mGluRs can be distributed in a target-specific manner, such that synaptic input from one presynaptic neuron can be modulated by different receptors at each of its postsynaptic targets. Consequently, mGluRs provide a mechanism for synaptic specialization of glutamatergic transmission in the hippocampus. This review will highlight the variability in mGluR modulation of excitatory transmission within area CA3 with an emphasis on how these receptors contribute to the strength and timing of network activity within pyramidal cells and interneurons.