Blockade Of Action Potentials Research Articles

A review of the bioeffects of low-intensity focused ultrasound and the benefits of a cellular approach.

This review article highlights the historical developments and current state of knowledge of an important neuromodulation technology: low-intensity focused ultrasound. Because compelling studies have shown that focused ultrasound can modulate neuronal activity non-invasively, especially in deep brain structures with high spatial specificity, there has been a renewed interest in attempting to understand the specific bioeffects of focused ultrasound at the cellular level. Such information is needed to facilitate the safe and effective use of focused ultrasound to treat a number of brain and nervous system disorders in humans. Unfortunately, to date, there appears to be no singular biological mechanism to account for the actions of focused ultrasound, and it is becoming increasingly clear that different types of nerve cells will respond to focused ultrasound differentially based on the complement of their ion channels, other membrane biophysical properties, and arrangement of synaptic connections. Furthermore, neurons are apparently not equally susceptible to the mechanical, thermal and cavitation-related consequences of focused ultrasound application-to complicate matters further, many studies often use distinctly different focused ultrasound stimulus parameters to achieve a reliable response in neural activity. In this review, we consider the benefits of studying more experimentally tractable invertebrate preparations, with an emphasis on the medicinal leech, where neurons can be studied as unique individual cells and be synaptically isolated from the indirect effects of focused ultrasound stimulation on mechanosensitive afferents. In the leech, we have concluded that heat is the primary effector of focused ultrasound neuromodulation, especially on motoneurons in which we observed a focused ultrasound-mediated blockade of action potentials. We discuss that the mechanical bioeffects of focused ultrasound, which are frequently described in the literature, are less reliably achieved as compared to thermal ones, and that observations ascribed to mechanical responses may be confounded by activation of synaptically-coupled sensory structures or artifacts associated with electrode resonance. Ultimately, both the mechanical and thermal components of focused ultrasound have significant potential to contribute to the sculpting of specific neural outcomes. Because focused ultrasound can generate significant modulation at a temperature <5°C, which is believed to be safe for moderate durations, we support the idea that focused ultrasound should be considered as a thermal neuromodulation technology for clinical use, especially targeting neural pathways in the peripheral nervous system.

Synapse and Active Zone Assembly in the Absence of Presynaptic Ca2+ Channels and Ca2+ Entry

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of β subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

Monoterpenoid terpinen-4-ol inhibits voltage-dependent Na+ channels of small dorsal root ganglia rat neurons

The monoterpenoid terpinen-4-ol (4TERP) is known to inhibit cell excitability, has low toxicity and important pharmacological activities, which are likely related to neural excitability, such as anti-inflammatory, antiepileptic and antinociceptive effects. However, the pharmacological characteristics and mechanisms underlying the effects of 4TERP on blockade of neural action potential are not completely elucidated. Since Na+ current (INa) through voltage-dependent Na+ channels (NaV) is a major mechanism for excitability, the present study investigated the pharmacological characteristics and mechanisms of the action of 4TERP on INa through NaV. For this aim, dissociated small neurons of dorsal root ganglia of adult rats were used for whole cell patch-clamp recordings. 4TERP concentration-dependently inhibits INa (IC50 0.8 ± 0.3 mM; pharmacological efficacy 42.89 ± 5.54%). 4TERP interfered with INa through a mechanism with various components, which includes predominantly channel pore block and sensitivity to frequency of use. In presence of 4TERP (3 mM), decreasing stimulation from 5 Hz to very low frequency (75 s of quiescence previously to stimulation) induced INa decrease to 65.17 ± 5.86% of control. 4TERP also altered (left shift) voltage sensitivity of the steady state activation of NaV. Data are discussed aiming to interpret the importance of blockade of INa through NaV as participant of 4TERP-induced inhibition of membrane excitability.

Direct activation of G-protein-gated inward rectifying K+ channels promotes nonrapid eye movement sleep.

A major challenge in treating insomnia is to find effective medicines with fewer side effects. Activation of G-protein-gated inward rectifying K+ channels (GIRKs) by GABAB agonists baclofen or γ-hydroxybutyric acid (GHB) promotes nonrapid eye movement (NREM) sleep and consolidates sleep. However, baclofen has poor brain penetration, GHB possesses abuse liability, and in rodents both drugs cause spike-wave discharges (SWDs), an absence seizure activity. We tested the hypothesis that direct GIRK activation promotes sleep without inducing SWD using ML297, a potent and selective GIRK activator. Whole-cell patch-clamp recordings from hypocretin/orexin or hippocampal neurons in mouse brain slices were made to study neuronal excitability and synaptic activity; spontaneous activity, locomotion, contextual and tone-conditioned memory, and novel object recognition were assessed. Electroencephalogram/electromyogram (EEG/EMG) recordings were used to study GIRK modulation of sleep. ML297, like baclofen, caused membrane hyperpolarization, decreased input resistance, and blockade of spontaneous action potentials. Unlike baclofen, ML297 (5-10 µM) did not cause significant depression of postsynaptic excitatory and inhibitory currents (EPSCs-IPSCs), indicating preferential postsynaptic inhibition. ML297 (30 mg/kg, i.p.) inhibited wake activity and locomotion, and preferentially increased NREM sleep without altering EEG delta power, REM sleep, inducing SWDs, or impairing conditioned memory and novel object recognition. This study finds that direct activation of neuronal GIRK channels modulates postsynaptic membrane excitability and prolongs NREM sleep without changing sleep intensity, inducing SWDs, or impairing memory in rodents. These results suggest that direct GIRK activation with a selective compound may present an innovative approach for the treatment of chronic insomnia.

Spontaneous astrocytic Ca2+ activity abounds in electrically suppressed ischemic penumbra of aged mice.

Experimental focal cortical ischemic lesions consist of an ischemic core and a potentially salvageable peri-ischemic region, the ischemic penumbra. The activity of neurons and astrocytes is assumed to be suppressed in the penumbra because the electrical function is interrupted, but this is incompletely elucidated. Most experimental stroke studies used young adult animals, whereas stroke is prevalent in the elderly population. Using two-photon imaging in vivo, we here demonstrate extensive but electrically silent, spontaneous Ca2+ activity in neurons and astrocytes in the ischemic penumbra of 18- to 24-month-old mice 2-4 hr after middle cerebral artery occlusion. In comparison, stroke reduced spontaneous Ca2+ activity in neurons and astrocytes in adult mice (3-4 months of age). In aged mice, stroke increased astrocytic spontaneous Ca2+ activity considerably while neuronal spontaneous Ca2+ activity was unchanged. Blockade of action potentials and of purinergic receptors strongly reduced spontaneous Ca2+ activity in both neurons and astrocytes in the penumbra of old stroke mice. This indicates that stroke had a direct influence on mechanisms in presynaptic terminals and on purinergic signaling. Thus, highly dynamic variations in spontaneous Ca2+ activity characterize the electrically compromised penumbra, with remarkable differences between adult and old mice. The data are consistent with the notion that aged neurons and astrocytes take on a different phenotype than young mice. The increased activity of the aged astrocyte phenotype may be harmful to neurons. We suggest that the abundant spontaneous Ca2+ activity in astrocytes in the ischemic penumbra of old mice may be a novel target for neuroprotection strategies. A video abstract of this article can be found at https://youtu.be/AKlwKFsz1qE.

Direct versus indirect actions of ghrelin on hypothalamic NPY neurons.

ObjectivesAssess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons.Materials and methodsElectrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca2+ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus.ResultsAcylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca2+]c, with an EC50 ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP3) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na+]o suppressed activation, suggesting a role for Na+-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons in situ. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca2+]c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca2+ channels, respectively, while Ni2+, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca2+ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca2+ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action in vivo.ConclusionsNPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons in situ. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na+ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca2+ channels, which could contribute to NPY neuronal activity in situ.

The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility. To generate an improved understanding of zDHHC2 trafficking and how this might be regulated by neuronal activity, we searched for intramolecular signals that regulate enzyme localisation. Two signals were mapped to the C-terminal tail of zDHHC2: a non-canonical dileucine motif [SxxxLL] and a downstream NP motif. Mutation of these signals enhanced plasma membrane accumulation of zDHHC2 in both neuroendocrine PC12 cells and rat hippocampal neurons, consistent with reduced endocytic retrieval. Furthermore, mutation of these signals also increased accumulation of the enzyme in neurites. Interestingly, several threonine and serine residues are adjacent to these sorting motifs and analysis of phospho-mimetic mutants highlighted a potential role for phosphorylation in regulating the efficacy of these signals. This study offers new molecular insight into the signals that determine zDHHC2 localisation and highlights a potential mechanism to regulate these trafficking signals.

Effects of K+ channel openers on spontaneous action potentials in detrusor smooth muscle of the guinea-pig urinary bladder

The modulation of spontaneous excitability in detrusor smooth muscle (DSM) upon the pharmacological activation of different populations of K+ channels was investigated. Effects of distinct K+ channel openers on spontaneous action potentials in DSM of the guinea-pig bladder were examined using intracellular microelectrode techniques. NS1619 (10μM), a large conductance Ca2+-activated K+ (BK) channel opener, transiently increased action potential frequency and then prevented their generation without hyperpolarizing the membrane in a manner sensitive to iberiotoxin (IbTX, 100nM). A higher concentration of NS1619 (30μM) hyperpolarized the membrane and abolished action potential firing. NS309 (10μM) and SKA31 (100μM), small conductance Ca2+-activated K+ (SK) channel openers, dramatically increased the duration of the after-hyperpolarization and then abolished action potential firing in an apamin (100nM)-sensitive manner. Flupirtine (10μM), a Kv7 channel opener, inhibited action potential firing without hyperpolarizing the membrane in a manner sensitive to XE991 (10μM), a Kv7 channel blocker. BRL37344 (10μM), a β3-adrenceptor agonist, or rolipram (10nM), a phosphodiesterase 4 inhibitor, also inhibited action potential firing. A higher concentration of rolipram (100nM) hyperpolarized the DSM and abolished the action potentials. IbTX (100nM) prevented the rolipram-induced blockade of action potentials but not the hyperpolarization. BK and Kv7 channels appear to predominantly contribute to the stabilization of DSM excitability. Spare SK channels could be pharmacologically activated to suppress DSM excitability. BK channels appear to be involved in the cyclic AMP-induced inhibition of action potentials but not the membrane hyperpolarization.

High-Frequency Stimulation of Dorsal Column Axons: Potential Underlying Mechanism of Paresthesia-Free Neuropathic Pain Relief

Spinal cord stimulation (SCS) treats neuropathic pain through retrograde stimulation of dorsal column axons and their inhibitory effects on wide dynamic range (WDR) neurons. Typical SCS uses frequencies from 50-100 Hz. Newer stimulation paradigms use high-frequency stimulation (HFS) up to 10 kHz and produce pain relief but without paresthesia. Our hypothesis is that HFS preferentially blocks larger diameter axons (12-15 µm) based on dynamics of ion channel gates and the electric potential gradient seen along the axon, resulting in inhibition of WDR cells without paresthesia. We input field potential values from a finite element model of SCS into an active axon model with ion channel subcomponents for fiber diameters 1-20 µm and simulated dynamics on a 0.001 msec time scale. Assuming some degree of wave rectification seen at the axon, action potential (AP) blockade occurs as hypothesized, preferentially in larger over smaller diameters with blockade in most medium and large diameters occurring between 4.5 and 10 kHz. Simulations show both ion channel gate and virtual anode dynamics are necessary. At clinical HFS frequencies and pulse widths, HFS preferentially blocks larger-diameter fibers and concomitantly recruits medium and smaller fibers. These effects are a result of interaction between ion gate dynamics and the "activating function" (AF) deriving from current distribution over the axon. The larger fibers that cause paresthesia in low-frequency simulation are blocked, while medium and smaller fibers are recruited, leading to paresthesia-free neuropathic pain relief by inhibiting WDR cells.

Electrophysiological properties of abdominal muscle‐projecting expiratory motoneurons of rats

Expiration is considered a passive process during resting breathing condition. There is a lack of information about the intrinsic electrophysiological properties of expiratory motoneurons and their possible changes in conditions in which the expiration becomes an active process, such as hypoxia. Considering that abdominal expiratory muscle activity depends on excitability of these motoneurons, here we aimed to characterize the electrophysiological properties of abdominal muscle‐projecting expiratory motoneurons combining retrograde tracer, immunofluorescence and whole cell patch clamp, in thoraco‐lumbar spinal cord slices from juvenile Wistar rats. Three different types of cholinergic expiratory motoneurons with similar capacitance were recorded: neurons with spontaneous action potentials whose resting potential hyperpolarizes (−65 ± 5 mV vs −69.7 ± 4 mV, n=9) and firing frequency decreases (8.5 ± 2 Hz vs 1.7 ± 1.3 Hz, n=9) after synaptic blockade. The passive properties of these motoneurons, such as input resistance and excitability, did not change after synaptic blockade (Ri = 0.16 ± 0.03 GΩ vs 0.13 ± 0.03 GΩ; Excitability = 40 ± 10 Hz vs 32 ± 11 Hz, n=9). The second group of motoneurons also exhibited spontaneous action potentials, but showed spike frequency adaptation, slowing or ceasing firing frequency during a maintained stimulus. Their membrane potential also hyperpolarized (−60 ± 1 mV vs −66 ± 1 mV, n=4) and firing frequency was abolished by synaptic blockade. On the other hand, no differences in the input resistance was observed (0.13 ± 0.01 GΩ vs 0.11 ± 0.01 GΩ, n=4). The third group did not show spontaneous action potential and synaptic blockade did not change membrane potential (−72.2 ± 2 mV vs −76 ± 3 mV, n=6) or input resistance (0.1 ± 0.04 GΩ vs 0.06 ± 0.02 GΩ, n=6). However, synaptic blockade decreased their intrinsic excitability. Overall, our data show that there are three different populations of abdominal muscle‐projecting expiratory motoneurons in rats, which may contribute to recruitment of expiratory muscles in different physiological and pathophysiological conditions.Support or Funding InformationFAPESP, CAPES, CNPQ

Dual action of leptin on rest-firing and stimulated catecholamine release via phosphoinositide 3-kinase-driven BK channel up-regulation in mouse chromaffin cells.

Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect and, as such, exerts a relevant action on the adipo-adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3-kinase signalling cascade. This inhibitory effect is absent in db(-) /db(-) mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well-adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready-releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release. Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect. Besides being expressed in the hypothalamus and hippocampus, leptin receptors (ObRs) are also present in chromaffin cells of the adrenal medulla. In the present study, we report the effect of leptin on mouse chromaffin cell (MCC) functionality, focusing on cell excitability and catecholamine secretion. Acute application of leptin (1nm) on spontaneously firing MCCs caused a slowly developing membrane hyperpolarization followed by complete blockade of action potential (AP) firing. This inhibitory effect at rest was abolished by the BK channel blocker paxilline (1μm), suggesting the involvement of BK potassium channels. Single-channel recordings in 'perforated microvesicles' confirmed that leptin increased BK channel open probability without altering its unitary conductance. BK channel up-regulation was associated with the phosphoinositide 3-kinase (PI3K) signalling cascade because the PI3K specific inhibitor wortmannin (100nm) fully prevented BK current increase. We also tested the effect of leptin on evoked AP firing and Ca(2+) -driven exocytosis. Although leptin preserves well-adapted AP trains of lower frequency, APs are broader and depolarization-evoked exocytosis is increased as a result of the larger size of the ready-releasable pool and higher frequency of vesicle release. The kinetics and quantal size of single secretory events remained unaltered. Leptin had no effect on firing and secretion in db(-) /db(-) mice lacking the ObR gene, confirming its specificity. In conclusion, leptin exhibits a dual action on MCC activity. It dampens AP firing at rest but preserves AP firing and increases catecholamine secretion during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release.

Activity-dependent synaptic GRIP1 accumulation drives synaptic scaling up in response to action potential blockade

Synaptic scaling is a form of homeostatic plasticity that stabilizes neuronal firing in response to changes in synapse number and strength. Scaling up in response to action-potential blockade is accomplished through increased synaptic accumulation of GluA2-containing AMPA receptors (AMPAR), but the receptor trafficking steps that drive this process remain largely obscure. Here, we show that the AMPAR-binding protein glutamate receptor-interacting protein-1 (GRIP1) is essential for regulated synaptic AMPAR accumulation during scaling up. Synaptic abundance of GRIP1 was enhanced by activity deprivation, directly increasing synaptic GRIP1 abundance through overexpression increased the amplitude of AMPA miniature excitatory postsynaptic currents (mEPSCs), and shRNA-mediated GRIP1 knockdown prevented scaling up of AMPA mEPSCs. Furthermore, knockdown and replace experiments targeting either GRIP1 or GluA2 revealed that scaling up requires the interaction between GRIP1 and GluA2. Finally, GRIP1 synaptic accumulation during scaling up did not require GluA2 binding. Taken together, our data support a model in which activity-dependent trafficking of GRIP1 to synaptic sites drives the forward trafficking and enhanced synaptic accumulation of GluA2-containing AMPAR during synaptic scaling up.

Physiological temperatures drive glutamate release onto trigeminal superficial dorsal horn neurons

Trigeminal sensory afferent fibers terminating in nucleus caudalis (Vc) relay sensory information from craniofacial regions to the brain and are known to express transient receptor potential (TRP) ion channels. TRP channels are activated by H(+), thermal, and chemical stimuli. The present study investigated the relationships among the spontaneous release of glutamate, temperature, and TRPV1 localization at synapses in the Vc. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from Vc neurons (n = 151) in horizontal brain-stem slices obtained from Sprague-Dawley rats. Neurons had basal sEPSC rates that fell into two distinct frequency categories: High (≥10 Hz) or Low (<10 Hz) at 35°C. Of all recorded neurons, those with High basal release rates (67%) at near-physiological temperatures greatly reduced their sEPSC rate when cooled to 30°C without amplitude changes. Such responses persisted during blockade of action potentials indicating that the High rate of glutamate release arises from presynaptic thermal mechanisms. Neurons with Low basal frequencies (33%) showed minor thermal changes in sEPSC rate that were abolished after addition of TTX, suggesting these responses were indirect and required local circuits. Activation of TRPV1 with capsaicin (100 nM) increased miniature EPSC (mEPSC) frequency in 70% of neurons, but half of these neurons had Low basal mEPSC rates and no temperature sensitivity. Our evidence indicates that normal temperatures (35-37°C) drive spontaneous excitatory synaptic activity within superficial Vc by a mechanism independent of presynaptic action potentials. Thus thermally sensitive inputs on superficial Vc neurons may tonically activate these neurons without afferent stimulation.

Presynaptic NMDA receptors - dynamics and distribution in developing axons in vitro and in vivo.

During cortical development, N-methyl-D-aspartate (NMDA) receptors (NMDARs) facilitate presynaptic terminal formation, enhance neurotransmitter release and are required in presynaptic neurons for spike-timing-dependent long-term depression (tLTD). However, the extent to which NMDARs are found within cortical presynaptic terminals has remained controversial, and the sub-synaptic localization and dynamics of axonal NMDARs are unknown. Here, using live confocal imaging and biochemical purification of presynaptic membranes, we provide strong evidence that NMDARs localize to presynaptic terminals in vitro and in vivo in a developmentally regulated manner. The NR1 and NR2B subunits (also known as GRIN1 and GRIN2B, respectively) were found within the active zone membrane, where they could respond to synaptic glutamate release. Surprisingly, NR1 also appeared in glutamatergic and GABAergic synaptic vesicles. During synaptogenesis, NR1 was mobile throughout axons - including growth cones and filopodia, structures that are involved in synaptogenesis. Upon synaptogenic contact, NMDA receptors were quickly recruited to terminals by neuroligin-1 signaling. Unlike dendrites, the trafficking and distribution of axonal NR1 were insensitive to activity changes, including NMDA exposure, local glutamate uncaging or action potential blockade. These results support the idea that presynaptic NMDARs play an early role in presynaptic development.

Activity-dependent coordinated mobility of hippocampal inhibitory synapses visualized with presynaptic and postsynaptic tagged-molecular markers

Axonal varicosities and dendritic spines at excitatory synapses are dynamic structures essential for synaptic plasticity, whereas the behavior of inhibitory synapses during development and plasticity remains largely unknown. To investigate the morphology and dynamics of inhibitory synapses, we used two distinct pre- and postsynaptic fluorescent probes: one is a yellow fluorescent protein, Venus, incorporated into vesicular GABA transporter (VGAT) gene as a specific marker of presynaptic inhibitory neurons and the other red fluorescent protein (mCherry)-tagged gephyrin, a postsynaptic scaffolding protein, as a postsynaptic marker. Using primary culture of mouse hippocampal neurons and confocal laser-scanning microscopy, we established a system by which close contacts of Venus-positive axonal varicosities with mCherry-labeled gephyrin clusters in the dendritic shafts of dissociated hippocampal pyramidal neurons could be clearly visualized. Time-lapse imaging revealed that: (1) the presynaptic varicosities actively moved with marked changes in their shapes, and the postsynaptic scaffolding protein gephyrin clusters underwent coordinated movements in a tight association with the presynaptic varicosities, (2) the extents of morphological changes and movements depended on the developmental stages, reaching a stable level as the inhibitory synaptic connections matured, and (3) the motility indexes of the varicosity and its counterpart gephyrin cluster were well correlated. Furthermore, action potential blockade with tetrodotoxin treatment reduced the varicosity size, gephyrin cluster mobility as well as the amplitude of GABAergic synaptic currents in pyramidal neurons. Such a neural activity-dependent dynamic change in GABAergic synaptic morphology is likely to play a critical role in the regulatory mechanism underlying the formation and plasticity of inhibitory synapses.

Functional Characterization of the Dendritically Localized mRNA Neuronatin in Hippocampal Neurons

Local translation of dendritic mRNAs plays an important role in neuronal development and synaptic plasticity. Although several hundred putative dendritic transcripts have been identified in the hippocampus, relatively few have been verified by in situ hybridization and thus remain uncharacterized. One such transcript encodes the protein neuronatin. Neuronatin has been shown to regulate calcium levels in non-neuronal cells such as pancreatic or embryonic stem cells, but its function in mature neurons remains unclear. Here we report that neuronatin is translated in hippocampal dendrites in response to blockade of action potentials and NMDA-receptor dependent synaptic transmission by TTX and APV. Our study also reveals that neuronatin can adjust dendritic calcium levels by regulating intracellular calcium storage. We propose that neuronatin may impact synaptic plasticity by modulating dendritic calcium levels during homeostatic plasticity, thereby potentially regulating neuronal excitability, receptor trafficking, and calcium dependent signaling.

Direction-Selective Circuitry in Rat Retina Develops Independently of GABAergic, Cholinergic and Action Potential Activity

The ON-OFF direction selective ganglion cells (DSGCs) in the mammalian retina code image motion by responding much more strongly to movement in one direction. They do so by receiving inhibitory inputs selectively from a particular sector of processes of the overlapping starburst amacrine cells, a type of retinal interneuron. The mechanisms of establishment and regulation of this selective connection are unknown. Here, we report that in the rat retina, the morphology, physiology of the ON-OFF DSGCs and the circuitry for coding motion directions develop normally with pharmacological blockade of GABAergic, cholinergic activity and/or action potentials for over two weeks from birth. With recent results demonstrating light independent formation of the retinal DS circuitry, our results strongly suggest the formation of the circuitry, i.e., the connections between the second and third order neurons in the visual system, can be genetically programmed, although emergence of direction selectivity in the visual cortex appears to require visual experience.

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1 μM tetrodotoxin decreased TH expression beginning around 20 h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300 nM apamin increased TH mRNA and protein between 12 and 24 h later in slices of adult midbrain. Two-week infusions of 300 nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ∼20 h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.

Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.

Reduced GABAergic Inhibition Explains Cortical Hyperexcitability in the Wobbler Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of the central nervous system. Symptomatic and presymptomatic ALS patients demonstrate cortical hyperexcitability, which raises the possibility that alterations in inhibitory gamma-aminobutyric acid (GABA)ergic system could underlie this dysfunction. Here, we studied the GABAergic system in cortex using patch-clamp recordings in the wobbler mouse, a model of ALS. In layer 5 pyramidal neurons of motor cortex, the frequency of GABA(A) receptor-mediated spontaneous inhibitory postsynaptic currents was reduced by 72% in wobbler mice. Also, miniature inhibitory postsynaptic currents recorded under blockade of action potentials were decreased by 64%. Tonic inhibition mediated by extrasynaptic GABA(A) receptors was reduced by 87%. In agreement, we found a decreased density of parvalbumin- and somatostatin-positive inhibitory interneurons and reduced vesicular GABA transporter immunoreactivity in the neuropil. Finally, we observed an increased input resistance and excitability of wobbler excitatory neurons, which could be explained by lack of GABA(A) receptor-mediated influences. In conclusion, we demonstrate decreases in GABAergic inhibition, which might explain the cortical hyperexcitability in wobbler mice.