Postsynaptic Responses Research Articles

Generation and Characterization of the First Murine Model of Alzheimer's Disease with Mutated AβPP Inserted in a BALB/c Background (C.B6/J-APPswe).

Numerous mouse models of Alzheimer's disease (AD) are available, but all suffer from certain limitations, thus prompting further attempts. To date, no one model exists with amyloidopathy in a BALB/c strain. To generate and characterize the C.B6/J-APPswe mouse, a model of AD with a mutated human gene for the amyloid-β protein precursor (AβPP) inserted in a BALB/c background. We analyzed five groups at different ages (3, 6, 9, 12, and 16-18 months) of C.B6/J-APPswe and wild-type mice (50% males and 50% females) for the main hallmarks of AD by western blotting, amyloid-β (Aβ) ELISA, immunocytochemistry, electrophysiology, and behavioral tests. The C.B6/J-APPswe mouse displays early AβPP and Aβ production, late amyloid plaques formation, high level of Tau phosphorylation, synaptic deficits (reduced density and functional impairment due to a reduced post-synaptic responsiveness), neurodegeneration caused by apoptosis and necroptosis/necrosis, microgliosis, astrocytic abnormalities, and sex-related differences in explorative behavior, anxiety-like behavior, and spatial long-term and working memories. Social housing is feasible despite the intra-cage aggressiveness of male animals. C.B6/J-APPswe mice develop most of the distinctive features of AD and is a suitable model for the study of brain atrophy mechanisms and of the differences between males and females in the onset of cognitive/non-cognitive deficits.

Use-dependent facilitation of electrical transmission involves changes to postsynaptic K+ current.

Activity-dependent modulation of electrical transmission typically involves Ca2+ influx acting directly on gap junctions or initiating Ca2+-dependent pathways that in turn modulate coupling. We now describe short-term use-dependent facilitation of electrical transmission between bag cell neurons from the hermaphroditic snail, Aplysia californica, that is instead mediated by changes in postsynaptic responsiveness. Bag cell neurons secrete reproductive hormone during a synchronous afterdischarge of action potentials coordinated by electrical coupling. Here, recordings from pairs of coupled bag cell neurons in culture showed that nonjunctional currents influence electrical transmission in a dynamic manner. Under a dual whole cell voltage-clamp, the junctional current was linear and largely voltage-independent, while in current-clamp, the coupling coefficient was similar regardless of the extent of presynaptic hyperpolarization. Moreover, a train stimulus of action potential-like waveforms, in a voltage-clamped presynaptic neuron, elicited electrotonic potentials, in a current-clamped postsynaptic neuron, that facilitated over time when delivered at a frequency approximating the afterdischarge. Junctional current remained constant over the train stimulus, as did postsynaptic voltage-gated Ca2+ current. However, postsynaptic voltage-gated K+ current underwent cumulative inactivation, suggesting that K+ current run-down facilitates the electrotonic potential by boosting the response to successive junctional currents. Accordingly, preventing run-down by blocking postsynaptic K+ channels occluded facilitation. Finally, stimulation of bursts in coupled pairs resulted in synchronous firing, where active neurons could recruit silent partners through short-term use-dependent facilitation. Thus, potentiation of electrical transmission may promote synchrony in bag cell neurons and, by extension, reproductive function.NEW & NOTEWORTHY The understanding of how activity can facilitate electrical transmission is incomplete. We found that electrotonic potentials between electrically coupled neuroendocrine bag cell neurons facilitated in a use-dependent fashion. Rather than changes to the junctional current, facilitation was associated with cumulative inactivation of postsynaptic K+ current, presumably augmenting responsiveness. When made to burst, neurons synchronized their spiking, in part by use-dependent facilitation bringing quiescent cells to the threshold. Facilitation may foster en masse firing and neurosecretion.

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.

Rate-limiting recovery processes in neurotransmission under sustained stimulation

At active zones of chemical synapses, an arriving electric signal induces the fusion of vesicles with the presynaptic membrane, thereby releasing neurotransmitters into the synaptic cleft. After a fusion event, both the release site and the vesicle undergo a recovery process before becoming available for reuse again. Of central interest is the question which of the two restoration steps acts as the limiting factor during neurotransmission under high-frequency sustained stimulation. In order to investigate this problem, we introduce a non-linear reaction network which involves explicit recovery steps for both the vesicles and the release sites, and includes the induced time-dependent output current. The associated reaction dynamics are formulated by means of ordinary differential equations (ODEs), as well as via the associated stochastic jump process. While the stochastic jump model describes the dynamics at a single active zone, the average over many active zones is close to the ODE solution and shares its periodic structure. The reason for this can be traced back to the insight that recovery dynamics of vesicles and release sites are statistically almost independent. A sensitivity analysis on the recovery rates based on the ODE formulation reveals that neither the vesicle nor the release site recovery step can be identified as the essential rate-limiting step but that the rate-limiting feature changes over the course of stimulation. Under sustained stimulation, the dynamics given by the ODEs exhibit transient changes leading from an initial depression of the postsynaptic response to an asymptotic periodic orbit, while the individual trajectories of the stochastic jump model lack the oscillatory behavior and asymptotic periodicity of the ODE-solution.

Muscle-directed mechanosensory feedback activates egg-laying circuit activity and behavior in Caenorhabditis elegans

Mechanosensory feedback of the internal reproductive state drives decisions about when and where to reproduce.1 For instance, stretch in the Drosophila reproductive tract produced by artificial distention or from accumulated eggs regulates the attraction to acetic acid to ensure optimal oviposition.2 How such mechanosensoryfeedback modulates neural circuits to coordinate reproductive behaviors is incompletely understood. We previously identified a stretch-dependent homeostat that regulates egg laying in Caenorhabditis elegans. Sterilized animals lacking eggs show reduced Ca2+ transient activity in the presynaptic HSN command motoneurons that drive egg-laying behavior, while animals forced to accumulate extra eggs show dramatically increased circuit activity that restores egg laying.3 Interestingly, genetic ablation or electrical silencing of the HSNs delays, but does not abolish, the onset of egg laying,3,4,5 with animals recovering vulval muscle Ca2+ transient activity upon egg accumulation.6 Using an acute gonad microinjection technique to mimic changes in pressure and stretch resulting from germline activity and egg accumulation, we find that injection rapidly stimulates Ca2+ activity in both neurons and muscles of the egg-laying circuit. Injection-induced vulval muscle Ca2+ activity requires L-type Ca2+ channels but is independent of presynaptic input. Conversely, injection-induced neural activity is disrupted in mutants lacking the vulval muscles, suggesting "bottom-up" feedback from muscles to neurons. Direct mechanical prodding activates the vulval muscles, suggesting that they are the proximal targets of the stretch-dependent stimulus. Our results show that egg-laying behavior in C.elegans is regulated by a stretch-dependent homeostat that scales postsynaptic muscle responses with egg accumulation in the uterus.

Inhibiting neuronal AC1 for treating anxiety and headache in the animal model of migraine

Migraines are a common medical condition. From a basic science point of view, the central mechanism for migraine and headache is largely unknown. In the present study, we demonstrate that cortical excitatory transmission is significantly enhanced in the anterior cingulate cortex (ACC)-a brain region which is critical for pain perception. Biochemical studies found that the phosphorylation levels of both the NMDA receptor GluN2B and AMPA receptor GluA1 were enhanced in ACC of migraine rats. Both the presynaptic release of glutamate and postsynaptic responses of AMPA receptors and NMDA receptors were enhanced. Synaptic long-term potentiation (LTP) was occluded. Furthermore, behavioral anxiety and nociceptive responses were increased, which were reversed by application of AC1 inhibitor NB001 within ACC. Our results provide strong evidence that cortical LTPs contribute to migraine-related pain and anxiety. Drugs that inhibit cortical excitation such as NB001 may serve as potential medicines for treating migraine in the future.

Intracellular recordings reveal integrative function of the basolateral amygdala in acoustic communication.

The amygdala, a brain center of emotional expression, contributes to appropriate behavior responses during acoustic communication. In support of that role, the basolateral amygdala (BLA) analyzes the meaning of vocalizations through the integration of multiple acoustic inputs with information from other senses and an animal's internal state. The mechanisms underlying this integration are poorly understood. This study focuses on the integration of vocalization-related inputs to the BLA from auditory centers during this processing. We used intracellular recordings of BLA neurons in unanesthetized big brown bats that rely heavily on a complex vocal repertoire during social interactions. Postsynaptic and spiking responses of BLA neurons were recorded to three vocal sequences that are closely related to distinct behaviors (appeasement, low-level aggression, and high-level aggression) and have different emotional valence. Our novel findings are that most BLA neurons showed postsynaptic responses to one or more vocalizations (31 of 46) but that many fewer neurons showed spiking responses (8 of 46). The spiking responses were more selective than postsynaptic potential (PSP) responses. Furthermore, vocal stimuli associated with either positive or negative valence were similarly effective in eliciting excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), and spiking responses. This indicates that BLA neurons process both positive- and negative-valence vocal stimuli. The greater selectivity of spiking responses than PSP responses suggests an integrative role for processing within the BLA to enhance response specificity in acoustic communication.NEW & NOTEWORTHY The amygdala plays an important role in social communication by sound, but little is known about how it integrates diverse auditory inputs to form selective responses to social vocalizations. We show that BLA neurons receive inputs that are responsive to both negative- and positive-affect vocalizations but their spiking outputs are fewer and highly selective for vocalization type. Our work demonstrates that BLA neurons perform an integrative function in shaping appropriate behavioral responses to social vocalizations.

Voltage-gated calcium channel subunit α2δ-1 in spinal dorsal horn neurons contributes to aberrant excitatory synaptic transmission and mechanical hypersensitivity after peripheral nerve injury.

Neuropathic pain, an intractable pain symptom that occurs after nerve damage, is caused by the aberrant excitability of spinal dorsal horn (SDH) neurons. Gabapentinoids, the most commonly used drugs for neuropathic pain, inhibit spinal calcium-mediated neurotransmitter release by binding to α2δ-1, a subunit of voltage-gated calcium channels, and alleviate neuropathic pain. However, the exact contribution of α2δ-1 expressed in SDH neurons to the altered synaptic transmission and mechanical hypersensitivity following nerve injury is not fully understood. In this study, we investigated which types of SDH neurons express α2δ-1 and how α2δ-1 in SDH neurons contributes to the mechanical hypersensitivity and altered spinal synaptic transmission after nerve injury. Using in situ hybridization technique, we found that Cacna2d1, mRNA coding α2δ-1, was mainly colocalized with Slc17a6, an excitatory neuronal marker, but not with Slc32a1, an inhibitory neuronal marker in the SDH. To investigate the role of α2δ-1 in SDH neurons, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and showed that SDH neuron-specific ablation of Cacna2d1 alleviated mechanical hypersensitivity following nerve injury. We further found that excitatory post-synaptic responses evoked by electrical stimulation applied to the SDH were significantly enhanced after nerve injury, and that these enhanced responses were significantly decreased by application of mirogabalin, a potent α2δ-1 inhibitor, and by SDH neuron-specific ablation of Cacna2d1. These results suggest that α2δ-1 expressed in SDH excitatory neurons facilitates spinal nociceptive synaptic transmission and contributes to the development of mechanical hypersensitivity after nerve injury.

S-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin invivo and invitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.

Imaging Voltage Globally and in Isofrequency Lamina in Slices of Mouse Ventral Cochlear Nucleus.

The cochlear nuclei (CNs) receive sensory information from the ear and perform fundamental computations before relaying this information to higher processing centers. These computations are performed by distinct types of neurons interconnected in circuits dedicated to the specialized roles of the auditory system. In the present study, we explored the use of voltage imaging to investigate CN circuitry. We tested two approaches based on fundamentally different voltage sensing technologies. Using a voltage-sensitive dye we recorded glutamate receptor-independent signals arising predominantly from axons. The mean conduction velocity of these fibers of 0.27 m/s was rapid but in range with other unmyelinated axons. We then used a genetically-encoded hybrid voltage sensor (hVOS) to image voltage from a specific population of neurons. Probe expression was controlled using Cre recombinase linked to c-fos activation. This activity-induced gene enabled targeting of neurons that are activated when a mouse hears a pure 15-kHz tone. In CN slices from these animals auditory nerve fiber stimulation elicited a glutamate receptor-dependent depolarization in hVOS probe-labeled neurons. These cells resided within a band corresponding to an isofrequency lamina, and responded with a high degree of synchrony. In contrast to the axonal origin of voltage-sensitive dye signals, hVOS signals represent predominantly postsynaptic responses. The introduction of voltage imaging to the CN creates the opportunity to investigate auditory processing circuitry in populations of neurons targeted on the basis of their genetic identity and their roles in sensory processing.

Neuroligin-2 controls the establishment of fast GABAergic transmission in adult-born granule cells.

GABAergic inhibition is critical for the precision of neuronal spiking and the homeostatic regulation of network activity in the brain. Adult neurogenesis challenges network homeostasis because new granule cells (GCs) integrate continuously in the functional dentate gyrus. While developing, adult-born GCs undergo a transient state of enhanced excitability due to the delayed maturation of perisomatic GABAergic inhibition by parvalbumin interneurons (PV-INs). The mechanisms underlying this delayed synaptic maturation remain unknown. We examined the morphology and function of synapses formed by PV-INs onto new GCs over a 2-month interval in young adult mice, and investigated the influence of the synaptic adhesion molecule neuroligin-2 (NL2). Perisomatic appositions of PV-IN terminals onto new GCs were conspicuous at 2 weeks and continued to grow in size to reach a plateau over the fourth week. Postsynaptic knockdown of NL2 by expression of a short-hairpin RNA (shNL2) in new GCs resulted in smaller size of synaptic contacts, reduced area of perisomatic appositions of the vesicular GABA transporter VGAT, and the number of presynaptic active sites. GCs expressing shNL2 displayed spontaneous GABAergic responses with decreased frequency and amplitude, as well as slower kinetics compared to control GCs. In addition, postsynaptic responses evoked by optogenetic stimulation of PV-INs exhibited slow kinetics, increased paired-pulse ratio and coefficient of variation in GCs with NL2 knockdown, suggesting a reduction in the number of active synapses as well as in the probability of neurotransmitter release (Pr ). Our results demonstrate that synapses formed by PV-INs on adult-born GCs continue to develop beyond the point of anatomical growth, and require NL2 for the structural and functional maturation that accompanies the conversion into fast GABAergic transmission.

Chlorpromazine, an Inverse Agonist of D1R-Like, Differentially Targets Voltage-Gated Calcium Channel (CaV) Subtypes in mPFC Neurons.

The dopamine receptor type 1 (D1R) and the dopamine receptor type 5 (D5R), which are often grouped as D1R-like due to their sequence and signaling similarities, exhibit high levels of constitutive activity. The molecular basis for this agonist-independent activation has been well characterized through biochemical and mutagenesis in vitro studies. In this regard, it was reported that many antipsychotic drugs act as inverse agonists of D1R-like constitutive activity. On the other hand, D1R is highly expressed in the medial prefrontal cortex (mPFC), a brain area with important functions such as working memory. Here, we studied the impact of D1R-like constitutive activity and chlorpromazine (CPZ), an antipsychotic drug and D1R-like inverse agonist, on various neuronal CaV conductances, and we explored its effect on calcium-dependent neuronal functions in the mouse medial mPFC. Using ex vivo brain slices containing the mPFC and transfected HEK293T cells, we found that CPZ reduces CaV2.2 currents by occluding D1R-like constitutive activity, in agreement with a mechanism previously reported by our lab, whereas CPZ directly inhibits CaV1 currents in a D1R-like activity independent manner. In contrast, CPZ and D1R constitutive activity did not affect CaV2.1, CaV2.3, or CaV3 currents. Finally, we found that CPZ reduces excitatory postsynaptic responses in mPFC neurons. Our results contribute to understanding CPZ molecular targets in neurons and describe a novel physiological consequence of CPZ non-canonical action as a D1R-like inverse agonist in the mouse brain.

Targeted sensors for glutamatergic neurotransmission.

Optical report of neurotransmitter release allows visualisation of excitatory synaptic transmission. Sensitive genetically-encoded fluorescent glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-γ2 and SnFR-γ8, were enriched at synapses, retained function and reported spontaneous glutamate release in rat hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic mouse neurons cultured on astrocytic microislands, evoked neurotransmitter release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal neurotransmitter release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-γ2 is a linear reporter of the global quantal parameters and short-term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest (ROIs) showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-γ2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

PS-C20-5: WARM IV INFUSION IN HYPERTENSIVE URGENCY

Background: Hypertensive Urgency is defined as marked elevation in systemic blood pressure without any evidence of end organ damage. At present, the condition is managed with intravenous (IV) antihypertensives, of which most common is Sodium Nitroprusside. Aim: In this abstract we explore the possibility of slow IV infusion of warm (37–38oC) Normal Saline along with IV antihypertensives for a better and faster management of Hypertensive Urgency. Content: The slow IV will gradually increase the core body temperature of the patient while preventing fluid overload. Homeostatic regulation of Systemic Blood Pressure is maintained by an intricate network of correlated mechanisms, most of which are temperature dependent. The raised body temperature will be useful to control the Blood Pressure through the following mechanisms - Cardiac Output: reduces as core body temperature increases. Baroreflex: decreased post-synaptic adrenergic vasoconstrictor responses. Chemoreceptors: vascular responsiveness to adrenergic receptor stimulation reduces at higher temperatures. RAAS: Kosunen et al recorded reduced Systolic and Diastolic Blood Pressures at raised body temperatures despite increased levels of Angiotensin II & Aldosterone. Endothelin: Serum concentration of Endothelin-I reduces at higher temperatures. Sweat: Thermal stimulation of sweat gland will help excretion of both salt and water, thus maintaining the sodium balance. VEGF: VEGF-A, a potent vasodilator, is increased during elevated body temperature. However, apart from Blood Pressure, a change in core body temperature causes change in many other parameters as well, which must be considered during this discussion. Coagulation profile - Valeri et al noted no change in in-vivo coagulation states till 100oF Effect on other visceral organs - Internal visceral organs like Liver, Kidneys and Brain were not affected till 100oF Infection Control - Core body temperature close to 100oF has also been found to have a protective role against invasive fungal infections in the ICU Discussion: Though the effects of various antihypertensive drugs to raised body temperatures need to be tested, the above points prove that introducing a protocol of warm IV saline Infusion in patients of Hypertensive Urgency may be extremely beneficial, and also prevent clinical inertia.

Insulin facilitates synaptic transmission via gap junctions between fast-spiking interneurons in the rat insular cortex.

Inhibitory synaptic currents from fast-spiking neurons (FSNs), a typical gamma-aminobutyric acid (GABA)ergic interneuron in the cerebral cortex, to pyramidal neurons are facilitated by insulin. FSNs frequently show electrical synapses to FSNs, however, the effect of insulin on these electrical synapses is unknown. The aim of this study was to evaluate effects of insulin on electrical synaptic potentials between FSNs. Electrical synaptic potentials via gap junctions between FSNs were recorded to examine how insulin modulates these potentials in the rat insular cortex (IC). Bath application of insulin (10 nM), which increases the spike firing rate of pyramidal neurons and unitary inhibitory postsynaptic currents recorded from FSN to pyramidal neuron connections, slightly but significantly increased electrical synaptic currents. The mean ratio of electrical synapses, the coupling coefficient that is obtained by postsynaptic voltage responses divided by presynaptic voltage amplitude, was 8.3 ± 1.1% in control and 9.2 ± 1.1% (n = 14) during 10 nM insulin application. Input resistance and voltage responses to large hyperpolarizing currents (-140 pA) were not changed by insulin. These results suggest that insulin facilitates spike synchronization by increasing electrical synaptic currents via gap junctions of GABAergic FSNs in the IC.

Optogenetic stimulation reveals a latent tipping point in cortical networks during ictogenesis.

Brain-state transitions are readily apparent from changes in brain rhythms,1 but are difficult to predict, suggestive that the underlying cause is latent to passive recording methods. Among the most important transitions, clinically, are the starts of seizures. We here show that an 'active probing' approach may have several important benefits for epileptic management, including by helping predict these transitions. We used mice expressing the optogenetic actuator, channelrhodopsin, in pyramidal cells, allowing this population to be stimulated in isolation. Intermittent stimulation at frequencies as low as 0.033 Hz (period = 30 s) delayed the onset of seizure-like events in an acute brain slice model of ictogenesis, but the effect was lost if stimulation was delivered at even lower frequencies (1/min). Notably, active probing additionally provides advance indication of when seizure-like activity is imminent, revealed by monitoring the postsynaptic response to stimulation. The postsynaptic response, recorded extracellularly, showed an all-or-nothing change in both amplitude and duration, a few hundred seconds before seizure-like activity began-a sufficient length of time to provide a helpful warning of an impending seizure. The change in the postsynaptic response then persisted for the remainder of the recording, indicative of a state change from a pre-epileptic to a pro-epileptic network. This occurred in parallel with a large increase in the stimulation-triggered Ca2+ entry into pyramidal dendrites, and a step increase in the number of evoked postsynaptic action potentials, both consistent with a reduction in the threshold for dendritic action potentials. In 0 Mg2+ bathing media, the reduced threshold was not associated with changes in glutamatergic synaptic function, nor of GABAergic release from either parvalbumin or somatostatin interneurons, but simulations indicate that the step change in the optogenetic response can instead arise from incremental increases in intracellular [Cl-]. The change in the response to stimulation was replicated by artificially raising intracellular [Cl-], using the optogenetic chloride pump, halorhodopsin. By contrast, increases in extracellular [K+] cannot account for the firing patterns in the response to stimulation, although this, and other cellular changes, may contribute to ictal initiation in other circumstances. We describe how these various cellular changes form a synergistic network of positive feedback mechanisms, which may explain the precipitous nature of seizure onset. This model of seizure initiation draws together several major lines of epilepsy research as well as providing an important proof-of-principle regarding the utility of open-loop brain stimulation for clinical management of the condition.

Quantifying postsynaptic receptor dynamics: insights into synaptic function.

The molecular composition of presynaptic and postsynaptic neuronal terminals is dynamic, and yet long-term stabilizations in postsynaptic responses are necessary for synaptic development and long-term plasticity. The need to reconcile these concepts is further complicated by learning- and memory-related plastic changes in the molecular make-up of synapses. Advances in single-particle tracking mean that we can now quantify the number and diffusive properties of specific synaptic molecules, while statistical thermodynamics provides a framework to analyse these molecular fluctuations. In this Review, we discuss the use of these approaches to gain quantitative descriptions of the processes underlying the turnover, long-term stability and plasticity of postsynaptic receptors and show how these can help us to understand the balance between local molecular turnover and synaptic structural identity and integrity.

Chronic, episodic nicotine exposure alters GABAergic synaptic transmission to hypoglossal motor neurons and genioglossus muscle function at a critical developmental age.

Regulation of GABAergic signaling through nicotinic acetylcholine receptor (nAChR) activation is critical for neuronal development. Here, we test the hypothesis that chronic episodic developmental nicotine exposure (eDNE) disrupts GABAergic signaling, leading to dysfunction of hypoglossal motor neurons (XIIMNs), which innervate the tongue muscles. We studied control and eDNE pups at two developmentally vulnerable age ranges: postnatal days (P)1-5 and P10-12. The amplitude and frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs, mIPSCs) at baseline were not altered by eDNE at either age. In contrast, eDNE increased GABAAR-α1 receptor expression on XIIMNs and, in the older group, the postsynaptic response to muscimol (GABAA receptor agonist). Activation of nAChRs with exogenous nicotine increased the frequency of GABAergic sIPSCs in control and eDNE neurons at P1-5. By P10-12, acute nicotine increased sIPSC frequency in eDNE but not control neurons. In vivo experiments showed that the breathing-related activation of tongue muscles, which are innervated by XIIMNs, is reduced at P10-12. This effect was partially mitigated by subcutaneous muscimol, but only in the eDNE pups. Taken together, these data indicate that eDNE alters GABAergic transmission to XIIMNs at a critical developmental age, and this is expressed as reduced breathing-related drive to XIIMNs in vivo.NEW & NOTEWORTHY Here, we provide a thorough assessment of the effects of nicotine exposure on GABAergic synaptic transmission, from the cellular to the systems level. This work makes significant advances in our understanding of the impact of nicotine exposure during development on GABAergic neurotransmission within the respiratory network and the potential role this plays in the excitatory/inhibitory imbalance that is thought to be an important mechanism underlying neonatal breathing disorders, including sudden infant death syndrome.

Depression of Synaptic N-methyl-D-Aspartate Responses by Xenon and Nitrous Oxide

In "synapse bouton preparation" of rat hippocampal CA3 neurons, we examined how Xe and N2O modulate N-methyl-D-aspartate (NMDA) receptor-mediated spontaneous and evoked excitatory post-synaptic currents (sEPSCNMDA and eEPSCNMDA) excluded extrasynaptic, glial and other neuronal tonic currents. This preparation is a mechanically isolated single neuron attached with nerve endings (boutons) preserving normal physiological function and promotes the exact evaluation of sEPSCNMDA and eEPSCNMDA These sEPSC and eEPSC are elicited by spontaneous glutamate (Glu) release from many homologous glutamatergic boutons and by focal paired-pulse electric stimulation of a single bouton, respectively. The s/eEPSCAMPA/KA and s/eEPSCNMDA were isolated pharmacologically by their specific antagonists. Thus, independent contributions of pre- and postsynaptic responses be could also quantified. All kinetic properties of s/eEPSCAMPA/KA and s/eEPSCNMDA were detected clearly. The s/eEPSCNMDA showed smaller amplitude, slower rise and 1/e decay time constant (τDecay) than s/eEPSCAMPA/KA Xe (70%) and N2O (70%) significantly decreased the frequency and amplitude without altering the τDecay of sEPSCNMDA They also decreased the amplitude but increased the Rf and PPR without altering the τDecay of the eEPSCNMDA These data show clearly that "synapse bouton preparation" can be an accurate model for evaluating s/eEPSCNMDA Such inhibitory effects of gas anesthetics are primarily due to presynaptic mechanisms. Present results may explain partially the powerful analgesic effects of Xe and N2O. Significance Statement We could record pharmacologically isolated sEPSCNMDA and eEPSCNMDA and clearly detect all kinetic parameters of sEPSCNMDA and eEPSCNMDA at synaptic levels by using "synapse bouton preparation" of rat hippocampal CA3 neurons. We found that Xe and N2O clearly suppressed both sEPSCNMDA and eEPSCNMDA Different from previous studies, present results suggest that Xe and N2O predominantly inhibit the NMDA responses by presynaptic mechanisms.

Sleep need-dependent changes in functional connectivity facilitate transmission of homeostatic sleep drive

How the homeostatic drive for sleep accumulates over time and is released remains poorly understood. In Drosophila, we previously identified the R5 ellipsoid body (EB) neurons as putative sleep drive neurons1 and recently described a mechanism by which astrocytes signal to these cells to convey sleep need.2 Here, we examine the mechanisms acting downstream of the R5 neurons to promote sleep. EM connectome data demonstrate that R5 neurons project to EPG neurons.3 Broad thermogenetic activation of EPG neurons promotes sleep, whereas inhibiting these cells reduces homeostatic sleep rebound. Perforated patch-clamp recordings reveal that EPG neurons exhibit elevated spontaneous firing following sleep deprivation, which likely depends on an increase in extrinsic excitatory inputs. Our data suggest that cholinergic R5 neurons participate in the homeostatic regulation of sleep, and epistasis experiments indicate that the R5 neurons act upstream of EPG neurons to promote sleep. Finally, we show that the physical and functional connectivity between the R5 and EPG neurons increases with greater sleep need. Importantly, dual patch-clamp recordings demonstrate that activating R5 neurons induces cholinergic-dependent excitatory postsynaptic responses in EPG neurons. Moreover, sleep loss triggers an increase in the amplitude of these responses, as well as in the proportion of EPG neurons that respond. Together, our data support a model whereby sleep drive strengthens the functional connectivity between R5 and EPG neurons, triggering sleep when a sufficient number of EPG neurons are activated. This process could enable the proper timing of the accumulation and release of sleep drive.