Simulations predict differing phase responses to excitation vs. inhibition in theta-resonant pyramidal neurons.

Rhythmic activity is ubiquitous in neural systems, with theta-resonant pyramidal neurons integrating rhythmic inputs in many cortical structures. Impedance analysis has been widely used to examine frequency-dependent responses of neuronal membranes to rhythmic inputs, but it assumes that the neuronal membrane is a linear system, requiring the use of small signals to stay in a near-linear regime. However, postsynaptic potentials are often large and trigger nonlinear mechanisms (voltage-gated ion channels). The goals of this work were to 1) develop an analysis method to evaluate membrane responses in this nonlinear domain and 2) explore phase relationships between rhythmic stimuli and subthreshold and spiking membrane potential (Vmemb) responses in models of theta-resonant pyramidal neurons. Responses in these output regimes were asymmetrical, with different phase shifts during hyperpolarizing and depolarizing half-cycles. Suprathreshold theta-rhythmic stimuli produced nonstationary Vmemb responses. Sinusoidal inputs produced "phase retreat": action potentials occurred progressively later in cycles of the input stimulus, resulting from adaptation. Sinusoidal current with increasing amplitude over cycles produced "phase advance": action potentials occurred progressively earlier. Phase retreat, phase advance, and subthreshold phase shifts were modulated by multiple ion channel conductances. Our results suggest differential responses of cortical neurons depending on the frequency of oscillatory input, which will play a role in neuronal responses to shifts in network state. We hypothesize that intrinsic cellular properties complement network properties and contribute to in vivo phase-shift phenomena such as phase precession, seen in place and grid cells, and phase roll, also observed in hippocampal CA1 neurons.NEW & NOTEWORTHY We augmented electrical impedance analysis to characterize phase shifts between large-amplitude current stimuli and nonlinear, asymmetric membrane potential responses. We predict different frequency-dependent phase shifts in response excitation vs. inhibition, as well as shifts in spike timing over multiple input cycles, in theta-resonant pyramidal neurons. We hypothesize that these effects contribute to navigation-related phenomena such as phase precession and phase roll. Our neuron-level hypothesis complements, rather than falsifies, prior network-level explanations of these phenomena.

Imaging of Evoked Cortical Depolarizations Using Either ASAP2s, or chi-VSFP, or Di-4-Anepps, or Autofluorescence Optical Signals.

BACKGROUND: Population voltage imaging is used for studying brain physiology and brain circuits. Using a genetically encoded voltage indicator (GEVI), "VSFP" or "ASAP2s", or a voltage-sensitive dye, Di-4-Anepps, we conducted population voltage imaging in brain slices. The resulting optical signals, optical local field potentials (LFPs), were used to evaluate the performances of the 3 voltage indicators. METHODS: In brain slices prepared from VSFP-transgenic or ASAP2s-transgenic mice, we performed multi-site optical imaging of evoked cortical depolarizations - compound excitatory postsynaptic potentials (cEPSPs). Optical signal amplitudes (ΔF/F) and cEPSP decay rates (OFF rates) were compared using analysis of variance (ANOVA) followed by unpaired Student's t test (31-104 data points per voltage indicator). RESULTS: The ASAP2s signal amplitude (ΔF/F) was on average 3 times greater than Di-4-Anepps, and 7 times greater than VSFP. The optical cEPSP decay (OFF rate) was the slowest in Di-4-Anepps and fastest in ASAP2s. When ASAP2s expression was weak, we observed slow, label-free (autofluorescence, metabolic) optical signals mixed into the ASAP2s traces. Fast hyperpolarizations, that typically follow depolarizing cortical transients (afterhyperpolarizations), were prominent in ASAP2s but not present in the VSFP and Di-4-Anepps experiments. CONCLUSIONS: Experimental applications for ASAP2s may potentially include systems neuroscience studies that require voltage indicators with large signal amplitude (ΔF/F), fast decay times (fast response time is needed for monitoring high frequency brain oscillations), and/or detection of brain patches in transiently hyperpolarized states (afterhyperpolarization).

Effects of Ih and TASK-like shunting current on dendritic impedance in layer 5 pyramidal-tract neurons.

Pyramidal neurons in neocortex have complex input-output relationships that depend on their morphologies, ion channel distributions, and the nature of their inputs, but which cannot be replicated by simple integrate-and-fire models. The impedance properties of their dendritic arbors, such as resonance and phase shift, shape neuronal responses to synaptic inputs and provide intraneuronal functional maps reflecting their intrinsic dynamics and excitability. Experimental studies of dendritic impedance have shown that neocortical pyramidal tract neurons exhibit distance-dependent changes in resonance and impedance phase with respect to the soma. We, therefore, investigated how well several biophysically detailed multicompartment models of neocortical layer 5 pyramidal tract neurons reproduce the location-dependent impedance profiles observed experimentally. Each model tested here exhibited location-dependent impedance profiles, but most captured either the observed impedance amplitude or phase, not both. The only model that captured features from both incorporates hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and a shunting current, such as that produced by Twik-related acid-sensitive K+ (TASK) channels. TASK-like channel density in this model was proportional to local HCN channel density. We found that although this shunting current alone is insufficient to produce resonance or realistic phase response, it modulates all features of dendritic impedance, including resonance frequencies, resonance strength, synchronous frequencies, and total inductive phase. We also explored how the interaction of HCN channel current (Ih) and a TASK-like shunting current shape synaptic potentials and produce degeneracy in dendritic impedance profiles, wherein different combinations of Ih and shunting current can produce the same impedance profile.NEW & NOTEWORTHY We simulated chirp current stimulation in the apical dendrites of 5 biophysically detailed multicompartment models of neocortical pyramidal tract neurons and found that a combination of HCN channels and TASK-like channels produced the best fit to experimental measurements of dendritic impedance. We then explored how HCN and TASK-like channels can shape the dendritic impedance as well as the voltage response to synaptic currents.

Local glutamate-mediated dendritic plateau potentials change the state of the cortical pyramidal neuron.

Dendritic spikes in thin dendritic branches (basal and oblique dendrites) are traditionally inferred from spikelets measured in the cell body. Here, we used laser-spot voltage-sensitive dye imaging in cortical pyramidal neurons (rat brain slices) to investigate the voltage waveforms of dendritic potentials occurring in response to spatially restricted glutamatergic inputs. Local dendritic potentials lasted 200-500 ms and propagated to the cell body, where they caused sustained 10- to 20-mV depolarizations. Plateau potentials propagating from dendrite to soma and action potentials propagating from soma to dendrite created complex voltage waveforms in the middle of the thin basal dendrite, comprised of local sodium spikelets, local plateau potentials, and backpropagating action potentials, superimposed on each other. Our model replicated these voltage waveforms across a gradient of glutamatergic stimulation intensities. The model then predicted that somatic input resistance (Rin) and membrane time constant (tau) may be reduced during dendritic plateau potential. We then tested these model predictions in real neurons and found that the model correctly predicted the direction of Rin and tau change but not the magnitude. In summary, dendritic plateau potentials occurring in basal and oblique branches put pyramidal neurons into an activated neuronal state ("prepared state"), characterized by depolarized membrane potential and smaller but faster membrane responses. The prepared state provides a time window of 200-500 ms, during which cortical neurons are particularly excitable and capable of following afferent inputs. At the network level, this predicts that sets of cells with simultaneous plateaus would provide cellular substrate for the formation of functional neuronal ensembles.NEW & NOTEWORTHY In cortical pyramidal neurons, we recorded glutamate-mediated dendritic plateau potentials with voltage imaging and created a computer model that recreated experimental measures from dendrite and cell body. Our model made new predictions, which were then tested in experiments. Plateau potentials profoundly change neuronal state: a plateau potential triggered in one basal dendrite depolarizes the soma and shortens membrane time constant, making the cell more susceptible to firing triggered by other afferent inputs.

Cadmium versus Lanthanum Effects on Spontaneous Electrical Activity and Expression of Connexin Isoforms Cx26, Cx36, and Cx45 in the Human Fetal Cortex.

Electrical activity is important for brain development. In brain slices, human subplate neurons exhibit spontaneous electrical activity that is highly sensitive to lanthanum. Based on the results of pharmacological experiments in human fetal tissue, we hypothesized that hemichannel-forming connexin (Cx) isoforms 26, 36, and 45 would be expressed on neurons in the subplate (SP) zone. RNA sequencing of dissected human cortical mantles at ages of 17-23 gestational weeks revealed that Cx45 has the highest expression, followed by Cx36 and Cx26. The levels of Cx and pannexin expression between male and female fetal cortices were not significantly different. Immunohistochemical analysis detected Cx45- and Cx26-expressing neurons in the upper segment of the SP zone. Cx45 was present on the cell bodies of human SP neurons, while Cx26 was found on both cell bodies and dendrites. Cx45, Cx36, and Cx26 were strongly expressed in the cortical plate, where newborn migrating neurons line up to form cortical layers. New information about the expression of 3 "neuronal" Cx isoforms in each cortical layer/zone (e.g., SP, cortical plate) and pharmacological data with cadmium and lanthanum may improve our understanding of the cellular mechanisms underlying neuronal development in human fetuses and potential vulnerabilities.

Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex-Mouse Subplate Zone.

Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1-P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 µM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1-P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.

Embedded ensemble encoding hypothesis: The role of the "Prepared" cell.

We here reconsider current theories of neural ensembles in the context of recent discoveries about neuronal dendritic physiology. The key physiological observation is that the dendritic plateau potential produces sustained depolarization of the cell body (amplitude 10-20 mV, duration 200-500 ms). Our central hypothesis is that synaptically-evoked dendritic plateau potentials lead to a prepared state of a neuron that favors spike generation. The plateau both depolarizes the cell toward spike threshold, and provides faster response to inputs through a shortened membrane time constant. As a result, the speed of synaptic-to-action potential (AP) transfer is faster during the plateau phase. Our hypothesis relates the changes from "resting" to "depolarized" neuronal state to changes in ensemble dynamics and in network information flow. The plateau provides the Prepared state (sustained depolarization of the cell body) with a time window of 200-500 ms. During this time, a neuron can tune into ongoing network activity and synchronize spiking with other neurons to provide a coordinated Active state (robust firing of somatic APs), which would permit "binding" of signals through coordination of neural activity across a population. The transient Active ensemble of neurons is embedded in the longer-lasting Prepared ensemble of neurons. We hypothesize that "embedded ensemble encoding" may be an important organizing principle in networks of neurons.

The stochastic nature of action potential backpropagation in apical tuft dendrites.

In cortical pyramidal neurons, backpropagating action potentials (bAPs) supply Ca2+ to synaptic contacts on dendrites. To determine whether the efficacy of AP backpropagation into apical tuft dendrites is stable over time, we performed dendritic Ca2+ and voltage imaging in rat brain slices. We found that the amplitude of bAP-Ca2+ in apical tuft branches was unstable, given that it varied from trial to trial (termed "bAP-Ca2+ flickering"). Small perturbations in dendritic physiology, such as spontaneous synaptic inputs, channel inactivation, or temperature-induced changes in channel kinetics, can cause bAP flickering. In the tuft branches, the density of Na+ and K+ channels was sufficient to support local initiation of fast spikelets by glutamate iontophoresis. We quantified the time delay between the somatic AP burst and the peak of dendritic Ca2+ transient in the apical tuft, because this delay is important for induction of spike-timing dependent plasticity. Depending on the frequency of the somatic AP triplets, Ca2+ signals peaked in the apical tuft 20-50 ms after the 1st AP in the soma. Interestingly, at low frequency (<20 Hz), the Ca2+ peaked sooner than at high frequency, because only the 1st AP invaded tuft. Activation of dendritic voltage-gated Ca2+ channels is sensitive to the duration of the dendritic voltage transient. In apical tuft branches, small changes in the duration of bAP voltage waveforms cause disproportionately large increases in dendritic Ca2+ influx (bAP-Ca2+ flickering). The stochastic nature of bAP-Ca2+ adds a new perspective on the mechanisms by which pyramidal neurons combine inputs arriving at different cortical layers.NEW & NOTEWORTHY The bAP-Ca2+ signal amplitudes in some apical tuft branches randomly vary from moment to moment. In repetitive measurements, successful AP invasions are followed by complete failures. Passive spread of voltage from the apical trunk into the tuft occasionally reaches the threshold for local Na+ spike, resulting in stronger Ca2+ influx. During a burst of three somatic APs, the peak of dendritic Ca2+ in the apical tuft occurs with a delay of 20-50 ms depending on AP frequency.

Connexin hemichannels contribute to spontaneous electrical activity in the human fetal cortex.

Before the human cortex is able to process sensory information, young postmitotic neurons must maintain occasional bursts of action-potential firing to attract and keep synaptic contacts, to drive gene expression, and to transition to mature membrane properties. Before birth, human subplate (SP) neurons are spontaneously active, displaying bursts of electrical activity (plateau depolarizations with action potentials). Using whole-cell recordings in acute cortical slices, we investigated the source of this early activity. The spontaneous depolarizations in human SP neurons at midgestation (17-23 gestational weeks) were not completely eliminated by tetrodotoxin--a drug that blocks action potential firing and network activity--or by antagonists of glutamatergic, GABAergic, or glycinergic synaptic transmission. We then turned our focus away from standard chemical synapses to connexin-based gap junctions and hemichannels. PCR and immunohistochemical analysis identified the presence of connexins (Cx26/Cx32/Cx36) in the human fetal cortex. However, the connexin-positive cells were not found in clusters but, rather, were dispersed in the SP zone. Also, gap junction-permeable dyes did not diffuse to neighboring cells, suggesting that SP neurons were not strongly coupled to other cells at this age. Application of the gap junction and hemichannel inhibitors octanol, flufenamic acid, and carbenoxolone significantly blocked spontaneous activity. The putative hemichannel antagonist lanthanum alone was a potent inhibitor of the spontaneous activity. Together, these data suggest that connexin hemichannels contribute to spontaneous depolarizations in the human fetal cortex during the second trimester of gestation.

Transgenic Strategies for Sparse but Strong Expression of Genetically Encoded Voltage and Calcium Indicators.

Rapidly progressing development of optogenetic tools, particularly genetically encoded optical indicators, enables monitoring activities of neuronal circuits of identified cell populations in longitudinal in vivo studies. Recently developed advanced transgenic approaches achieve high levels of indicator expression. However, targeting non-sparse cell populations leads to dense expression patterns such that optical signals from neuronal processes cannot be allocated to individual neurons. This issue is particularly pertinent for the use of genetically encoded voltage indicators whose membrane-delimited signals arise largely from the neuropil where dendritic and axonal membranes of many cells intermingle. Here we address this need for sparse but strong expression of genetically encoded optical indicators using a titratable recombination-activated transgene transcription to achieve a Golgi staining-type indicator expression pattern in vivo. Using different transgenic strategies, we also illustrate that co-expression of genetically encoded voltage and calcium indicators can be achieved in vivo for studying neuronal circuit input-output relationships.

Voltage imaging to understand connections and functions of neuronal circuits.

Understanding of the cellular mechanisms underlying brain functions such as cognition and emotions requires monitoring of membrane voltage at the cellular, circuit, and system levels. Seminal voltage-sensitive dye and calcium-sensitive dye imaging studies have demonstrated parallel detection of electrical activity across populations of interconnected neurons in a variety of preparations. A game-changing advance made in recent years has been the conceptualization and development of optogenetic tools, including genetically encoded indicators of voltage (GEVIs) or calcium (GECIs) and genetically encoded light-gated ion channels (actuators, e.g., channelrhodopsin2). Compared with low-molecular-weight calcium and voltage indicators (dyes), the optogenetic imaging approaches are 1) cell type specific, 2) less invasive, 3) able to relate activity and anatomy, and 4) facilitate long-term recordings of individual cells' activities over weeks, thereby allowing direct monitoring of the emergence of learned behaviors and underlying circuit mechanisms. We highlight the potential of novel approaches based on GEVIs and compare those to calcium imaging approaches. We also discuss how novel approaches based on GEVIs (and GECIs) coupled with genetically encoded actuators will promote progress in our knowledge of brain circuits and systems.

Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines.

A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly signals are integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes which determine the input-output function of any neuron are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin (synaptic contacts on distal dendrites) and summate at particular locations to influence action potential initiation. It became possible recently to carry out this type of measurement using high-resolution multisite recording of membrane potential changes with intracellular voltage-sensitive dyes. This chapter reviews the development and foundation of the method of voltage-sensitive dye recording from individual neurons. Presently, this approach allows monitoring membrane potential transients from all parts of the dendritic tree as well as from axon collaterals and individual dendritic spines.

Palette of fluorinated voltage-sensitive hemicyanine dyes.

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440-670 nm; the two-photon excitation range is 900-1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Characterization of two near-infrared genetically encoded voltage indicators.

Significance: Efforts starting more than 20 years ago led to increasingly well performing genetically encoded voltage indicators (GEVIs) for optical imaging at wavelengths <600 nm. Although optical imaging in the >600 nm wavelength range has many advantages over shorter wavelength approaches for mesoscopic in vivo monitoring of neuronal activity in the mammalian brain, the availability and evaluation of well performing near-infrared GEVIs are still limited. Aim: Here, we characterized two recent near-infrared GEVIs, Archon1 and nirButterfly, to support interested tool users in selecting a suitable near-infrared GEVI for their specific research question requirements. Approach: We characterized side-by-side the brightness, sensitivity, and kinetics of both near-infrared GEVIs in a setting focused on population imaging. Results: We found that nirButterfly shows seven-fold higher brightness than Archon1 under the same conditions and faster kinetics than Archon1 for population imaging without cellular resolution. But Archon1 showed larger signals than nirButterfly. Conclusions: Neither GEVI characterized here surpasses in all three key parameters (brightness, kinetics, and sensitivity), so there is no unequivocal preference for one of the two. Our side-by-side characterization presented here provides new information for future in vitro and ex vivo experimental designs.

Physiological features of parvalbumin-expressing GABAergic interneurons contributing to high-frequency oscillations in the cerebral cortex.

Parvalbumin-expressing (PV+) inhibitory interneurons drive gamma oscillations (30-80 Hz), which underlie higher cognitive functions. In this review, we discuss two groups/aspects of fundamental properties of PV+ interneurons. In the first group (dubbed Before Axon), we list properties representing optimal synaptic integration in PV+ interneurons designed to support fast oscillations. For example: [i] Information can neither enter nor leave the neocortex without the engagement of fast PV+ -mediated inhibition; [ii] Voltage responses in PV+ interneuron dendrites integrate linearly to reduce impact of the fluctuations in the afferent drive; and [iii] Reversed somatodendritic Rm gradient accelerates the time courses of synaptic potentials arriving at the soma. In the second group (dubbed After Axon), we list morphological and biophysical properties responsible for (a) short synaptic delays, and (b) efficient postsynaptic outcomes. For example: [i] Fast-spiking ability that allows PV+ interneurons to outpace other cortical neurons (pyramidal neurons). [ii] Myelinated axon (which is only found in the PV+ subclass of interneurons) to secure fast-spiking at the initial axon segment; and [iii] Inhibitory autapses - autoinhibition, which assures brief biphasic voltage transients and supports postinhibitory rebounds. Recent advent of scientific tools, such as viral strategies to target PV cells and the ability to monitor PV cells via in vivo imaging during behavior, will aid in defining the role of PV cells in the CNS. Given the link between PV+ interneurons and cognition, in the future, it would be useful to carry out physiological recordings in the PV+ cell type selectively and characterize if and how psychiatric and neurological diseases affect initiation and propagation of electrical signals in this cortical sub-circuit. Voltage imaging may allow fast recordings of electrical signals from many PV+ interneurons simultaneously.

Spontaneous electrical activity in the human fetal cortex in vitro.

Our knowledge about the developing human cerebral cortex is based on the analysis of fixed postmortem material. Here we use electrical recordings from unfixed human postmortem tissue to characterize the synaptic physiology and spontaneous network activity of pioneer cortical neurons ("subplate neurons"). Our electrophysiological experiments show that functional glutamate or GABA ionotropic receptors are expressed on human subplate (SP) neurons as early as 20 gestational weeks. Extracellular (synaptic) stimulations evoked postsynaptic potentials in a very small fraction of SP neurons, suggesting that functional synaptic contacts are rare at midgestation. Although synaptic inputs were scarce, we regularly observed spontaneous (unprovoked) electrical activity among human SP neurons, comprised of sustained plateau depolarizations and bursts of action potential firing, which resembled cortical UP and DOWN states in the adult neocortex. Plateau depolarizations and bursts of action potential firing are thought to depend on the mature morphology and physiology of adult cortical network. However, our current data reveal that similar cortical rhythm is generated by a very immature ensemble of human fetal neurons. In the relative absence of sensory inputs, as in development in utero, or in slow-wave sleep (i.e., throughout the entire lifespan), the spontaneous slow oscillatory pattern (UP and DOWN states) is a fundamental aspect of human cortical physiology.

Rapid dopaminergic and GABAergic modulation of calcium and voltage transients in dendrites of prefrontal cortex pyramidal neurons.

The physiological responses of dendrites to dopaminergic inputs are poorly understood and controversial. We applied dopamine on one dendritic branch while simultaneously monitoring action potentials (APs) from multiple dendrites using either calcium-sensitive dye, voltage-sensitive dye or both. Dopaminergic suppression of dendritic calcium transients was rapid (<0.5 s) and restricted to the site of dopamine application. Voltage waveforms of backpropagating APs were minimally altered in the same dendrites where dopamine was confirmed to cause large suppression of calcium signals, as determined by dual voltage and calcium imaging. The dopamine effects on dendritic calcium transients were fully mimicked by D1 agonists, partially reduced by D1 antagonist and completely insensitive to protein kinase blockade; consistent with a membrane delimited mechanism. This dopamine effect was unaltered in the presence of L-, R- and T-type calcium channel blockers. The somatic excitability (i.e. AP firing) was not affected by strong dopaminergic stimulation of dendrites. Dopamine and GABA were then sequentially applied on the same dendrite. In contrast to dopamine, the pulses of GABA prohibited AP backpropagation distally from the application site, even in neurons with natural Cl− concentration (patch pipette removed). Thus, the neocortex employs at least two distinct mechanisms (dopamine and GABA) for rapid modulation of dendritic calcium influx. The spatio-temporal pattern of dendritic calcium suppression described in this paper is expected to occur during phasic dopaminergic signalling, when midbrain dopaminergic neurons generate a transient (0.5 s) burst of APs in response to a salient event.

Evoked Cortical Depolarizations Before and After the Amyloid Plaque Accumulation: Voltage Imaging Study.

BACKGROUND: In Alzheimer's disease (AD), synaptic dysfunction is thought to occur many years before the onset of cognitive decline. OBJECTIVE: Detecting synaptic dysfunctions at the earliest stage of AD would be desirable in both clinic and research settings. METHODS: Population voltage imaging allows monitoring of synaptic depolarizations, to which calcium imaging is relatively blind. We developed an AD mouse model (APPswe/PS1dE9 background) expressing a genetically-encoded voltage indicator (GEVI) in the neocortex. GEVI was restricted to the excitatory pyramidal neurons (unlike the voltage-sensitive dyes). RESULTS: Expression of GEVI did not disrupt AD model formation of amyloid plaques. GEVI expression was stable in both AD model mice and Control (healthy) littermates (CTRL) over 247 days postnatal. Brain slices were stimulated in layer 2/3. From the evoked voltage waveforms, we extracted several parameters for comparison AD versus CTRL. Some parameters (e.g., temporal summation, refractoriness, and peak latency) were weak predictors, while other parameters (e.g., signal amplitude, attenuation with distance, and duration (half-width) of the evoked transients) were stronger predictors of the AD condition. Around postnatal age 150 days (P150) and especially at P200, synaptically-evoked voltage signals in brain slices were weaker in the AD groups versus the age- and sex-matched CTRL groups, suggesting an AD-mediated synaptic weakening that coincides with the accumulation of plaques. However, at the youngest ages examined, P40 and P80, the AD groups showed differentially stronger signals, suggesting "hyperexcitability" prior to the formation of plaques. CONCLUSION: Our results indicate bidirectional alterations in cortical physiology in AD model mice; occurring both prior (P40-80), and after (P150-200) the amyloid deposition.

Studying Synaptically Evoked Cortical Responses ex vivo With Combination of a Single Neuron Recording (Whole-Cell) and Population Voltage Imaging (Genetically Encoded Voltage Indicator).

In a typical electrophysiology experiment, synaptic stimulus is delivered in a cortical layer (1-6) and neuronal responses are recorded intracellularly in individual neurons. We recreated this standard electrophysiological paradigm in brain slices of mice expressing genetically encoded voltage indicators (GEVIs). This allowed us to monitor membrane voltages in the target pyramidal neurons (whole-cell), and population voltages in the surrounding neuropil (optical imaging), simultaneously. Pyramidal neurons have complex dendritic trees that span multiple cortical layers. GEVI imaging revealed areas of the brain slice that experienced the strongest depolarization on a specific synaptic stimulus (location and intensity), thus identifying cortical layers that contribute the most afferent activity to the recorded somatic voltage waveform. By combining whole-cell with GEVI imaging, we obtained a crude distribution of activated synaptic afferents in respect to the dendritic tree of a pyramidal cell. Synaptically evoked voltage waves propagating through the cortical neuropil (dendrites and axons) were not static but rather they changed on a millisecond scale. Voltage imaging can identify areas of brain slices in which the neuropil was in a sustained depolarization (plateau), long after the stimulus onset. Upon a barrage of synaptic inputs, a cortical pyramidal neuron experiences: (a) weak temporal summation of evoked voltage transients (EPSPs); and (b) afterhyperpolarization (intracellular recording), which are not represented in the GEVI population imaging signal (optical signal). To explain these findings [(a) and (b)], we used four voltage indicators (ArcLightD, chi-VSFP, Archon1, and di-4-ANEPPS) with different optical sensitivity, optical response speed, labeling strategy, and a target neuron type. All four imaging methods were used in an identical experimental paradigm: layer 1 (L1) synaptic stimulation, to allow direct comparisons. The population voltage signal showed paired-pulse facilitation, caused in part by additional recruitment of new neurons and dendrites. "Synaptic stimulation" delivered in L1 depolarizes almost an entire cortical column to some degree.