Neural correlates of single-vessel haemodynamic responses in vivo.

Neural activation increases blood flow locally. This vascular signal is used by functional imaging techniques to infer the location and strength of neural activity. However, the precise spatial scale over which neural and vascular signals are correlated is unknown. Furthermore, the relative role of synaptic and spiking activity in driving haemodynamic signals is controversial. Previous studies recorded local field potentials as a measure of synaptic activity together with spiking activity and low-resolution haemodynamic imaging. Here we used two-photon microscopy to measure sensory-evoked responses of individual blood vessels (dilation, blood velocity) while imaging synaptic and spiking activity in the surrounding tissue using fluorescent glutamate and calcium sensors. In cat primary visual cortex, where neurons are clustered by their preference for stimulus orientation, we discovered new maps for excitatory synaptic activity, which were organized similarly to those for spiking activity but were less selective for stimulus orientation and direction. We generated tuning curves for individual vessel responses for the first time and found that parenchymal vessels in cortical layer 2/3 were orientation selective. Neighbouring penetrating arterioles had different orientation preferences. Pial surface arteries in cats, as well as surface arteries and penetrating arterioles in rat visual cortex (where orientation maps do not exist), responded to visual stimuli but had no orientation selectivity. We integrated synaptic or spiking responses around individual parenchymal vessels in cats and established that the vascular and neural responses had the same orientation preference. However, synaptic and spiking responses were more selective than vascular responses--vessels frequently responded robustly to stimuli that evoked little to no neural activity in the surrounding tissue. Thus, local neural and haemodynamic signals were partly decoupled. Together, these results indicate that intrinsic cortical properties, such as propagation of vascular dilation between neighbouring columns, need to be accounted for when decoding haemodynamic signals.

Cortical neurons and networks are dormant but fully responsive during isoelectric brain state.

A continuous isoelectric electroencephalogram reflects an interruption of endogenously-generated activity in cortical networks and systematically results in a complete dissolution of conscious processes. This electro-cerebral inactivity occurs during various brain disorders, including hypothermia, drug intoxication, long-lasting anoxia and brain trauma. It can also be induced in a therapeutic context, following the administration of high doses of barbiturate-derived compounds, to interrupt a hyper-refractory status epilepticus. Although altered sensory responses can be occasionally observed on an isoelectric electroencephalogram, the electrical membrane properties and synaptic responses of individual neurons during this cerebral state remain largely unknown. The aim of the present study was to characterize the intracellular correlates of a barbiturate-induced isoelectric electroencephalogram and to analyse the sensory-evoked synaptic responses that can emerge from a brain deprived of spontaneous electrical activity. We first examined the sensory responsiveness from patients suffering from intractable status epilepticus and treated by administration of thiopental. Multimodal sensory responses could be evoked on the flat electroencephalogram, including visually-evoked potentials that were significantly amplified and delayed, with a high trial-to-trial reproducibility compared to awake healthy subjects. Using an analogous pharmacological procedure to induce prolonged electro-cerebral inactivity in the rat, we could describe its cortical and subcortical intracellular counterparts. Neocortical, hippocampal and thalamo-cortical neurons were all silent during the isoelectric state and displayed a flat membrane potential significantly hyperpolarized compared with spontaneously active control states. Nonetheless, all recorded neurons could fire action potentials in response to intracellularly injected depolarizing current pulses and their specific intrinsic electrophysiological features were preserved. Manipulations of the membrane potential and intracellular injection of chloride in neocortical neurons failed to reveal an augmented synaptic inhibition during the isoelectric condition. Consistent with the sensory responses recorded from comatose patients, large and highly reproducible somatosensory-evoked potentials could be generated on the inactive electrocorticogram in rats. Intracellular recordings revealed that the underlying neocortical pyramidal cells responded to sensory stimuli by complex synaptic potentials able to trigger action potentials. As in patients, sensory responses in the isoelectric state were delayed compared to control responses and exhibited an elevated reliability during repeated stimuli. Our findings demonstrate that during prolonged isoelectric brain state neurons and synaptic networks are dormant rather than excessively inhibited, conserving their intrinsic properties and their ability to integrate and propagate environmental stimuli.

Identifying neuronal correlates of dying and resuscitation in a model of reversible brain anoxia.

We developed a new rodent model of reversible brain anoxia and performed continuous electrocorticographic (ECoG) and intracellular recordings of neocortical neurons to identify in real-time the cellular and network dynamics that successively emerge throughout the dying-to-recovery process. Along with a global decrease in ECoG amplitude, deprivation of oxygen supply resulted in an early surge of beta-gamma activities, accompanied by rhythmic membrane depolarizations and regular firing in pyramidal neurons. ECoG and intracellular signals were then dominated by low-frequency activities which progressively declined towards isoelectric levels. Cortical neurons during the isoelectric state underwent a massive membrane potential depolarizing shift, captured in the ECoG as a large amplitude triphasic wave known as the "wave-of-death" (WoD). This neuronal anoxic depolarization, associated with a block of action potentials and a loss of cell integrative properties, could however be reversed if brain re-oxygenation was rapidly restored (within 2-3.5 min). The subsequent slow repolarization of neocortical neurons resulted in a second identifiable ECoG wave we termed "wave-of-resuscitation" since it inaugurated the progressive regaining of pre-anoxic synaptic and firing activities. These results demonstrate that the WoD is not a biomarker of an irremediable death and unveil the cellular correlates of a novel ECoG wave that may be predictive of a successful recovery. The identification of real-time biomarkers of onset and termination of cell anoxic insult could benefit research on interventional strategies to optimize resuscitation procedures.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo.

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons' integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

The Touch and Zap method for in vivo whole-cell patch recording of intrinsic and visual responses of cortical neurons and glial cells.

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe "Touch and Zap", an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the "Touch". By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or "Zap", as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique.

Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording, and pharmacology.

Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1) hyperpolarization by intracellular current injection, (2) pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3) GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5-10 nA), which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be impractical.

Targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation(1-4), direction(5-7), ocular dominance(8,9) and binocular disparity(9). Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex(10,11). Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined(12). The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines(13) or record activity simultaneously from hundreds of individual neuronal cell bodies(6,14). Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.