Federico Esposti

Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

Spikes in Retinal Bipolar Cells Phase-Lock to Visual Stimuli with Millisecond Precision

Summary Background The conversion of an analog stimulus into the digital form of spikes is a fundamental step in encoding sensory information. Here, we investigate this transformation in the visual system of fish by in vivo calcium imaging and electrophysiology of retinal bipolar cells, which have been assumed to be purely graded neurons. Results Synapses of all major classes of retinal bipolar cell encode visual information by using a combination of spikes and graded signals. Spikes are triggered within the synaptic terminal and, although sparse, phase-lock to a stimulus with a jitter as low as 2–3 ms. Spikes in bipolar cells encode a visual stimulus less reliably than spikes in ganglion cells but with similar temporal precision. The spike-generating mechanism does not alter the temporal filtering of a stimulus compared with the generator potential. The amplitude of the graded component of the presynaptic calcium signal can vary in time, and small fluctuations in resting membrane potential alter spike frequency and even switch spiking on and off. Conclusions In the retina of fish, the millisecond precision of spike coding begins in the synaptic terminal of bipolar cells. This neural compartment regulates the frequency of digital signals transmitted to the inner retina as well as the strength of graded signals.

Long-term invariant parameters obtained from 24-h Holter recordings: A comparison between different analysis techniques

Over the last two decades, a large number of different methods had been used to study the fractal-like behavior of the heart rate variability (HRV). In this paper some of the most used techniques were reviewed. In particular, the focus is set on those methods which characterize the long memory behavior of time series (in particular, periodogram, detrended fluctuation analysis, rescale range analysis, scaled window variance, Higuchi dimension, wavelet-transform modulus maxima, and generalized structure functions). The performances of the different techniques were tested on simulated self-similar noises (fBm and fGn) for values of alpha, the slope of the spectral density for very small frequency, ranging from -1 to 3 with a 0.05 step. The check was performed using the scaling relationships between the various indices. DFA and periodogram showed the smallest mean square error from the expected values in the range of interest for HRV. Building on the results obtained from these tests, the effective ability of the different methods in discriminating different populations of patients from RR series derived from Holter recordings, was assessed. To this extent, the Noltisalis database was used. It consists of a set of 30, 24-h Holter recordings collected from healthy subjects, patients suffering from congestive heart failure, and heart transplanted patients. All the methods, with the exception at most of rescale range analysis, were almost equivalent in distinguish between the three groups of patients. Finally, the scaling relationships, valid for fBm and fGn, when empirically used on HRV series, also approximately held.

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity

Summary Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. Video Abstract

Olfactory stimulation selectively modulates the OFF pathway in the retina of zebrafish.

Summary Cross-modal regulation of visual performance by olfactory stimuli begins in the retina, where dopaminergic interneurons receive projections from the olfactory bulb. However, we do not understand how olfactory stimuli alter the processing of visual signals within the retina. We investigated this question by in vivo imaging activity in transgenic zebrafish expressing SyGCaMP2 in bipolar cell terminals and GCaMP3.5 in ganglion cells. The food-related amino acid methionine reduced the gain and increased sensitivity of responses to luminance and contrast transmitted through OFF bipolar cells but not ON. The effects of olfactory stimulus were blocked by inhibiting dopamine uptake and release. Activation of dopamine receptors increased the gain of synaptic transmission in vivo and potentiated synaptic calcium currents in isolated bipolar cells. These results indicate that olfactory stimuli alter the sensitivity of the retina through the dopaminergic regulation of presynaptic calcium channels that control the gain of synaptic transmission through OFF bipolar cells.

A Synaptic Mechanism for Temporal Filtering of Visual Signals

Synaptic volume matters! The size of the presynaptic compartment of retinal bipolar cells controls the amplitude, speed, and adaptation of synaptic transmission.

Statistical Long-Term Correlations in Dissociated Cortical Neuron Recordings

The study of nonlinear long-term correlations in neuronal signals is a central topic for advanced neural signal processing. In particular, the existence of long-term correlations in neural signals recorded via multielectrode array (MEA) could provide interesting information about changes in interneuron communications. In this study we propose a new method for long-term correlation analysis of neuronal burst activity based on the periodogram alpha slope estimation of the MEA signal. We applied our method to recordings taken from cultured networks of dissociated rat cortical neurons. We show the effectiveness of the method in analyzing the activity changes as well as the temporal dynamics that take place during the development of such cultures. Results demonstrate that the alpha parameter is able to divide the network development in three well-defined stages, showing pronounced variations in the long-term correlation among bursts.

Rapid mapping of visual receptive fields by filtered back projection: application to multi‐neuronal electrophysiology and imaging

To understand vision, we must measure the spatio‐temporal receptive field of neurons in the visual system. We describe how the filtered back projection can be used to map the receptive fields of many neurons simultaneously, within a few minutes. This method can also reveal complex features of visual receptive fields such as the tuning of orientation selective neurons and the contributions from separate ON and OFF components. We demonstrate that the filtered back projection is suited to mapping receptive fields from populations of neurons recorded with imaging or electrophysiology and should therefore prove useful for investigations of visual processing throughout the visual pathway.

How do TTX and AP5 affect the post-recovery neuronal network activity synchronization?

A lot of methods were created in last decade for the spatio-temporal analysis of multi-electrode array (MEA) neuronal data sets. The greater part of these methods does not consider the network as a whole but performs an analysis channel by channel. In this paper we illustrate how a very simple approach that considers the total network activity, is able to show interesting neuronal network features. In particular we perform two different analyses: a connectivity examination studying networks at different days in vitro and an analysis of the long period effects of the administration of two common neuro-active drugs, i.e. TTX and AP5. Our analysis is performed considering burst topology, i.e. cataloguing network bursts as Global (if they involve more than the 25% of the MEA channels) or Local (if less that 25%). This division allows, in the first analysis, to understand the network connectivity (increasing from div 1 to 6) and decreasing till reaching a plateau (from div 6 to 10). The second analysis highlights a substantial difference between the long period effects of TTX and AP5. While TTX induces a massive Global activity explosion, sign of a prolonged inhibitory synapse depression, AP5 shows only a modest Local activity increase, mark of the low effect of NMDA receptors on a mature neuronal network without inputs.

The estimation of long-term memory characteristics in MEA neuronal culture recordings

The nonlinear analysis of multichannel MEA recordings from neuronal networks is becoming a central topic in Neuroengineering. Up-to-date these kind of analyses required complex ad hoc methods. In this paper we introduce a new approach that allows the analysis of the whole-neuronal-network-activity with well-established nonlinear signal processing methods. In particular, we show here the estimation of long-term-memory behaviors through the Periodogram method in the bursting activity of cortical neuron cultures during development. Moreover, we show how this method is able to highlight structural activity changes of the network.