Riccardo Beltramo

Layer-specific excitatory circuits differentially control recurrent network dynamics in the neocortex

In the absence of external stimuli, the mammalian neocortex shows intrinsic network oscillations. These dynamics are characterized by translaminar assemblies of neurons whose activity synchronizes rhythmically in space and time. How different cortical layers influence the formation of these spontaneous cellular assemblies is poorly understood. We found that excitatory neurons in supragranular and infragranular layers have distinct roles in the regulation of intrinsic low-frequency oscillations in mice in vivo. Optogenetic activation of infragranular neurons generated network activity that resembled spontaneous events, whereas photoinhibition of these same neurons substantially attenuated slow ongoing dynamics. In contrast, light activation and inhibition of supragranular cells had modest effects on spontaneous slow activity. This study represents, to the best of our knowledge, the first causal demonstration that excitatory circuits located in distinct cortical layers differentially control spontaneous low-frequency dynamics.

An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo.

Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source.

Integrated brain circuits: neuron-astrocyte interaction in sleep-related rhythmogenesis.

Although astrocytes are increasingly recognized as important modulators of neuronal excitability and information transfer at the synapse, whether these cells regulate neuronal network activity has only recently started to be investigated. In this article, we highlight the role of astrocytes in the modulation of circuit function with particular focus on sleep-related rhythmogenesis. We discuss recent data showing that these glial cells regulate slow oscillations, a specific thalamocortical activity that characterizes non-REM sleep, and sleep-associated behaviors. Based on these findings, we predict that our understanding of the genesis and tuning of thalamocortical rhythms will necessarily go through an integrated view of brain circuits in which non-neuronal cells can play important neuromodulatory roles.

Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence

Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.

Two-Photon Calcium Imaging in the Intact Brain

The calcium ion is a fundamental second messenger that plays crucial roles in the pathophysiology of brain cells. In this chapter, we will focus on the measurement of calcium fluctuations as a reporter of cellular excitability of both neurons and glial cells in the intact central nervous system. We will first describe the methodological aspects of in vivo two-photon fluorescence calcium imaging and then review recent data highlighting the ways in which this technique is revolutionizing our understanding of brain circuits at the cellular level. Finally, we will discuss recent technical advancements that promise to open new horizons in the optical investigation of brain function in awake, behaving animals.

Characterisation of white vinegars of different sources with artificial neural networks

Wine and cider vinegars currently attract growing interest from consumers, giving rise to a consequent increase in supply. A full appreciation of their quality is only possible, however, through recognition of their superior quality when compared with vinegars produced from raw materials of inferior quality. Therefore, it is necessary to pinpoint the parameters that define the quality of these products. Chemico-physical and sensory analysis has been used to draw up artificial neural networks (ANNs), on the basis of a vast sampling of vinegars from various countries, produced from a variety of raw materials, that was already subjected to multivariate statistical analysis. Among the chemical parameters, polyalcohols and other elements such as pH, tartaric acid and proline proved to be highly reliable, whereas other volatile substances and the results of sensory analysis were not very discriminating and could not be used to re-classify samples of unknown origin. The positive results obtained indicate that ANNs are a powerful mathematical tool, since they can be used to construct models that predict the botanical origin of the product and to re-classify samples of unknown origin, without any initial restrictive hypothesis.

Optical Investigation of Brain Networks Using Structured Illumination

In microscopy, the projection of complex light patterns, also called structured illumination, is instrumental for many important applications. In this chapter, we first introduce some of the most common applications of structured light in neuroscience and survey different techniques used to generate patterned illumination. Then we discuss methods for creating structured light based on phase modulation by means of liquid crystal on silicon spatial light modulators (SLMs). We provide a detailed description of the optical setup, and finally, we describe current applications of this technique in the investigation of brain microcircuits.

Correlating Anatomy and Function with Gene Expression in Individual Neurons by Combining in Vivo Labeling, Patch Clamp, and Single Cell RNA-seq

The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation. Here we describe a method to link the gene expression of individual neurons with their position, axonal projection, or sensory response properties. Neurons are labeled in vivo based on their anatomical or functional properties and, using patch clamp pipettes, their RNA individually harvested in vitro for RNAseq. We validate the methodology using multiple established molecularly and anatomically distinct cell populations and explore molecular differences between uncharacterized neurons in mouse visual cortex. Gene expression patterns between L5 neurons projecting to frontal or contralateral cortex are distinct while L2 neurons differing in position, projection, or function are molecularly similar. With this method we can determine the genetic expression pattern of functionally and anatomically identified individual neurons.