Paolo Medini

Physiology and Plasticity of Cortical Microcircuits in Vivo

The cerebral cortex is the part of our brain where
sensory information is processed so to guide our daily motor behaviour, and where the most complicated cognitive tasks are performed. The brain cortex is composed by morphologically and functionally distinct cell types that connect with each other and with other brain areas in a very precise way. Connectivity in the cortex is highly layer- and cell-type specific, and this is reflected in the different way a sensory (e.g. visual) stimulus is represented in different cell types (Medini, Neuroscience, 2011). This is also reflected in the fact that the different cell types composing cortical circuits reacts differently to lack of a sensory input (experience-dependent plasticity -e.g. in response to closure of one eye in the part of the visual cortex receiving inputs from both eyes –Medini, Journal of Neuroscience, 2011).

Figure 1. Layer 4 Pyramids (L4P) and layer 2/3 pyramids (L2/3P) have different spike responses but similar synaptic responses to a visual stimulus in rat primary visual cortex. Top: in vivo whole-cell recordings from a L2/3P (left) and from a L4P (right) from the rat binocular visual cortex. The neuron was filled with byocitin during the recording and the dendrites have been reconstructed in 3D. Bottom (A): synaptic responses (top traces) and spike counts (bottom histograms) of the two neurons stimulated with an optimally oriented moving light bar. Note the similar amplitude of the synaptic (subthreshold) responses but the higher spike (suprathreshold) response of the L4P. From Medini, Neuroscience, 2011.

In the lab we investigate how sensory information is processed by the different cell types composing cortical microcircuits (e.g. excitatory vs inhibitory; superficial or deep cortical layers) and how these circuits combine different sensory inputs to guide motor behaviour (multisensory integration) in specialized “association” cortices –whose functioning is still scarcely understood. In particular, we focus on the cellular microcircuits responsible for how different sensory inputs (e.g. visual, tactile and auditory) interact already in primary sensory cortices (that were classically thought to process only one sensory modality –Iurilli et al, Neuron, 2012) and how they are integrated in truly multisensory association cortices (Olcese et al, Neuron, 2013). Importantly, we next aim at understanding the synaptic and molecular mechanisms that modify this circuit organization after brain lesions or sensory deprivations (e.g. blindness or deafness). This knowledge is necessary to promote brain repair as in case of lesions, it is not enough to transplant differentiated neuronal cells, but the latter have to integrate or even re-create this precise microcircuit organization. Importantly, there are different forms of lesion-driven plasticity in the brain: those leading to function recovery (which is adaptive) and those leading to maladaptive consequences (e.g. cortical circuits may become hyperexcitable to some extent after cortical lesions up to the point to trigger seizures). Dissecting the molecular and cellular mediators of “good” and “bad” plasticity after lesions is important because, if the mechanisms are different it will be possible to promote targeted cellular and molecular interventions aimed at promoting “good” plasticity on one side and to counteract “bad”, maladaptive plastic changes on the other hand.

Figure 2. Optically- (two-photon)-targeted recordings from inhibitory and excitatory cells in a mouse visuo-tactile cortical area. A. Electrophysiological recordings from parvalbumin-positive cells in the visuo-tactile area RL in mouse cortex. The red fluorescent protein tdTomato is selectively expressed in parvalbumin-positive neurons in transgenic mice and a glass micropipette filled with the green fluorescent indicator Alexa488 is slowly advanced in the living cortical tissue until a physical contact with the red cell is established (iuxtasomal recording). B. Spike responses to a tactile stimulus (whisker movement, blue), to a visual stimulus (flash, red) and the simultaneous presentation of the two stimuli (multimodal stimulation, green). Note the scarce increase of the spike response of the inhibitory cell upon multimodal stimulation. C. Targeted iuxtasomal recordings from excitatory cells are done after ejecting some green Alexa in the extracellular space to visualize and target with the recording pipette the somata of the pyramidal, excitatory cells. D. As in B, but note the marked increase of responsiveness after multimodal stimulation compared to unimodal stimulation. From Olcese et al, Neuron, 2013.

Thus, in extreme summary the two topics of interest in the Lab are:
a) connectivity of cortical microcircuits, at both short-range level (within one cortex) and at long-term level (communication between different cortical areas)
b) synaptic and molecular mechanisms of plasticity of these circuits after lesions/sensory deprivations/lack-of-use
To shy new light onto these still elusive but fascinating phenomena, we use a combination of state of the art techniques to investigate cortical circuits in the intact, living brain. In particular, we use and variably combine in vivo patch clamp recordings (Figure 1) with two-photon calcium or cellular imaging as well as optogenetics. We use genetically modified strains that express fluorescent proteins selectively in excitatory or inhibitory cells so to target electrophysiological recordings to identified cell types (Figure 2), and organic or genetically-encoded calcium indicators to perform population functional imaging with cellular resolution (Figure 3), by means of in vivo two-photon microscopy. To understand the circuit function, we need to manipulate the neuronal activity of identified cell types bidirectionally: to this purpose we use viral vectors as well as transgenic strains to attain cell-type specific expression of molecules that allow activation or inhibition of genetically identified neurons with lights of different wavelengths (optogenetics -Figure 4).

Figure 3. Two-photon population calcium imaging in the visuo-tactile area RL located between the primary somatosensory cortex (S1) and the primary visual cortex (V1). A. Targeting of the calcium indicator injection between S1 and V1. B. Calcium imaging of neurons loaded with the green fluorescent calcium indicator OGB-1. Neurons responding to the sole visual stimulation (V-stim) are labelled in red, those responding to sole tactile stimulation (T-stim) are labelled in blue, and those responding to both are labelled in green (M-stim). C. Example of calcium fluorescence transients driven by the three kind os sensory stimuli. From Olcese et al, Neuron, 2013.

The Lab is in a unique position to fully accomplish these tasks as it has a Physiology Section physically located at the Institute of Medical Biology-Physiology, as well as a Molecular Neurobiology Section at the Molecular Biology Department.

Figure 4. Sound inhibits V1 and this is mimicked by optogenetic stimulation of layer 5 neurons in the auditory cortex (A1). A. Left: Blue laser photostimulation of layer 5 neurons in mice expressing channelrhodopsin selectively in layer 5: the spiking of layer 5 cells was monitored with a juxtasomal electrode (JS). Middle: note the immediate spiking of A1 neurons a few ms after blue laser photostimulation. Right: simultaneous in vivo whole-cell recordings in V1 show that A1 photstimulation evokes a membrane potential hyperpolarization in V1 neurons. B. The upper, black trace shows that both A1 photostimulation and sound presentation (noise burst) hyperpolarize the V1 neuron. The bottom, green and red traces show the excitatory (red) and inhibitory (red) conductances evoked by A1 photostimualtion and sound presentation. Please note that both stimuli evoke an increase of inhibition in the V1 neuron. From Iurilli et al, Neuron, 2012.

Master Thesis Students, PhD and PostDoc applicants are welcome in the Lab!

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