Together, these observations DZNeP concentration support the idea that, at least under the conditions of anesthesia, sleep, or perhaps quiet wakefulness (Poulet and Petersen, 2008), activity

that is generated locally in a small cortical area can spread over long distances and recruit large corticothalamic regions into an event that has a unitary character. During a period, lasting for about a second, a large group of neurons throughout most of the cortex and thalamus is coactive during an Up state. On average, the number of neurons that are active during the Up state appears to be largely constant. These observations assign a new meaning to the notion that Up states represent “windows of opportunity” for cortical signaling (Compte et al., 2008), by identifying network Ca2+ waves as stereotypic periods of global corticothalamic recruitment in vivo, during which locally generated neuronal activity is transmitted and computed in large-scale circuits. All experiments were carried out according to

institutional animal welfare guidelines and were approved by the government of Bavaria, Germany. Adult C57/Bl6 mice were anesthetized with an intraperitoneal bolus injection of a mixture of ketamine and xylazine and placed PD0325901 chemical structure in a stereotaxic frame. Above the primary visual cortex (V1), a craniotomy was made 3.8 mm posterior to bregma and 2.0 mm lateral to the midline. Viral constructs were delivered through a small durotomy by a glass micropipette with an outer tip diameter of 45 μm and an inner diameter of 15 μm. The micropipette was slowly inserted 600 μm below the

pia for targeting of cortical layer 5 and 100 μm for targeting of layer 2/3. Two adeno-associated virus (AAV) preparations, serotype 2, were mixed at a ratio of 1:4: AAV-CAG-Cre and AAV-EF1A-DIO-hChR2(H134R)-mCherry. We injected 350 nl of the viral solution into V1 at a rate of 0.1 μl/min (Cardin et al., 2009). After the injection, the pipette was held in place for 5 min before slowly retracting it from the brain. The scalp incision was closed with tissue adhesive (Vetbond, 3M Animal Care Products), and postinjection analgesics were given to aid recovery. Optical recordings were carried out after a minimum of 10 days after MTMR9 viral construct injection. For characterization of ChR2 expression, animals were perfused transcardially with 4% PFA 10 days postinjection and the brains were postfixed for 24 hr. We cut 70- to 80-μm-thick sections with a vibratome (Leica), stored them in PBS, and mounted them in Vectashield (Vector Laboratories) containing media for confocal imaging. Tissue sections were analyzed with an Olympus Fluoview confocal microscope (FV 1000) equipped with 20× (oil) and 10× objectives (UPlanSAPO, Olympus), with numerical apertures of 0.85 and 0.4, respectively. A custom-built set-up was used for combined optical fiber-based optogenetic stimulation and neuronal Ca2+ recordings (Figure 1A).