, 2009), and thus should generate a measurable Ca Ibrutinib cost transient in the postsynaptic compartment (Goldberg et al., 2003b). We recorded from fast-spiking interneurons (see Experimental Procedures) with patch pipettes containing the fluorescent Ca indicator Oregon Green BAPTA-1 (150 μM) in a slice preparation that preserves much of the thalamocortical fiber bundle (Agmon and Connors,

1991 and Porter et al., 2001), allowing a stimulation electrode to be placed in this pathway slightly ventral to the fimbria. We simultaneously imaged the dendritic arbor of the recorded neuron (Figure 1D) and recorded the electrophysiological response in voltage clamp to stimulation of thalamic afferents. We used three stimulation protocols: (1) bulk stimulation, in which multiple thalamic afferents are recruited; (2) threshold single fiber stimulation, in which a single afferent impinging on the recorded neuron or imaged dendrite is stimulated just at threshold, leading PI3K inhibitor to fluctuation between recruitment successes and failures; and (3) single fiber stimulation, in which a single fiber impinging on the recorded neuron or imaged dendrite is recruited reliably,

without failures, by the stimulation electrode (see Experimental Procedures). Bulk stimulation of thalamic afferents elicited a pattern of Ca hotspots—localized, transient postsynaptic increases in Ca concentration—decorating the dendritic arbor of cortical interneurons (Figure 1E). Addition of the AMPA-R antagonist NBQX reduced hotspot intensity by 58% ± 5% (n = 5), while further addition of the NMDA-R antagonist R-CPP eliminated hotspots entirely (to −1% ± 1%, n = 3; Figure 1F). Similarly, application of R-CPP reduced hotspot intensity by 59% ± 4% (n = 5; see Figure S1 available online), suggesting that Ca-permeable see more AMPA-R and NMDA-R contribute about equally to the postsynaptic Ca signal under our recording conditions. Do hotspots mark the location

of a synaptic contact? In the absence of perfect voltage clamp, hotspots could in principle result from the activation of voltage-gated Ca channels (VGCCs) not necessarily colocalized with the synaptic contact. To test this possibility, we monitored the spatial distribution of Ca transients in response to depolarizing voltage steps (Goldberg et al., 2003b). The resulting Ca transient was spread throughout the dendritic arbor (Figure 1G), indicating that VGCCs are distributed broadly and therefore unlikely to produce hotspots at significant distance from the site of synaptic contact. Thus, thalamic stimulation generates a spatial pattern of Ca transients that corresponds to the location of glutamate receptor-mediated thalamic inputs. To ascertain whether an individual hotspot corresponded to the input of a single thalamic fiber, we used a threshold single fiber stimulation paradigm.