Thalamocortical And Corticocortial Innervation Pattern In Mouse Cortex:Laminar And Cell-Type Specificity

1.Thalamocortical Innervation Pattern in Mouse Auditory Cortex:Laminar and Cell-Type Specificity.In mammals, sensory information is relayed by the thalamus into the primary sensory cortex of the corresponding modality. In the cortex, it is generally proposed that information is processed in a serial, vertical manner across cortical laminae (Callaway,1998; Douglas and Martin,2004). L4 is the major thalamorecipient layer, and relays thalamic signals to superficial layers (L2/3) before the signals are further transmitted to deep layers (L5/6). This hierarchical model is supported by anatomical evidence that axons of L4 principal neurons primarily innervate L2/3 and that axons of L2/3 pyramidal neurons arborize extensively in L5/6 (Gilbert et al., 1979; Feldmeyer,2012). However, recent quantitative measurements of reconstructed thalamocortical axons suggest that innervation of L5/6 may also be extensive (Oberlaender et al.,2012), and indeed in vivo whole-cell recordings in the barrel and primary auditory cortex have demonstrated that neurons in L5/6 can also receive direct thalamic inputs (Zhou et al.,2010; Constantinople et al.,2013; Sun et al.,2013). These recent data suggest that the thalamic innervation of sensory cortical neurons may be more extensive than previously thought.Thalamic inputs to L4 neurons have been extensively studied in vitro(Cruikshank et al.,2002; Gabernet et al.,2005; Inoue and Imoto,2006; Sun et al., 2006; Barkat et al.,2011) and in vivo (Chung and Ferster,1998; Liu et al.,2007; Li et al.,2013; Lien and Scanziani,2013). In those studies, excitatory and inhibitory neurons in this thalamorecipient layer were most often distinguished by their different firing patterns to current injections. Only recently, genetically identified inhibitory neurons are beginning to be examined (Tan et al.,2008; Schiff and Reyes,2012). In view of the limited results from those previous studies, a comprehensive characterization of thalamic innervation of cortical neurons across laminae and of identified cells types is necessary for a deeper understanding of thalamocortical transformation.Conventionally, thalamocortical responses were studied in thalamocortical slice preparations. The thalamus (or thalamocortical axon tracts) has been activated electrically, and the resulting cortical responses have been recorded with electrophysiological or optical techniques. A concern over electrical stimulation is that it may activate non-targeted neurons and axonal/dendritic processes near the stimulation electrodes. Recently, optogenetic techniques have been introduced in studies of long-range axonal projections (Petreanu et al.,2007; Petreanu et al.,2009; Cruishank et al.,2010). By expressing channelrhodopsin 2 (ChR2), a light-sensitive cation channel (Nagel et al.,2003), mammalian neurons could be excited by blue light with high temporal precision (Boyden et al.,2005; Cardin et al.,2009; Gradinaru et al.,2009). More importantly, enough ChR2 could be expressed in the axons and terminals of those neurons that the terminal arbors themselves could be directly excited by light, triggering transmitter release without the need for illumination of parent somata (Petreanu et al.,2007). Therefore, with optogenetics, long-range axonal projections can be examined in a more specific manner. In this study, by expressing ChR2 in the thalamus in various cell-type specific mouse lines, we examined thalamocortical innervation patterns of different cortical cell classes across laminae.Here, in primary auditory cortical slices, by optogenetically stimulating axons from the medial geniculate body, we found that excitatory cells and parvalbumin (PV)-expressing inhibitory neurons across L2/3 to L6 are broadly innervated by thalamic projections with a similar laminar pattern and the strongest innervation occurring in L4. The innervation of PV neurons is stronger than excitatory neurons in the same layer, but a relatively constant ratio between their innervation strengths was observed across layers. For somatostatin (SOM) and vasoactive intestinal peptide (VIP) inhibitory neurons, essentially only a subset of L4 neurons were weakly innervated by thalamic axons. In addition, about half of inhibitory neurons in L1 were innervated, relatively strongly, by thalamic axons. Similar innervation patterns were also observed in the primary visual cortex. Thus, thalamic information can be processed independently by different cortical layers in parallel, which is likely shaped by feedforward inhibition from PV neurons in each lamina. Such parallel processing may largely facilitate the hierarchical processing.2. Intracortical Innervation Pattern in Mouse Auditory Cortex:Laminar and Cell-Type Specificity.There are several types of inhibitory neurons in the cerebral cortex.We could classify them according to their morphology, molecular marker, electrophysiological properties and projection pattern. There are three major types:PV, SOM and VIP.PV interneurons account for 40% of the total inhibitory neurons, including the basket and chandelier cells. PV interneurons have short spike width, high firing rate, fast hyperpolarization, low input resistance, showing fast spiking mode. It is believed to provide major inhibitory input in local circuits. In cortex circuit, PV interneurons mainly inhibitory other PV inhibitory neurons, and also innervate local excitatory neurons.SOM interneurons are located from L2/3 to L6 in the cortex, and the density increased with the cortical depth. It has regular spiking, low input resistance, and relative fast hyperpolarization. SOM interneurons serve as "switch" in mice somatosensory cortex. When the mice are quiet, SOM silently innervate distal dendrite of local L2/3 pyramidal cell. When the mice starting moving, it starts giving inhibitory input to the excitatory neurons. Thus, SOM interneurons have important role in cortical information processing.VIP interneurons locate mostly in L2/3, and they have varied firing patterns, such as regular or bust firing, and high excitability. VIP interneurons modulate various GABAergic interneurons via di-synaptic inhibition in local circuits. It innervate SOM cells preferentially.Before wide application of optogenetics, researchers usually speculate the neuronal network connectivity from in vivo experiments without direct evidence. Recently, with the wide use of optogenetics and transgenic animals, the research about neuronal network connectivity became more and more accurate. However, people would like to study more on the excitatory source of inhibitory neurons or the connection among them without enough study on output of different inhibitory neurons to excitatory neurons in the auditory cortex. As we know, this study is thefirst one to report the subtypes and laminar distribution of different inhibitory neuron in the auditory local circuits.In this study, we injected virus to different transgenic mice to express ChR2 in different interneurons to study the laminar specific and cell-type selectivity in local circuits. cally to stimulate inhibitory neurons in different layers in auditory cortex, and record the IPSC of excitatory neurons. The result shows that PV mainly innervate pyramidal cell in the same layer and has the least projection range. In comparison, SOM and VIP neurons have wide interlaminar input. Among all the interlaminar inputs, excitatory neurons in L4 and L5 receive the most wide inhibitory inputs. ThisWe used the whole-cell slice recording combined with ChR2-EYFP expression in PV, SOM and VIP inhibitory, by moving the blue light with 80 um diameter verti provides us a new information that L5 not only takes part in feedback but also in integration of intracortical signal processing. By comparing the IPSC amplitude of different inhibitory neurons, we also found inhibitory neurons provide strongest input to excitatory neurons in the same layers, which is consistent with previous reports. By moving blue light horizontally to stimulate excitatory neurons with different distance from the inhibitory neurons, we found PV neurons provide the shortest inhibitory input to excitatory neurons in the same layer. Neurons do not receive any IPSC from PV neurons 100um away. While SOM neurons provide the mose wide inhibitory input. Even neurons 300um away could receive input from SOM neurons. VIP projection range is up to 200um. Compared with SOM and VIP neurons, PV provide strongest inhibitory inputs, which is 1.5 times the SOM input and 3 times the VIP inputs.Taken together with the result from part one, we propose that PV,SOM and VIP have significant difference in modulating sensory information processing.PV interneurons, as the major receiver of thalamocortical input, are mainly involved long range sensory information processing.While, within the cortex, PV provided very little intralaminar or interlaminar inhibitory output, which demonstrated that PV has weak role in cortical processing.In comparison, SOM and VIP almost have no effect in long range integration of information. But within auditory cortex, both have strong interlaminar output and have longer range of output than PV within the same layer. This suggested that PV and SOM inhibitory interneurons mainly took part in the intracortical intergration of information. In addition, compared with PV interneurons, SOM and VIP provided weaker inhibition, and we think this kind of weak output is beneficial for the accurate modulation of the intracortcial sensory information.Base on the results above,we draw the follow conclusion:1. In the auditory cortex, PV neurons have small interlaminar projection range, and only provide inhibition to the excitatory pyramidal neurons in the same layer. The amplitude of PV output is very strong, which is 1.5 times the SOM input and 3 times the VIP inputs.2. SOM and VIP interneurons have wide projection range in Al. L4 and L5 excitatory neurons receive the most interlaminar inhibition.3. Within the same layers, PV provided shortest range of inhibition, as far as 100um.SOM provided the widest range of inhibition, as far as 300um; VIP neurons has intermediate projection range, as far as 200um.4. PV neurons take part in more in the long range sensory information input, and exert little effect in intracortical integration of information. But it has strongest inhibitory output. In contrary to PV, SOM and VIP neurons mainly take part in the intracortcial processing of auditory information,which has relatively weak inhibitory output, and wide interlaminar projection.This suggested that SOM and VIP involve very little in long range integration of sensory information.