Supplemental Material: Annu. Rev. Neurosci. 2021.

44:221-252
https://doi.org/10.1146/annurev-neuro-100520-012117
Neocortical Layer 1: An Elegant Solution to Top-Down and Bottom-Up Integration
Schuman, Dellal, Prönneke, Machold, and Rudy

Supplemental Appendix

Zona incerta and amygdala inputs to layer 1

While the main inputs to neocortical L1 are from higher-order thalamic nuclei,

neocortical areas, and neuromodulatory centers, other subcortical structures have been

shown to innervate L1. Of particular interest are zona incerta, which forms a long-range

GABAergic input to L1 early in development, and the amygdala, which forms a

bidirectional circuit with the PFC and recruits superficial INs. Here, we briefly discuss

these two inputs to L1.

Zona incerta (ZI). ZI is a mysterious region in the subthalamus whose functions,

not fully known, are thought to range from influencing arousal to locomotion (Mitrofanis

2005). ZI not only projects to the neocortex (Divac et al. 1978, Lin et al. 1990, Porter &

White 1983), but largely targets L1, where it is the only known extracortical long-range

GABAergic input (Chen & Kriegstein 2015, Dammerman et al. 2000, Lin et al. 1990,

Shiosaka et al. 1984). Fibers from ZI contact the tuft dendrites of L4-5 PCs, where they

account for the majority of GABAergic input to L1 during the first post-natal week (Chen

& Kriegstein 2015). Furthermore, when ZI axon activity is blocked, PC tuft dendrites fail

to develop normally and have fewer spines (Chen & Kriegstein 2015), suggesting that ZI-

mediated GABAergic input to L1 is important for the morphological, and presumably

synaptic, development of PCs. Their functional role in the adult is unknown, but might be

important since the projections are maintained during adulthood.

Amygdala. The amygdala is a heterogeneous structure comprised of different

nuclei in the temporal lobe; it is involved in the processing of many emotions, but has
been most extensively studied in the context of fear associations (Janak & Tye 2015,

LeDoux 2007). The amygdala forms a bidirectional circuit with the neocortex, receiving

input from L5 and L2/3 cortico-amygdala PCs (Ghashghaei et al. 2007, Janak & Tye

2015). The most studied amygdalo-cortical circuit is the one formed with PFC, although

sensory areas are innervated as well (Amaral & Price 1984, Janak & Tye 2015). The most

common motif for amygdalo-cortical circuits is innervation of neocortical L1/2 and L5/6,

with the highest density near the L1/2 border (Amaral & Price 1984, Bacon et al. 1996,

Ghashghaei et al. 2007, Krettek & Price 1974, Krettek & Price 1977, Porrino et al. 1981).

In PFC, amygdala projections are thought to elicit a primarily inhibitory response (Dilgen

et al. 2013, Perez-Jaranay & Vives 1991) and, indeed, they activate superficial

GABAergic INs, promoting feedforward inhibition (McGarry & Carter 2016). However, the

complete structure of the amygdalo-cortical circuit and the role of L1 are yet to be fully

understood.

Interneuron circuits in neocortical layer 1

To understand how L1 processes its diverse inputs, we must better understand the

output connectivity of the INs that contribute to L1 circuits and the input connectivity of

long-range fibers. While the set of long-range inputs to L1 has been well-studied across

cortical areas in anatomical work (see Part II of this review), only a few relatively recent

papers have explored some of the input connectivity to INs in L1 (Abs et al. 2018,

Anastasiades et al. 2020, Chou et al. 2020, Cruikshank et al. 2012, Ibrahim et al. 2016,

Palmer et al. 2012, Lee et al. 2013). Importantly, as we discussed, the output connectivity

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of most INs in L1 remains unknown. Here, we discuss three IN-mediated L1 circuits that

have been identified.

VIP IN disinhibitory circuit. VIP INs preferentially target SST INs in L2/3 of S1, V1,

A1, and prefrontal cortices, strongly suggesting that VIP IN-mediated disinhibition of PCs

is a common motif in the upper layers of the neocortex (Lee et al. 2013, Pfeffer et al.

2013, Pi et al. 2013; reviewed in Tremblay et al. 2016). Lee et al. (2013), working in the

mouse whisker system, observed that L2/3 VIP INs in S1 received much stronger vM1

feedback than other L2/3 cell types and suggested this disinhibitory circuit is involved in

sensorimotor integration. They predicted, and confirmed in vivo that when M1 PCs are

active, VIP and SST IN activity increases and decreases respectively. It has been

proposed that the VIP disinhibitory circuit mediates several additional feedback actions,

including the effect of cingulate cortex on L2/3 PCs in V1 to influence visual selective

attention, and the effect of locomotion on gain modulation in V1 via nicotinic activation of

VIP INs (see Part III, Layer 1 and neocortical function section in main text). Furthermore,

a VIP disinhibitory circuit was suggested to mediate working memory in prefrontal cortex

(Kamigaki & Dan 2017). Other L1 INs may also mediate disinhibition of PCs. Jiang et al.

(2013) suggested that SBCs, which possibly includes VIP and α7 cells, mainly inhibit L2/3

INs. In addition, Letzkus et al. (2011) reported that unidentified L1 INs (perhaps α7 cells)

inhibited L2/3 PV INs, producing disinhibition of L2/3 PCs (see L1 circuits and learning

section in main text).

Inhibitory circuits. Based on their known connectivity, NGFCs, L2 chandelier cells,

and VIP-CCK cells are good candidates as mediators of L1 feedback-evoked PC

inhibition. An NGFC-mediated feedforward inhibitory circuit has been suggested to

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mediate GABAB-dependent interhemispheric inhibition of L5 PCs (Palmer et al. 2012). A

similar circuit might be involved in the action of some higher-order thalamic nuclei, which

have been shown to activate L1 NGFCs in the medial prefrontal cortex (mPFC) and, in

turn, produce feedforward inhibition of L2/3 PCs (Cruikshank et al. 2012, Anastasiades

et al. 2020).

Martinotti cell-mediated inhibition of NDNF INs. Abs et al. (2018) showed in slice

recordings that the L1 axon of Martinotti type SST INs produces powerful inhibition of L1

NDNF cells. Although this study did not establish whether Martinotti cells inhibited

NGFCs, canopy cells, or both, this circuit may provide a means to control the powerful

inhibition of PC distal dendrites by NGFCs. Interestingly, Abs et al. (2018) observed in

vivo that trains of increasingly intense auditory stimuli elicited progressively stronger

responses from Martinotti cells, as expected given that SST INs are strongly recruited by

the local PC network as described earlier. On the other hand, and consistent with the

connectivity observed in slices, the responses of L1 NDNF INs were successively smaller,

a pattern they showed was due to Martinotti cell-mediated inhibition of the NDNF INs.

Diversity of pyramidal cell dendrites and dendritic integration

Diversity in the morphology and ion channel expression of the dendritic arbor of

PC populations can contribute to how distinct long-range inputs impact specific PC

ensembles. While this area is largely unexplored, emerging evidence suggests the

existence of location- and cell type–specific differences in the active electrical properties

of dendrites. For instance, a recent study showed that dendrites of L5 PCs in the rostral

area of rodent V1 generate Ca2+ channel-dependent plateau potentials, whereas those

4
more caudal do not, perhaps because the apical dendrite is shorter (Fletcher & Williams

2019). Guest, Bast et al. (2019) reported that the primary bifurcation of the apical dendrite

of L5 pyramidal tract neurons in rat S1 varied across neurons, occurring anywhere

between L4 and upper L2. Interestingly, the density of thalamocortical synapses from

VPm peaked around the primary bifurcation. Thus, the location of distal input amplification

by primary sensory thalamus will vary between neurons. These observations suggest that

coupling between top-down and bottom-up information may vary among different PT

neurons. In another interesting example, afferents arising from dLGN, LP, and cortical

feedback are segregated into patches in L1, resulting in patch and interpatch subnetworks

within L2/3 PCs and PV INs (D'Souza et al. 2019, Ji et al. 2015).

Lastly, differences in the processing of distinct inputs by INs and unexplored fine

diversity among IN subgroups may contribute to the specificity of actions elicited by

different inputs. Evidence in support of this notion comes from L5 Martinotti cells, which

have distinct patterns of in vivo activity and axons that specifically target upper or lower

L1, possibly inhibiting specific aspects of dendritic electrogenesis (see The interneurons

of neocortical layer 1 section in main text). In addition, a recent study demonstrated that

GABAergic inputs to PC tufts in L1 are not distributed in a random fashion. Instead, the

ratio of excitatory vs. inhibitory synapses, determined for various dendritic compartments,

differed significantly between PC types (Karimi et al. 2020).

Informational content of corticocortical feedback in Layer 1

As discussed in detail in the main text, there is overwhelming anatomical evidence

that L1 receives feedback input from downstream cortical areas. However, the precise

5
information content of this feedback input is largely unknown. Here, we discuss several

recent studies that explore the type of information contained in feedback fibers in L1 and

the role they may play in sensory perception.

Some of the most studied feedback circuits are those formed from higher-order

visual cortices to V1. For instance, secondary visual cortex (V2) receives input from V1

and provides feedback to V1 in L1, as well as deeper layers. Work in marmosets using

optogenetic inhibition of V2 feedback to V1 suggests that V2 feedback increases

receptive field (RF) size and reduces surround suppression in V1 (Nurminen et al. 2018).

In addition, it has been demonstrated in monkeys that cells in V2 are activated more

strongly by naturalistic images than cells in V1, responses presumably generated at least

in part by integrating information from multiple RFs in V1 (Freeman et al. 2013).

Interestingly, V1 was also shown to receive input activated by the same naturalistic stimuli

at a location (superficial neocortex and deeper layers) and a time (~10ms after activity in

V2) strongly suggesting input of V2 origin (Ziemba et al. 2019). These findings and others

(Chen et al. 2017) are evidence that one function of feedback is to provide more

structured information to lower cortices and perhaps facilitate earlier processing.

Similarly, in the somatosensory system, S1 forms a bidirectional circuit with the

secondary somatosensory cortex (S2), with S2 providing feedback input to L1 and deeper

layers of S1. To explore this feedback, Kwon et al. (2016) used calcium imaging in L1 to

record the activity of S1-projecting feedback axons from S2 during a whisker-touch

detection task. The S1-projecting S2 axons were more modulated by behavioral choice

than the S2-projecting S1 axons, although both S1 and S2 neurons responded to stimuli

and choice. In Minamisawa et al. (2018), the same group uncovered that feedback

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projections from L4 of S2 to L1 of S1 are both somatotopic and enhance tuning curves

for some neurons in S1. Combined, these results suggest that somatomotor cortical

feedback likely refines responses very early in cortical processing and may influence

behavior. Zagha et al. (2013) showed that V1 feedback modulated network states in S1,

which could be another mechanism by which this feedback could regulate sensory

processing.

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