Chrna5 and lynx prototoxins identify acetylcholine super-responder subplate neurons

Summary Attention depends on cholinergic excitation of prefrontal neurons but is sensitive to perturbation of α5-containing nicotinic receptors encoded by Chrna5. However, Chrna5-expressing (Chrna5+) neurons remain enigmatic, despite their potential as a target to improve attention. Here, we generate complex transgenic mice to probe Chrna5+ neurons and their sensitivity to endogenous acetylcholine. Through opto-physiological experiments, we discover that Chrna5+ neurons contain a distinct population of acetylcholine super-responders. Leveraging single-cell transcriptomics, we discover molecular markers conferring subplate identity on this subset. We determine that Chrna5+ super-responders express a unique complement of GPI-anchored lynx prototoxin genes (Lypd1, Ly6g6e, and Lypd6b), predicting distinct nicotinic receptor regulation. To manipulate lynx regulation of endogenous nicotinic responses, we developed a pharmacological strategy guided by transcriptomic predictions. Overall, we reveal Chrna5-Cre mice as a transgenic tool to target the diversity of subplate neurons in adulthood, yielding new molecular strategies to manipulate their cholinergic activation relevant to attention disorders.


INTRODUCTION
Cholinergic modulation of the medial prefrontal cortex (mPFC) is essential for attention and detection of sensory cues. [1][2][3][4] Deep-layer pyramidal neurons in the prefrontal cortex (PFC) are critically involved in such executive function [5][6][7] and are robustly excited by acetylcholine, 8,9 via nicotinic and muscarinic receptor activation. 10 The a5 nicotinic receptor subunit encoded by Chrna5 is specifically expressed in deep-layer pyramidal neurons, 11,12 forming high-affinity nicotinic receptors in combination with a4 and b2 subunits. Electrophysiological, behavioral, and genetic evidence in both rodents and humans points to an important role of Chrna5 expression for nicotinic receptor function, attention, and executive function.
Constitutive deletion of Chrna5 in mice or knockdown in the adult rat PFC disrupts attention and reduces nicotinic receptor activation by exogenous acetylcholine stimulation in layer 6 neurons. [13][14][15] Optogenetic experiments in Chrna5 À/À mice show that Chrna5 is required for rapid onset of postsynaptic cholinergic activation and prevents desensitization of the endogenous cholinergic response during prolonged stimulation. 16 In humans, the non-synonymous rs16969968 (D398N) polymorphism in Chrna5 is associated with nicotine dependence, schizophrenia, and cognitive impairment. [17][18][19] Nicotinic a4b2a5 receptors with the D398N polymorphism have partial loss of function, attributed to changes in receptor desensitization, calcium permeability, or membrane trafficking. [20][21][22] Despite a5 nicotinic receptor involvement in attention and prefrontal cholinergic activation, systematic characterization of Chrna5-expressing (Chrna5+) neurons using modern genetic tools is lacking. Deeplayer neurons include diverse corticothalamic (L6CT), corticocortical, and layer 6b (L6b) populations. [23][24][25] Chrna5 is predicted to be expressed in L6CT neurons, 8,26 which are usually identified by their expression of Syt6, a conserved L6CT neuronal marker. Syt6-Cre mice have been widely used to characterize prefrontal L6CT neurons and their cholinergic properties. [27][28][29] However, it is unclear whether these are the same neurons expressing Chrna5. Characterization of Chrna5+ neurons has been limited by the lack of verified antibodies for the a5 subunit that could be used for post hoc immunostaining. Previous bacterial artificial chromosome (BAC)-transgenic mice labeling Chrna5+ neurons had altered expression of other genes in   (Table S1). In addition, we determined that Chrna5+ neurons are under presynaptic muscarinic autoinhibitory control, which can be relieved by atropine, resulting in even larger responses (post-atropine response: 194 G 42% of baseline, n = 6 cells). Our application of the nicotinic antagonist dihydro-b-erythroidine (DHBE) eliminated the optogenetic cholinergic response (post-DHBE response: 3 G 2% of baseline, n = 4 cells), indicating the relevant nicotinic receptors contain b2 subunits. This characterization of Chrna5+ neurons revealed them to be acetylcholine ''super-responders'' with stronger and faster onset cholinergic responses distinct from other deep-layer pyramidal neurons. We next examined whether Chrna5+ neurons in other cortical regions are also acetylcholine super-responders. Chrna5+-labeled neurons deep in layer 6 of primary somatosensory cortex (SSp) were also found to show significantly stronger and faster onset nicotinic responses to optogenetic acetylcholine release ( Figure S2) confirming that Chrna5-labeling identifies acetylcholine super-responders in multiple cortical areas.
To further probe the distinct cholinergic responses of prefrontal Chrna5+ neurons, we examined factors differentiating these cells from neurons labeled by Syt6 expression (Syt6+), which has hereto been the predominant marker of layer 6 corticothalamic neurons used to characterize their function during PFC-dependent tasks. 27,28,42,43 Because the extent of overlap between Chrna5+ and Syt6+ deep-layer pyramidal neuron populations was unclear, we adopted an imaging strategy to visualize the distribution of Chrna5+ and Syt6+ neurons in the PFC and determine the exact proportion of distinctive nonoverlapping Chrna5+ neurons. We generated a compound transgenic Chrna5-Cre /+ Ai14 /+ Syt6-EGFP /+ mouse to simultaneously express tdTomato in Chrna5+ neurons and EGFP in Syt6+ neurons and performed confocal and twophoton imaging of the endogenous fluorescence of these reporters in mPFC brain slices. Chrna5+ and Syt6+ neurons were both present primarily in layer 6, with a few Chrna5+ neurons in layer 5 ( Figures 1G  and 1I). Figure 1J (left) shows the proportion of all cells expressing tdTomato (Chrna5), GFP (Syt6), or both, as a percentage of the total number of fluorescent cells in that region. Closer investigation confirmed the existence of a substantial proportion of exclusively Chrna5+ neurons (37% of all labeled cells) which do not express Syt6, in addition to overlapping Chrna5+Syt6+ neurons (39%) which express both markers, and exclusively Syt6+ neurons (24%) which do not express Chrna5 (N = 4 mice, Figures 1H and 1J). Thus, nearly half of all Chrna5+ neurons are not labeled by Syt6 expression and would have been excluded in previous studies using Syt6-Cre mice. Furthermore, Chrna5+ neurons are found even in primary visual cortex, where Syt6 labeling is greatly reduced 28,44 ( Figure S3).
To address the hypothesis of higher-affinity nicotinic binding and its consequences for spiking in these -neurons, we switched to whole-cell recording in individual Chrna5+ and Syt6+ neurons. We recorded current clamp responses to acetylcholine (1 mM, 15 s) in labeled Chrna5+ and Syt6+ neurons from Chrna5-Cre /+ Ai14 /+ and Syt6-Cre /+ Ai14 /+ or Syt6-EGFP mice, respectively ( Figure 2E). Chrna5+ neurons showed stronger acetylcholine-evoked firing, attaining significantly higher peak firing frequency (29 G 6 Hz, n = 12 cells, 6 mice; t (24) = 2.74, p = 0.01, unpaired t test; Cohen's d: 1.08) compared to Syt6+ neurons (13 G 2 Hz, n = 14 cells, 5 mice; Figure 2E). The intrinsic electrophysiological properties of Chrna5+ and Syt6+ neurons did not show statistically significant differences (Table S2). We next examined the sensitivity of acetylcholine-evoked firing to competitive nicotinic receptor block by DHBE in the presence of atropine. Acetylcholine-evoked firing was completely eliminated in all Syt6+ neurons ( Figure 2F) whereas a large subset of Chrna5+ neurons (7/11) retained their ability to respond to acetylcholine (average peak firing rate in Chrna5+ neurons: 6 G 2 Hz; t (19) = 3.22, p = 0.004, unpaired t test) demonstrating similar resilience to competitive nicotinic receptor block as observed with Ca 2+ imaging. We used the noncompetitive nicotinic receptor blocker mecamylamine 45,46 to test our hypothesis that nicotinic receptors in this Chrna5+ subset were higher affinity and therefore allowed exogenous acetylcholine to outcompete DHBE. The addition of 5 mM mecamylamine was sufficient to eliminate acetylcholine-evoked firing in all the Chrna5+ neurons that were resilient to competitive nicotinic block (t (4) = 5.14, p = 0.007, paired t test; Figure 2F). Together, our Ca 2+ imaging and electrophysiology experiments revealed the existence of a distinct subset of Chrna5+ neurons dissimilar to Syt6+ neurons, with high-affinity nicotinic responses resilient to competitive nicotinic antagonism.

Single-cell transcriptomics identifies Chrna5+ subplate neurons with lynx genes
To determine the molecular identity of distinct Chrna5+ neurons with enhanced cholinergic responses, we pursued single-cell RNA-seq. We extracted gene expression data of L5-6 glutamatergic neurons (n = 2422 cells) in the mouse anterior cingulate cortex from the Allen Institute single-cell RNA-seq databases. [47][48][49] We classified these deep-layer pyramidal neurons into 3 groups: those expressing only Chrna5 (Chrna5+,  16,50 We focused on the Chrna5+, Syt6+, and Chrna5+Syt6+ groups to examine their transcriptomic differences. Prior work has shown that deep cortical neurons can be separated into L6b, L5, and L6CT subclasses by the expression of characteristic markers and hierarchical clustering. 49 Single-cell analysis revealed that the Chrna5+ group primarily included L6b (44%), L5 near-projecting (L5NP, 19%), and L6CT neurons (30%), whereas the Chrna5+Syt6+ and Syt6+ groups were predominantly composed of L6CT neurons (>90%) ( Figure 3B). We further examined the expression of marker genes in these respective groups to validate our cell classification. Chrna5+ neurons showed distinctive expression of several marker genes, Ctgf, Cplx3, Kcnab1, and Lpar1 ( Figures 3B and 3C) associated with subplate neurons. 32,51 Subplate neurons are early born and vital for brain development, leaving L6b neurons as descendants in adulthood. 33,34,52 Notably, the highest fold enrichment among all differentially expressed genes in Chrna5+ neurons was found for subplate markers Ctgf (fold change, 5.69) and Cplx3 (3.81) ( Table 1). All differentially expressed genes are listed in Table S4. Overall, Chrna5+ neurons including both L5NP and L6b subpopulations highly express subplate-marker genes. In contrast, Syt6-expressing Chrna5+Syt6+ and Syt6+ groups are only enriched in the corticothalamic markers Foxp2 and Syt6, consistent with their corticothalamic subtype. These results support our imaging, electrophysiological, and pharmacological results suggesting the exclusive Chrna5+ population is a distinct cell type from typical L6CT Syt6+ neurons.
To identify molecular changes predictive of Chrna5+ nicotinic ''super-responders'', we examined differential expression of genes that exert effects on postsynaptic cholinergic responses ( Figures 3D and 3E). We selected cholinergic receptor genes (nicotinic Chrna5-2, Chrna7, Chrnb2-4, and muscarinic subunits Chrm1-4), acetylcholinesterase (Ache), and members of the family of genes that encode lynx proteins (Ly6e, Ly6h, Ly6g6e, Lynx1, Lypd1, Lypd6, and Lypd6b) known to allosterically modulate nicotinic receptor  iScience Article responses. 35 We found substantial and highly significant changes in the expression of three lynx prototoxins Lypd1, Ly6g6e, and Lypd6b ( Figure 3D). While both Chrna5+ and Chrna5+Syt6+ populations express Chrna5, there was slightly higher expression of Chrna5 (25% increase) as well as lower expression of the inhibitory muscarinic receptor Chrm2 (20% decrease) in Chrna5+Syt6+ neurons. There were no significant differences in other nicotinic and muscarinic subunit expression between the two groups. Acetylcholinesterase, the enzyme that breaks down acetylcholine was also highly expressed (50% increase) in Chrna5+ neurons, which may benefit their nicotinic responses by protecting receptors from overactivation and desensitization. Of note, Chrnb2 expression does seem to be in fewer cells than expected ( Figure 3E), which can be attributed to sparse detection of Chrnb2 levels by scRNA-seq approaches. 53,54 The fold change of the genes in Figure 3E between Chrna5+ and Chrna5+Syt6+ neurons is shown in Table S3. Notably, the top three genes with the highest fold change in Chrna5+ neurons were the GPI-anchored lynx prototoxins: Lynx2 encoded by Lypd1 (fold change: 2.55), Ly6g6e (2.03), and Lypd6b (1.51). The distinct pattern of expression of specific lynx proteins in Chrna5+ neurons suggests unexpectedly complex endogenous control of nicotinic responses in these prefrontal subplate neurons.
Transcriptomic examination of Chrna5+ neurons in other cortical regions including primary motor (MOp), somatosensory (SSp), and visual cortices (VISp) also confirmed that Chrna5+ neurons consist of L6b and L5/ 6NP neurons expressing subplate-marker genes, in addition to the lynx modulatory proteins as described above in the PFC ( Figure S5). Therefore, Chrna5 expression identifies a conserved population of deep-layer 6 neurons with subplate identity across multiple cortical regions that display specialized expression of lynx proteins to modulate nicotinic receptors.

Perturbing native prefrontal cortical lynx-modulation of optogenetic nicotinic responses
To examine whether the molecular machinery of deep-layer prefrontal neurons endows them with greater dynamic range in responding to acetylcholine, we sought to experimentally perturb endogenous lynx modulation. Members of the lynx-family are GPI-anchored ( Figure 4A), and work on cell expression systems 36 suggests these anchors can be cleaved via activation of phospholipase C (PLC). These experiments are important because the potential impact of GPI cleavage on nicotinic responses in a native system is not well understood. We hypothesized that perturbing lynx-mediated control could affect endogenous nicotinic properties in a complex manner ( Figure 4A) since both positive (e.g., Ly6g6e) and negative (e.g., Lynx1) modulatory lynx proteins are expressed. To cleave GPI-anchored proteins, we used the PLC activator compound m-3M3FBS. [55][56][57][58] Nicotinic responses of deep-layer pyramidal neurons from ChAT-ChR2 mice to optogenetic acetylcholine release were recorded in the continuous presence of atropine before and after treatment with m-3M3FBS (25 mM, 5 min; Figure 4B). The rising slope of the nicotinic responses showed a significant increase after m-3M3FBS treatment ( Figure 4D), compared to baseline change (À9 G 6%). Thus, PLC activation causes a specific increase in nicotinic receptor responses, presumably due to cleavage of inhibitory GPI-anchored prototoxins such as Lynx1.
To test the transcriptomic prediction that cell type-specific differences in lynx modulation lead to different cholinergic properties, we obtained purified water-soluble recombinant Ly6g6e protein and examined its effects on optogenetic nicotinic responses in labeled Chrna5+ and Syt6+ neurons ( Figure 4F). These experiments were conducted in Chrna5-Cre /+ Ai14 /+ ChAT-ChR2 /+ and Syt6-Cre /+ Ai14 /+ ChAT-ChR2 /+ mice. We hypothesized that the modulation of Chrna5+ neuronal nicotinic receptors by endogenous Ly6g6e would occlude the effect of exogenous soluble Ly6g6e, whereas Syt6+ neurons would be altered by exposure to the exogenous Ly6g6e ( Figure 4G). Consistent with this hypothesis, we found that 10 min application of iScience Article soluble Ly6g6e did not significantly alter the amplitude of optogenetically evoked nicotinic responses in labeled Chrna5+ neurons (change in peak = À2.1 G 1.2 pA, t (6) = 1.79, p = 0.12, paired t test). However, in labeled Syt6+ neurons lacking endogenous expression of Ly6g6e, exogenous application of soluble Ly6g6e caused a significant decrease in the amplitude (change in peak = À10 G 1.8 pA, t (8) = 5.60, p < 0.001, paired t test; Figures 4H and 4I). The change in peak and area of the nicotinic responses caused by solube Ly6g6e was significantly different between Chrna5+ and Syt6+ neurons (change in peak: t (14) = 3.43, p = 0.004; change in area: t (14) = 2.53, p = 0.024, unpaired t test; Figures 4I and 4J). Of note, soluble and endogenous GPI-anchored prototoxins are known to have opposite effects on nicotinic receptors, and the exact direction of endogenous modulation of nicotinic receptors by different lynx proteins is still debated. 38,59,60 The key outcome of this experiment is the difference in the Ly6g6e modulation of Chrna5+ and Syt6+ neurons, not the direction. We conclude that Chrna5+ neurons exert specialized molecular control over their nicotinic receptors, shaping their fate as acetylcholine super-responders.

DISCUSSION
Our work examines the effects of GPI-anchored lynx prototoxins on native nicotinic receptor-mediated optogenetic responses, advancing from work on heterologous expression systems. These results are a first step in showing how endogenous lynx regulation of nicotinic responses can act in a complex cell type-specific fashion leading to specialized cholinergic properties in a subset of neurons. Overall, our study reveals a previously uncharacterized population of Chrna5+ subplate neurons in the prefrontal cortex that are exquisitely sensitive to acetylcholine, with differential expression of several lynx prototoxin genes that allow flexible tuning of their high-affinity nicotinic responses ( Figure 5).

Specialized cholinergic properties of Chrna5+ neurons
While important for attention, the neurophysiological impact of the auxiliary a5 nicotinic subunit in its native neuronal environment has remained beyond the reach of previous work. The contributions of a5 iScience Article to high-affinity nicotinic receptors have been extrapolated based on results of cell system experiments and work on rodents deleted for Chrna5. 13,15,16,22,61 Here, Chrna5-Cre mice allowed us to affirmatively demonstrate that neurons expressing the a5 nicotinic subunit respond faster and more strongly to endogenous acetylcholine. This cholinergic heterogeneity among layer 6 neurons prompted a larger-scale comparison between Chrna5+ neurons and a well-defined layer 6 population labeled by Syt6. 42 These experiments revealed a subset of Chrna5+ acetylcholine ''super-responders'' with high-affinity nicotinic responses that were not found in Syt6+ neurons.

Heterogeneity of cell types expressing Chrna5
Previously, the deep-layer cell types expressing Chrna5 were uncharacterized and generally thought to include L6CT neurons. 8 Investigation of L6CT neurons have relied on Syt6-Cre and Ntsr1-Cre mouse lines that label similar sets of neurons, 42,43,62 with only Syt6-Cre mice successfully labeling this population in PFC. 28 L6CT neurons labeled by Syt6 or Ntsr1 expression are excited by acetylcholine, 29,63 but the degree to which their nicotinic response relied on Chrna5 expression was unclear. Strikingly, we reveal that acetylcholine super-responders with high-affinity nicotinic receptors are from the population of Chrna5+ neurons without Syt6-expression. Transcriptomic analysis demonstrates that majority of these likely Chrna5+ ''super-responders'' arise from L5 near-projecting and L6b neurons, populations that express multiple markers linking them to the developmental subplate. These enigmatic cells are remnants of earliest-born cortical neurons that serve as a relay for establishing connections between cortex and thalamus. 64,65 Subplate neurons receive cholinergic inputs at birth, 66 highlighting their role in developmental cholinergic modulation.

Advantages of Chrna5 as a marker for subplate cells
In contrast to L6CT neurons, subplate neurons remain relatively uncharacterized due to the lack of transgenic mice to definitively label the diverse subtypes and inaccessibility of the available lines for in vivo targeting. Transcriptomic analysis ( Figure 5, Table 1) suggests that the Chrna5+ population is enriched for known subplate markers Ctgf (connective tissue growth factor), Cplx3 (complexin 3), Kcnab1, and Lpar1. 32,67,68 Significantly, the lynx prototoxin and nicotinic receptor modulator Ly6g6e, which is highly expressed in Chrna5+ neurons, is also a marker of subplate neurons. 69 Our study is the first to identify enhanced cholinergic activation regulated by Chrna5 and lynx-gene expression in subplate/L6b neurons. Subplate neurons have recently been found to strongly regulate cortical output through their intracortical connections. 70,71 Enhanced cholinergic activation in these neurons will have different consequences for prefrontal processing, challenging the popular conception that cholinergic modulation of attention occurs only through top-down control of thalamic input by L6CT neurons.

Molecular determinants of nicotinic receptor properties in Chrna5+ neurons
Our transcriptomic analysis revealed enhanced expression of GPI-anchored lynx prototoxin genes Ly6g6e, Lypd1, and Lypd6b in Chrna5+ neurons ( Figure 5). Lynx proteins are well-known modulators of nicotinic receptor properties and trafficking, 38,40 but most of the insight into their actions comes from heterologous cell systems, deletion, and overexpression experiments. Their effects on nicotinic receptors in their native environment are unclear. In expression systems, Ly6g6e potentiates a4b2 nicotinic responses, slowing their desensitization; 36 predicting cholinergic responses in Chrna5+ neurons would be resistant to desensitization as has been implied by Chrna5-deletion work. 16 In contrast, Lynx2 (Lypd1) is a predicted negative modulator that can increase desensitization of a4b2 nicotinic receptors. 72 Lynx2 acts intracellularly to reduce surface expression of a4b2 nicotinic receptors, 36 but may preferentially act on lower-affinity (a4) 3 (b2) 2 receptors 61,73 and indirectly promote expression of high-affinity (a4) 2 (b2) 2 a5 nicotinic receptors. The effect of Lypd6b on (a4) 2 (b2) 2 a5 nicotinic receptors is yet to be determined and may further contribute to the complex control of Chrna5+ nicotinic responses. 74 In addition, Lynx1, a well-known negative modulator of a4b2 nicotinic receptors, 39,[75][76][77] is also expressed in Chrna5+ neurons. Consistent with such complex lynx regulation, our experiments confirmed that removing GPI-anchored lynx proteins increases nicotinic response onset and amplitude potentially due to removal of Lynx1. In contrast, exogenous application of recombinant Ly6g6e had different effects in Chrna5+ and Syt6+ neurons, consistent with cell type-specific lynx modulation in Chrna5+ neurons predicted by transcriptomics.

Functional consequences
The effects of lynx proteins on nicotinic receptor function have so far been determined by heterologous expression systems, 36  iScience Article and viral manipulation of expression in the brain. 39,81 We advance this field by revealing, in native tissue, complex endogenous regulation of optogenetic nicotinic responses by multiple GPI-anchored lynx proteins. Inhibitory lynx expression and high levels of acetylcholinesterase in Chrna5+ neurons suggest that their responses are restrained and our experiments likely underestimated their nicotinic receptor function. These responses could be dramatically enhanced when acetylcholinesterase and inhibitory lynx modulation is reduced through other signaling mechanisms. Such flexible tuning of nicotinic responses by lynx prototoxins in Chrna5+ neurons can provide greater dynamic range and poises them to be key players during attentional processing ( Figure 5). A recent study found that preventing developmental increase in Lynx1 expression in corticocortical neurons by viral knockdown led to altered cortical connectivity and impaired attention. 39 Thus cell type-specific changes in lynx expression during development are critical for maturation of attention circuits. It is of interest to examine such changes during development in Chrna5+ neurons and how they differ from Syt6+ neurons.

Caveats and limitations of the study
Subplate-marker genes and localization of subplate cells can vary across cortical regions, and they have been better studied in sensory cortical regions. [82][83][84] Our optophysiological, anatomical, and transcriptomic data show Chrna5+ acetylcholine super-responder neurons to be found in layer 6b both in the primary somatosensory cortex and PFC ( Figures S2 and S3). However, an outstanding question is whether transcriptomic cell classes such as L5/6NP and L6b, which express multiple subplate-marker genes, correlate with early developmental subplate origin. Anatomical evidence alone is insufficient for this purpose. For example, while the distinct transcriptomic subclasses L6b and L5/6NP show overlapping spatial distribution in deep layers of the motor cortex, 85,86 these subclasses possess distinct epigenetic signatures 87 whose implications are yet to be understood. It will be necessary for future work to examine whether transcriptomic/epigenetically defined cell classes (L5/6NP, L6b) expressing subplate markers also show distinct early developmental origin. Lack of labeling strategies has limited efforts to characterize these neuronal subclasses and verify their developmental subplate identity. Chrna5-Cre mice will be an important tool to rectify this as they consistently label these conserved subclasses across multiple cortical regions. Another caveat of our study is the perturbation of lynx prototoxins using chemical strategies. While we have demonstrated cell type-specific effects of recombinant ly6g6e, it remains to be seen whether cell type-specific expression of ly6g6e or other lynx prototoxins is necessary for nicotinic modulation. Future work is needed to genetically manipulate lynx prototoxins in specified cell populations.

Summary of advances
Our study reveals a distinct group of ''acetylcholine super-responder'' neurons in the prefrontal cortex identified by Chrna5-expression that constitute subplate neurons vital for cortical development. We identify that their high-affinity a5 subunit-containing nicotinic receptors are under complex regulation by several lynx prototoxins and acetylcholinesterase. Chrna5-Cre mice are a valuable tool for future studies examining the in vivo role of these specialized neurons.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: