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Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1–3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFβ receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15–diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFβ-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8+ T cells in commonly used syngeneic tumor models, the coexpression of inhibitory receptors PD-1, LAG3, and TIM3 defined a group of highly activated and functional effector cells. Coexpression of these receptors further enriched for antigen-specific cells with increased T-cell receptor clonality. Anti–PD-L1 treatment increased the number and activation of these triple-positive CD8+ T cells without affecting the density of PD-1− cells. The intratumoral density of CD8+ T cells coexpressing inhibitory receptors negatively correlated with tumor burden. The density ratio and pretreatment phenotype of CD8+ T cells coexpressing inhibitory receptors was positively correlated with response across a variety of tumor models. Our results demonstrate that coexpression of inhibitory receptors is not a signifier of exhausted T cells, but rather can define a group of activated and functional effector cells in syngeneic tumor models. In the cancer setting, these cells could represent a heterogeneous population of not only exhausted but also highly activated cells responsive to treatment.

Cytotoxicity assay with sorted PD1+LAG3+TIM3+ CD8 T cells

Additional material and methods

Supplemental Figures Showing: Fig. S1: CD8 T cells co-expressing PD1, LAG3 and TIM3 are present at different frequencies in MC38 and EMT6 tumors and represent activated cells. Fig. S2: CD8 T cells co-expressing inhibitory receptors are enriched in antigen-experienced and activated cells relative to the PD1- subset. Fig. S3: Majority of CD8 T cells in blood of MC38 and EMT6 tumor bearing mice lack PD1 and T-bet expression. Fig. S4: Anti-PD-L1 treatment increases the activity and cytolytic potential of PD1+ CD8 T cell subsets in MC38 and EMT6 tumors. Fig. S5: JC and 4T1 tumors do not respond to anti-PD-L1 treatment. Fig. S6: CD8 T cells co-expressing PD1, LAG3 and TIM3 are highly activated in JC and 4T1 tumors. Table S1: List of flow cytometry markers used for univariate linear regression analysis in Supplementary table 2. Table S2: Top parameters correlated with response to anti-PD-L1.

Cytotoxicity assay with sorted PD1- CD8 T cells

FACS parameter database

Background and purpose Develop a translational assay of Transient Receptor Potential Ankyrin 1 (TRPA1) activity for use as a preclinical and clinical biomarker. Experimental approach Allyl isothiocyanate (AITC), capsaicin or citric acid were applied to ears of wildtype and Trpa1-knock out (Trpa1 KO) rats, and changes in dermal blood flow (DBF) were measured by laser speckle contrast imaging. In humans, the DBF, pain and itch responses to 5-20% AITC applied to the forearm were measured and safety was evaluated. Reproducibility of the DBF, pain and itch responses to topically applied 10% and 15% AITC were assessed at two visits separated by 13-15 days. DBF changes were summarized at 5-minute intervals as areas under the curve (AUC) and maxima. Intraclass correlation coefficient (ICC) was calculated to assess arm-arm and period-period reproducibility. Key results AITC- and citric acid-induced DBF were significantly reduced in Trpa1 KO rats compared to wildtype (90 ± 2% and 65 ± 11% reduction, respectively), whereas capsaicin response did not differ. In humans, each AITC concentration significantly increased DBF compared to vehicle with the maximal increase occurring 5 minutes post application. Ten percent and 15% AITC were selected as safe and effective stimuli. AUC from 0 to 5 minutes was the most reproducible metric of AITC-induced DBF across arms (ICC = 0.92) and periods (ICC = 0.85). Subject-reported pain was more reproducible than itch across visits (ICC = 0.76 vs 0.17, respectively). Conclusion and implications AITC-induced DBF is a suitable target engagement biomarker of TRPA1 activity for preclinical and clinical studies of TRPA1 antagonists.

TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of β-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2ko mice, with Trem2-deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE., TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of β-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2ko mice, with Trem2-deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2ko females at 6–7 months; but by 12 or 19–22 months of age, it was notably diminished in female and male PS2APP;Trem2ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2ko brains, and the Aβ42:Aβ40 ratio was elevated. The amount of soluble, fibrillar Aβ oligomers also increased in PS2APP;Trem2ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6 to 7 months onward in PS2APP;Trem2ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in CSF. Thus, aggravated neuritic dystrophy is a more consistent outcome of Trem2 deficiency than amyloid plaque load, suggesting that the microglial packing of Aβ into dense plaque is an important neuroprotective activity. SIGNIFICANCE STATEMENT Genetic studies indicate that TREM2 gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of Trem2 deletion in the PS2APP mouse AD model, in which overproduction of Aβ peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of Trem2-deficient mice, despite these mice displaying reduced plaque accumulation at later ages (12–22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aβ42:Aβ40 ratio and amount of soluble, fibrillar Aβ oligomers were elevated in Trem2-deficient brains. These results suggest that the Trem2-dependent compaction of Aβ into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aβ species.

SUMMARY NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely un-known. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer’s disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12–20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments., Graphical Abstract, In Brief Hanson et al. examine the therapeutic effects of enhancing GluN2A-subunit-containing NMDAR function in Dravet syndrome and Alzheimer’s disease mice. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in both disease models. GluN2A NMDAR enhancers may benefit brain disorders with network hypersynchrony and cognitive impairments.

Tissue regeneration after injury requires coordinated regulation of stem cell activation, division, and daughter cell differentiation, processes that are increasingly well understood in many regenerating tissues. How accurate stem cell positioning and localized integration of new cells into the damaged epithelium are achieved, however, remains unclear. Here, we show that enteroendocrine cells coordinate stem cell migration towards a wound in the Drosophila intestinal epithelium. In response to injury, enteroendocrine cells release the N-terminal domain of the PTK7 orthologue, Otk, which activates non-canonical Wnt signaling in intestinal stem cells, promoting actin-based protrusion formation and stem cell migration towards a wound. We find that this migratory behavior is closely linked to proliferation, and that it is required for efficient tissue repair during injury. Our findings highlight the role of non-canonical Wnt signaling in regeneration of the intestinal epithelium, and identify enteroendocrine cell-released ligands as critical coordinators of intestinal stem cell migration., Stem cell migration is critical during adult tissue regeneration. Here, the authors demonstrate that enteroendocrine cells coordinate stem cell migration towards sites of injury in the Drosophila intestine by activating non-canonical Wnt signaling.

Summary Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4β7 and αEβ7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4β7 and αEβ7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4β7 and αEβ7 reduces CD8+ T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEβ7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEβ7+ T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4β7 and αEβ7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention., Graphical abstract, Highlights Blockade of α4β7 and αEβ7 reduces CD8+ T cells in the gut mucosa more than α4β7 alone Anti-αEβ7 or -E-cadherin reduces retention and increases egress of T cells in the gut αEβ7+ intestinal T cells are proinflammatory and have little to no regulatory markers Etrolizumab reduces mucosal inflammatory T cell genes in patients with Crohn disease, Dai et al. demonstrate the cooperative roles α4β7 and αEβ7 integrins have in CD8+ T cell accumulation in the gut mucosa in a mouse model of trafficking. Intestinal biopsies from patients with Crohn disease that has been treated with etrolizumab (anti-β7) show treatment-specific reductions in gene expression associated with CD8+ cytotoxic T cells.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.

Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33+ Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.

Therapeutic approaches to treat inflammatory bowel diseases (IBD) include targeting integrins that mediate adhesion and migration of lymphocytes to the gastrointestinal (GI) tract. Etrolizumab is a humanized monoclonal antibody that selectively binds the β7 subunit of the heterodimeric integrins of α4β7 and αEβ7. The relative contribution of α4β7 and αEβ7 integrin family members to lymphocyte migration and retention in GI tractremains to be characterized. Here we applied single-cell technologies to healthy and IBD patient biopsies and found that αEβ7 is highly expressed in a subset of proinflammatory CD4+T cells and majority of cytotoxic intraepithelial CD8+T cells. In order to understand the role of αEβ7 in gut T cell accumulation, we used a photo-convertible reporter system to determine that concurrent inhibition of α4β7 and αEβ7 together led to a greater reduction of polyclonal or antigen specific T cell accumulation in the intestinal mucosa and epithelium compared to single blockade of either α4β7 or αEβ7. Further intra-vital two-photon microscopy and photo-specific labeling experiments revealed that blockade of αEβ7 reduces T cell interactions with basolateral epithelial surface, increases the migratory speed of activated T cells in the intestinal mucosa, and facilitates effector T cell egress from the intestinal mucosal through lymphatic vessels. Our data suggests co-blockade of α4β7 and αEβ7 together offers a superior inhibition of T cell accumulation in gastrointestinal tissues through a stepwise inhibition of T cell migration and subsequent tissue retention.

Exhausted T cells have been described in cancer patients and murine tumor models largely based on their expression of various inhibitory receptors. Understanding of the functional attributes of these cells is limited. Here, we report that among CD8

High-affinity transferrin receptor (TfR) bispecific antibodies facilitate trafficking of TfR to lysosomes and induce TfR degradation to decrease the ability of TfR to mediate BBB transcytosis., Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood–brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR antibody affinity alters the intracellular trafficking of TfR. Comparing high- and low-affinity TfR bispecific antibodies in vivo, we found that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-affinity anti-TfR bispecific. Thus, high-affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.

The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin-dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1-dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity-dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye-specific segregation in the SC and dorsal LGN.

SummarySensory experience drives robust plasticity of sensory maps in cerebral cortex, but the role of inhibitory circuits in this process is not fully understood. We show that classical deprivation-induced whisker map plasticity in layer 2/3 (L2/3) of rat somatosensory (S1) cortex involves robust weakening of L4-L2/3 feedforward inhibition. This weakening was caused by reduced L4 excitation onto L2/3 fast-spiking (FS) interneurons, which mediate sensitive feedforward inhibition and was partially offset by strengthening of unitary FS to L2/3 pyramidal cell synapses. Weakening of feedforward inhibition paralleled the known weakening of feedforward excitation. As a result, mean excitation-inhibition balance and timing onto L2/3 pyramidal cells were preserved. Thus, reduced feedforward inhibition is a covert compensatory process that can maintain excitatory-inhibitory balance during classical deprivation-induced Hebbian map plasticity.

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SummaryDSCAM and DSCAM-LIKE1 (DSCAML1) serve diverse neurodevelopmental functions, including axon guidance, synaptic adhesion, and self-avoidance, depending on the species, cell type, and gene family member studied. We examined the function of DSCAM and DSCAML1 in the developing mouse retina. In addition to a subset of amacrine cells, Dscam was expressed in most retinal ganglion cells (RGCs). RGCs had fasciculated dendrites and clumped cell bodies in Dscam−/− mice, suggesting a role in self-avoidance. Dscaml1 was expressed in the rod circuit, and mice lacking Dscaml1 had fasciculated rod bipolar cell dendrites and clumped AII amacrine cell bodies, also indicating a role in self-avoidance. Neurons in Dscam or Dscaml1 mutant retinas stratified their processes appropriately in synaptic laminae in the inner plexiform layer, and functional synapses formed in the rod circuit in mice lacking Dscaml1. Therefore, DSCAM and DSCAML1 function similarly in self-avoidance, and are not essential for synaptic specificity in the mouse retina.

SummaryDirection selectivity in the retina requires the asymmetric wiring of inhibitory inputs onto four subtypes of On-Off direction-selective ganglion cells (DSGCs), each preferring motion in one of four cardinal directions. The primary model for the development of direction selectivity is that patterned activity plays an instructive role. Here, we use a unique, large-scale multielectrode array to demonstrate that DSGCs are present at eye opening, in mice that have been reared in darkness and in mice that lack cholinergic retinal waves. These data suggest that direction selectivity in the retina is established largely independent of patterned activity and is therefore likely to emerge as a result of complex molecular interactions.

Ambient GABA modulates firing patterns in adult neural circuits by tonically activating extrasynaptic GABAAreceptors. Here, we demonstrate that during a developmental period when activation of GABAAreceptors causes membrane depolarization, tonic activation of GABAAreceptors blocks all spontaneous activity recorded in retinal ganglion cells (RGCs) and starburst amacrine cells (SACs). Bath application of the GABAAreceptor agonist muscimol blocked spontaneous correlated increases in intracellular calcium concentration and compound postsynaptic currents in RGCs associated with retinal waves. In addition, GABAAreceptor agonists activated a tonic current in RGCs that significantly reduced their excitability. Using a transgenic mouse in which green fluorescent protein is expressed under the metabotropic glutamate receptor subtype 2 promoter to target recordings from SACs, we found that GABAAreceptor agonists blocked compound postsynaptic currents and also activated a tonic current. GABAAreceptor antagonists reduced the holding current in SACs but not RGCs, indicating that ambient levels of GABA tonically activate GABAAreceptors in SACs. GABAAreceptor antagonists did not block retinal waves but did alter the frequency and correlation structure of spontaneous RGC firing. Interestingly, the drug aminophylline, a general adenosine receptor antagonist used to block retinal waves, induced a tonic GABAAreceptor antagonist-sensitive current in outside-out patches excised from RGCs, indicating that aminophylline exerts its action on retinal waves by direct activation of GABAAreceptors. These findings have implications for how various neuroactive drugs and neurohormones known to modulate extrasynaptic GABAAreceptors may influence spontaneous firing patterns that are critical for the establishment of adult neural circuits.

We measured available and actual habitat use, morphology, escape behaviour and clinging ability in a large sample(N = 242) of green anoles, Anolis carolinensis, in a habitat consisting primarily of segregated dense clumps of broadleaves, Aspidistra elatior (Tulane University campus, LA) to compare against similar data collected previously froma more typical habitat c. 30 km away, consisting of continuous strands of bushes and trees (Good Hope Field, St.Charles Parish, LA). At Tulane the anoles perched primarily on the broad, smooth leaves of broad leaves, whereasin Good Hope Field (GHF) they predominantly perched on branches and tree trunks. The two populations differedsignificantly in morphology. In Tulane, the anoles tended to have shorter distal hindlimb elements, longer forelimbelements, and were more ‘slender’ than those at GHF. A comparison of escape behaviour showed population and sexdifferences. In both populations, females had significantly longer approach distances (i.e. were more ‘wary’) thanmales. These distances were, in addition, significantly longer at GHF than at Tulane for both sexes; this may be dueto the potentially higher diversity and abundance of predators at GHF, although habituation to humans may alsoplay a role. Anoles at Tulane had significantly larger toepads and higher clinging abilities than those at GHF. Theenhanced clinging abilities of anoles at Tulane may have arisen due to their propensity to use smooth leaves as theirprimary substrate. Overall, our data reveal substantial ecological, behavioural, morphological, and functional dif-ferences among populations, some of which may be adaptive. © 2005 The Linnean Society of London, BiologicalJournal of the Linnean Society, 2005, 85, 223–234.

With the advent of transgenic mice, much has been learned about the expression and function of gap junctions. Previously, we reported that retinal ganglion cells in mice lacking the neuronal gap junction protein connexin 36 (Cx36) have nearly normal firing patterns at postnatal day 4 (P4) but many more asynchronous action potentials than wild-type mice at P10 (Torborg et al. [2005] Nat. Neurosci. 8:72–78). With the goal of understanding the origin of this increased activity in Cx36–/– mice, we used a transgenic mouse (Deans et al. [2001] Neuron 31:477–485) to characterize the developmental expression of a Cx36 reporter in the retina. We found that Cx36 was first detected weakly at P2 and gradually increased in expression until it reached an adult pattern at P14. Although the onset of expression varied by cell type, we identified Cx36 in the glycinergic AII amacrine cell, glutamatergic cone bipolar cell, and retinal ganglion cells (RGCs). In addition, we used calcium imaging and multielectrode array recording to characterize further the firing patterns in Cx36–/– mice. Both correlated and asynchronous action potentials in P10 Cx36–/– RGCs were significantly inhibited by bath application of an ionotropic glutamate receptor antagonist, indicating that the increase in activity was synaptically mediated. Hence, both the expression patterns and the physiology suggest an increasing role for Cx36-containing gap junctions in suppressing RGC firing between waves during postnatal retinal development. J. Comp. Neurol. 493:309–320, 2005. © 2005 Wiley-Liss, Inc.

A central issue in evolutionary biology concerns whether morphology, performance and habitat use have coevolved. We investigated evolutionary relationships among the size of the subdigital toepad, clinging ability and perch height in 12 species of Caribbean Anolis lizard. Specifically, we predicted that: (1) because larger anole species tend to perch high in the canopy, both toepad area and clinging ability should scale with positive allometry to enable small and large lizards to possess approximately similar ratios of both variables relative to mass; (2) anole species with relatively larger toepads (i.e. size-adjusted) should be relatively better clingers compared with species with relatively small toepads; (3) species that perch high in the canopy should possess relatively large clinging abilities (either on an absolute or a size-adjusted basis). Our first hypothesis was refuted, as both toepad area and clinging ability scaled close to isometry (0.67) relative to mass, indicating that large lizard species have low ratios of clinging ability to mass compared with small lizard species. However, our second and third predictions were confirmed. Anole species with relatively larger toepads were relatively better clingers compared with species with relatively smaller toepads. Anole species that perched high in the canopy (either on an absolute scale or relative to size) tended to have relatively larger toepads and greater clinging capacities compared with species that perched lower in the canopy. These data provide indirect comparative evidence that the evolution of increased toepad size in some anole species is adaptive, by facilitating the occupation of perches high in the canopy. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83, 389–398.

Whisker deflection evokes sparse, low-probability spiking among L2/3 pyramidal cells in rodent somatosensory cortex (S1), with spiking distributed nonuniformly between more and less responsive cells. The cellular and local circuit factors that determine whisker responsiveness across neurons are unclear. To identify these factors, we used two-photon calcium imaging and loose-seal recording to identify more and less responsive L2/3 neurons in S1 slices in vitro, during feedforward recruitment of the L2/3 network by L4 stimulation. We observed a broad gradient of spike recruitment thresholds within local L2/3 populations, with low- and high-threshold cells intermixed. This recruitment gradient was significantly correlated across different L4 stimulation sites, and between L4-evoked and whisker-evoked responses in vivo, indicating that a substantial component of responsiveness is independent of tuning to specific feedforward inputs. Lowand high-threshold L2/3 pyramidal cells differed in L4-evoked excitatory synaptic conductance and intrinsic excitability, including spike threshold and the likelihood of doublet spike bursts. A gradient of intrinsic excitability was observed across neurons. Cells that spiked most readily to L4 stimulation received the most synaptic excitation but had the lowest intrinsic excitability. Low- and high-threshold cells did not differ in dendritic morphology, passive membrane properties, or L4-evoked inhibitory conductance. Thus multiple gradients of physiological properties exist across L2/3 pyramidal cells, with excitatory synaptic input strength best predicting overall spiking responsiveness during network recruitment. © 2014 the American Physiological Society.

On-Off direction-selective retinal ganglion cells (DSGCs) encode the axis of visual motion. They respond strongly to an object moving in a preferred direction and weakly to an object moving in the opposite, "null," direction. Historically, On-Off DSGCs were classified into four subtypes according to their directional preference (anterior, posterior, superior, or inferior). Here, we compare two genetically identified populations of On-Off DSGCs: dopamine receptor 4 (DRD4)-DSGCs and thyrotropin-releasing hormone receptor (TRHR)-DSGCs. We find that although both populations are tuned for posterior motion, they can be distinguished by a variety of physiological and anatomical criteria. First, the directional tuning of TRHR-DSGCs is broader than that of DRD4-DSGCs. Second, whereas both populations project similarly to the dorsal lateral geniculate nucleus, they project differently to the ventral lateral geniculate nucleus and the superior colliculus. Moreover, TRHR-DSGCs, but not DRD4-DSGCs, also project to the zona incerta, a thalamic area not previously known to receive direction-tuned visual information. Our findings reveal unexpected diversity among mouse On-Off DSGC subtypes that uniquely process and convey image motion to the brain.

Direction-selective ganglion cells (DSGCs) fire robustly for stimuli moving along one direction of motion and are strongly inhibited by stimuli moving in the opposite, or null, direction. In contrast to direction-selective neurons in primary visual cortex, a role for neural activity in the development of direction-selective retinal circuits has not been established. Direction-selective responses are detected at eye opening, before which spontaneous correlated activity known as retinal waves provide directional input to ganglion cells. Indeed, we observed a significant bias in wave propagation along the nasal over temporal direction. Using simultaneous calcium imaging and cell-attached recordings from three genetically labeled DSGC types in mice, we observed that all three DSGC types fire action potentials during retinal waves. However, we found that the direction of wave propagation did not influence DSGC spiking. These results indicate that the mechanisms guiding the formation of the asymmetric inhibition underlying direction selectivity in the retina are not dependent upon the directional properties of retinal waves.

A characteristic feature of adult retina is mosaic organization: a spatial arrangement of cells of each morphological and functional type that produces uniform sampling of visual space. How the mosaics of visual receptive fields emerge in the retina during development is not fully understood. Here we use a large-scale multielectrode array to determine the mosaic organization of retinal ganglion cells (RGCs) in rats around the time of eye opening and in the adult. At the time of eye opening, we were able to reliably distinguish two types of ON RGCs and two types of OFF RGCs in rat retina based on their light response and intrinsic firing properties. Although the light responses of individual cells were not yet mature at this age, each of the identified functional RGC types formed a receptive field mosaic, where the spacing of the receptive field centers and the overlap of the receptive field extents were similar to those observed in the retinas of adult rats. These findings suggest that, although the light response properties of RGCs may need vision to reach full maturity, extensive visual experience is not required for individual RGC types to form a regular sensory map of visual space.

Summary Motion detection is an essential component of visual processing. On-Off direction-selective retinal ganglion cells (On-Off DSGCs) detect objects moving along specific axes of the visual field due to their precise retinal circuitry. The brain circuitry of On-Off DSGCs, however, is largely unknown. We report a mouse with GFP expressed selectively by the On-Off DSGCs that detect posterior motion (On-Off pDSGCs), allowing two-photon targeted recordings of their light responses and delineation of their complete map of central connections. On-Off pDSGCs project exclusively to the dorsal lateral geniculate nucleus and superior colliculus and in both targets form synaptic lamina that are separate from a lamina corresponding to non-DSGCs. Thus, individual On-Off DSGC subtypes are molecularly distinct and establish circuits that map specific qualities of directional motion to dedicated subcortical areas. This suggests that each RGC subtype represents a unique parallel pathway whose synaptic specificity in the retina is recapitulated in central targets.

Direction-selective neurons, which respond selectively to motion in one direction, have been characterized in visual circuits across many species. Recently, the development of these directional neurons has been explored in both retina and primary visual cortex. The development of direction-selective cells in primary visual cortex requires visual experience. In contrast, direction-selective ganglion cells in retina are present at the age of the earliest light responses. The vision-independent signals guiding the asymmetric wiring underlying retinal direction selectivity remain unknown. The details of how retinal and cortical circuits extract motion information could explain their differing requirements for visual experience in development.

A key assumption in ecomorphological studies is that morphology-function relationships are invariant due to underlying biomechanical principles. We tested the hypothesis that morphology-performance relationships are invariant across different seasons by examining how a key performance trait, bite force, and two aspects of morphology (head shape and dewlap size) changed seasonally in the field and in the laboratory in the green anole lizard Anolis carolinensis. We found that not only did bite force change seasonally (up to 80% within the same individual), but relationships between morphology and bite force are highly plastic. Of the three traits examined (bite force, head shape, and dewlap area), only head shape did not change seasonally. We noted opposing trends for how bite force and dewlap area changed seasonally; whereas dewlap areas were large in the spring, and small in the winter, bite forces were low in the spring and high in the winter. This pattern occurred because of a tradeoff at the individual level: individuals in the spring with large dewlaps and high bite forces diminish their dewlaps (but not bite force), whereas individuals with small dewlaps and low bite forces in the spring increase their bite forces (but not dewlap size). We also show that this trend was apparent both in the field (comparing different individuals) and the laboratory (comparing the same set of individuals under standardized conditions). Finally, seasonal changes were not consistent among individuals for either bite force or dewlap area, as individuals changed seasonally in proportion to their initial state. These findings cast doubt on the widely held view of invariant morphology-performance relationships, and offer a cautionary note for eco-morphological studies.