The hippocampus is integral to working and episodic memory and is a central region of interest in diseases affecting these processes. Pig models are widely used in translational research and may provide an excellent bridge between rodents and nonhuman primates for CNS disease models because of their gyrencephalic neuroanatomy and significant white matter composition. However, the laminar structure of the pig hippocampus has not been well characterized. Therefore, we histologically characterized the dorsal hippocampus of Yucatan miniature pigs and quantified the cytoarchitecture of the hippocampal layers. We then utilized stereotaxis combined with single-unit electrophysiological mapping to precisely place multichannel laminar silicon probes into the dorsal hippocampus without the need for image guidance. We used in vivo electrophysiological recordings of simultaneous laminar field potentials and single-unit activity in multiple layers of the dorsal hippocampus to physiologically identify and quantify these layers under anesthesia. Consistent with previous reports, we found the porcine hippocampus to have the expected archicortical laminar structure, with some anatomical and histological features comparable to the rodent and others to the primate hippocampus. Importantly, we found these distinct features to be reflected in the laminar electrophysiology. This characterization, as well as our electrophysiology-based methodology targeting the porcine hippocampal lamina combined with high-channel-count silicon probes, will allow for analysis of spike-field interactions during normal and disease states in both anesthetized and future awake behaving neurophysiology in this large animal.

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Alterations in the expression, molecular composition, and localization of voltage-gated sodium channels play major roles in a broad range of neurological disorders. Recent evidence identifies sodium channel proteolysis as a key early event after ischemia and traumatic brain injury, further expanding the role of the sodium channel in neurological diseases. In this study, we investigate the protease responsible for proteolytic cleavage of voltage-gated sodium channels (NaChs). NaCh proteolysis occurs after protease activation in rat brain homogenates, pharmacological disruption of ionic homeostasis in cortical cultures, and mechanical injury using an in vitro model of traumatic brain injury. Proteolysis requires Ca(2+) and calpain activation but is not influenced by caspase-3 or cathepsin inhibition. Proteolysis results in loss of the full-length alpha-subunits, and the creation of fragments comprising all domains of the channel that retain interaction even after proteolysis. Cell surface biotinylation after mechanical injury indicates that proteolyzed NaChs remain in the membrane before noticeable evidence of neuronal death, providing a mechanism for altered action potential initiation, propagation, and downstream signaling events after Ca(2+) elevation.

Since traumatic axonal injury (TAI) is implicated as a prominent pathology of concussion, we examined potential sex differences in axon structure and responses to TAI. Rat and human neurons were used to develop micropatterned axon tracts in vitro that were genetically either male or female. Ultrastructural analysis revealed for the first time that female axons were consistently smaller with fewer microtubules than male axons. Computational modeling of TAI showed that these structural differences place microtubules in female axons at greater risk of failure during trauma under the same applied loads than in male axons. Likewise, in an in vitro model of TAI, dynamic stretch-injury to axon tracts induced greater pathophysiology of female axons than male axons, including more extensive undulation formations resulting from mechanical breaking of microtubules, and greater calcium influx shortly after the same level of injury. At 24h post-injury, female axons exhibited significantly more swellings and greater loss of calcium signaling function than male axons. Accordingly, sexual dimorphism of axon structure in the brain may also contribute to more extensive axonal pathology in females compared to males exposed to the same mechanical injury.

Diffuse axonal injury is a primary neuropathological feature of concussion and is thought to greatly contribute to the classical symptoms of decreased processing speed and memory dysfunction. Although previous studies have investigated the injury biomechanics at the micro- and mesoscale of concussion, few have addressed the multiscale transmission of mechanical loading at thresholds that can induce diffuse axonal injury. Because it has been recognized that axonal pathology is commonly found at anatomic interfaces across all severities of traumatic brain injury, we combined computational, analytical, and experimental approaches to investigate the potential mechanical vulnerability of axons that span the gray-white tissue interface. Our computational models predict that material heterogeneities at the gray-white interface lead to a highly nonuniform distribution of stress in axons, which was most amplified in axonal regions near the interface. This mechanism was confirmed using an analytical model of an individual fiber in a strained bimaterial interface. Comparisons of these collective data with histopathological evaluation of a swine model of concussion demonstrated a notably similar pattern of axonal damage adjacent to the gray-white interface. The results suggest that the tissue property mismatch at the gray-white matter interface places axons crossing this region at greater risk of mechanical damage during brain tissue deformation from traumatic brain injury.

Functional restoration following major peripheral nerve injury (PNI) is challenging, given slow axon growth rates and eventual regenerative pathway degradation in the absence of axons. We are developing tissue-engineered nerve grafts (TENGs) to simultaneously "bridge" missing nerve segments and "babysit" regenerative capacity by providing living axons to guide host axons and maintain the distal pathway. TENGs were biofabricated using porcine neurons and "stretch-grown" axon tracts. TENG neurons survived and elicited axon-facilitated axon regeneration to accelerate regrowth across both short (1 cm) and long (5 cm) segmental nerve defects in pigs. TENG axons also closely interacted with host Schwann cells to maintain proregenerative capacity. TENGs drove regeneration across 5-cm defects in both motor and mixed motor-sensory nerves, resulting in dense axon regeneration and electrophysiological recovery at levels similar to autograft repairs. This approach of accelerating axon regeneration while maintaining the pathway for long-distance regeneration may achieve recovery after currently unrepairable PNIs.

Spatially invariant feature detection is a property of many visual systems that rely on visual information provided by two eyes. However, how information across both eyes is integrated for invariant feature detection is not fully understood. Here, we investigated spatial invariance of looming responses in descending neurons (DNs) of Drosophila melanogaster. We found that multiple looming responsive DNs integrate looming information across both eyes, even though their dendrites are restricted to a single visual hemisphere. One DN, the giant fiber (GF), responds invariantly to looming stimuli across tested azimuthal locations. We confirmed visual information propagates to the GF from the contralateral eye, through an unidentified pathway, and demonstrated that the absence of this pathway alters GF responses to looming stimuli presented to the ipsilateral eye. Our data highlight a role for bilateral visual integration in generating consistent, looming-evoked escape responses that are robust across different stimulus locations and parameters.

A major problem hindering the development of autograft alternatives for repairing peripheral nerve injuries is immunogenicity. We have previously shown successful regeneration in transected rat sciatic nerves using conduits filled with allogeneic dorsal root ganglion (DRG) cells without any immunosuppression. In this study, we re-examined the immunogenicity of our DRG neuron implanted conduits as a potential strategy to overcome transplant rejection. A biodegradable NeuraGen® tube was infused with pure DRG neurons or Schwann cells cultured from a rat strain differing from the host rats and used to repair 8 mm gaps in the sciatic nerve. We observed enhanced regeneration with allogeneic cells compared to empty conduits 16 weeks post-surgery, but morphological analyses suggest recovery comparable to the healthy nerves was not achieved. The degree of regeneration was indistinguishable between DRG and Schwann cell allografts although immunogenicity assessments revealed substantially increased presence of Interferon gamma (IFN-γ) in Schwann cell allografts compared to the DRG allografts by two weeks post-surgery. Macrophage infiltration of the regenerated nerve graft in the DRG group 16 weeks post-surgery was below the level of the empty conduit (0.56 fold change from NG; p<0.05) while the Schwann cell group revealed significantly higher counts (1.29 fold change from NG; p<0.001). Major histocompatibility complex I (MHC I) molecules were present in significantly increased levels in the DRG and Schwann cell allograft groups compared to the hollow NG conduit and the Sham healthy nerve. Our results confirmed previous studies that have reported Schwann cells as being immunogenic, likely due to MHC I expression. Nerve gap injuries are difficult to repair; our data suggest that DRG neurons are superior medium to implant inside conduit tubes due to reduced immunogenicity and represent a potential treatment strategy that could be preferable to the current gold standard of autologous nerve transplant.

The calpain family of calcium-dependent proteases has been implicated in a variety of diseases and neurodegenerative pathologies. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components and membrane receptors, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. Pharmacological and gene-knockout strategies have targeted calpains to determine their contribution to neurodegenerative pathology; however, limitations associated with treatment paradigms, drug specificity, and genetic disruptions have produced inconsistent results and complicated interpretation. Specific, targeted calpain inhibition achieved by enhancing endogenous calpastatin levels offers unique advantages in studying pathological calpain activation. We have characterized a novel calpastatin-overexpressing transgenic mouse model, demonstrating a substantial increase in calpastatin expression within nervous system and peripheral tissues and associated reduction in protease activity. Experimental activation of calpains via traumatic brain injury resulted in cleavage of α-spectrin, collapsin response mediator protein-2, and voltage-gated sodium channel, critical proteins for the maintenance of neuronal structure and function. Calpastatin overexpression significantly attenuated calpain-mediated proteolysis of these selected substrates acutely following severe controlled cortical impact injury, but with no effect on acute hippocampal neurodegeneration. Augmenting calpastatin levels may be an effective method for calpain inhibition in traumatic brain injury and neurodegenerative disorders.

Human recombinant activated factor-VII (rFVIIa) has been used successfully in the treatment of spontaneous intracerebral hemorrhage. In addition, there is increasing interest in its use to treat uncontrolled bleeding of other origins, including trauma. The aim of this study was to evaluate the safety and potential effectiveness of rFVIIa to mitigate bleeding using a clinically relevant model of traumatic brain injury (TBI) in the pig. A double injury model was chosen consisting of (1) an expanding cerebral contusion induced by the application of negative pressure to the exposed cortical surface and (2) a rapid rotational acceleration of the head to induce diffuse axonal injury (DAI). Injuries were performed on 10 anesthetized pigs. Five minutes after injury, 720 microg/kg rFVIIa (n=5) or vehicle control (n=5) was administered intravenously. Magnetic resonance imaging (MRI) studies were performed within 30 min and at 3 days post-TBI to determine the temporal expansion of the cerebral contusion. Euthanasia and histopathologic analysis were performed at day 3. This included observations for hippocampal neuronal degeneration, axonal pathology and microclot formation. The expansion of contusion volume over the 3 days post-injury period was reduced significantly in animals treated with rFVIIa compared to vehicle controls. Surprisingly, immunohistochemical analysis demonstrated that the number of dead/dying hippocampal neurons and axonal pathology was reduced substantially by rFVIIa treatment compared to vehicle. In addition, there was no difference in the extent of microthrombi between groups. rFVIIa treatment after TBI in the pig reduced expansion of hemorrhagic cerebral contusion volume without exacerbating the severity of microclot formation. Finally, rFVIIa treatment provided a surprising neuroprotective effect by reducing hippocampal neuron degeneration as well as the extent of DAI.

Current diagnostic criteria for the neuropathological evaluation of the traumatic brain injury-associated neurodegeneration, chronic traumatic encephalopathy, define the pathognomonic lesion as hyperphosphorylated tau-immunoreactive neuronal and astroglial profiles in a patchy cortical distribution, clustered around small vessels and showing preferential localization to the depths of sulci. However, despite adoption into diagnostic criteria, there has been no formal assessment of the cortical distribution of the specific cellular components defining chronic traumatic encephalopathy neuropathologic change. To address this, we performed comprehensive mapping of hyperphosphorylated tau-immunoreactive neurofibrillary tangles and thorn-shaped astrocytes contributing to chronic traumatic encephalopathy neuropathologic change. From the Glasgow Traumatic Brain Injury Archive and the University of Pennsylvania Center for Neurodegenerative Disease Research Brain Bank, material was selected from patients with known chronic traumatic encephalopathy neuropathologic change, either following exposure to repetitive mild (athletes n = 17; non-athletes n = 1) or to single moderate or severe traumatic brain injury (n = 4), together with material from patients with previously confirmed Alzheimer's disease neuropathologic changes (n = 6) and no known exposure to traumatic brain injury. Representative sections were stained for hyperphosphorylated or Alzheimer's disease conformation-selective tau, after which stereotypical neurofibrillary tangles and thorn-shaped astrocytes were identified and mapped. Thorn-shaped astrocytes in chronic traumatic encephalopathy neuropathologic change were preferentially distributed towards sulcal depths [sulcal depth to gyral crest ratio of thorn-shaped astrocytes 12.84 ± 15.47 (mean ± standard deviation)], with this pathology more evident in material from patients with a history of survival from non-sport injury than those exposed to sport-associated traumatic brain injury (P = 0.009). In contrast, neurofibrillary tangles in chronic traumatic encephalopathy neuropathologic change showed a more uniform distribution across the cortex in sections stained for either hyperphosphorylated (sulcal depth to gyral crest ratio of neurofibrillary tangles 1.40 ± 0.74) or Alzheimer's disease conformation tau (sulcal depth to gyral crest ratio 1.64 ± 1.05), which was comparable to that seen in material from patients with known Alzheimer's disease neuropathologic changes (P = 0.82 and P = 0.91, respectively). Our data demonstrate that in chronic traumatic encephalopathy neuropathologic change the astroglial component alone shows preferential distribution to the depths of cortical sulci. In contrast, the neuronal pathology of chronic traumatic encephalopathy neuropathologic change is distributed more uniformly from gyral crest to sulcal depth and echoes that of Alzheimer's disease. These observations provide new insight into the neuropathological features of chronic traumatic encephalopathy that distinguish it from other tau pathologies and suggest that current diagnostic criteria should perhaps be reviewed and refined.

Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses.

Strategies to accelerate the rate of axon regeneration would improve functional recovery following peripheral nerve injury, in particular for cases involving segmental nerve defects. We are advancing tissue engineered nerve grafts (TENGs) comprised of long, aligned, centimeter-scale axon tracts developed by the controlled process of axon "stretch-growth" in custom mechanobioreactors. The current study used a rat sciatic nerve model to investigate the mechanisms of axon regeneration across nerve gaps bridged by TENGs as well as the extent of functional recovery compared to nerve guidance tubes (NGT) or autografts. We established that host axon growth occurred directly along TENG axons, which mimicked the action of "pioneer" axons during development by providing directed cues for accelerated outgrowth. Indeed, axon regeneration rates across TENGs were 3-4 fold faster than NGTs and equivalent to autografts. The infiltration of host Schwann cells - traditional drivers of peripheral axon regeneration - was also accelerated and progressed directly along TENG axons. Moreover, TENG repairs resulted in functional recovery levels equivalent to autografts, with both several-fold superior to NGTs. These findings demonstrate that engineered axon tracts serve as "living scaffolds" to guide host axon outgrowth by a new mechanism - which we term "axon-facilitated axon regeneration" - that leads to enhanced functional recovery.

Mild traumatic brain injury (mTBI) causes persisting post-concussion syndrome for many patients without abnormalities on conventional neuroimaging. Currently, there is no method for identifying at-risk cases at an early stage for directing concussion management and treatment. SNTF is a calpain-derived N-terminal proteolytic fragment of spectrin (αII-spectrin1-1176) generated in damaged axons following mTBI. Preliminary human studies suggest that elevated blood SNTF on the day of mTBI correlates with white matter disruption and lasting brain dysfunction. Here, we further evaluated serum SNTF as a prognostic marker for persistent brain dysfunction in uncomplicated mTBI patients treated in a Level I trauma center emergency department. Compared with healthy controls (n = 40), serum SNTF increased by 92% within 24 h of mTBI (n = 95; p < 0.0001), and as a diagnostic marker exhibited 100% specificity and 37% sensitivity (AUC = 0.87). To determine whether the subset of mTBI cases positive for SNTF preferentially developed lasting brain dysfunction, serum levels on the day of mTBI were compared with multiple measures of brain performance at 90 days post-injury. Elevated serum SNTF correlated significantly with persistent impairments in cognition and sensory-motor integration, and predicted worse performance in each test on a case by case basis (AUC = 0.68 and 0.76, respectively). SNTF also predicted poorer recovery of cognitive stress function from 30 to 90 days (AUC = 0.79-0.90). These results suggest that serum SNTF, a surrogate marker for axonal injury after mTBI, may have potential for the rapid prognosis of lasting post-concussion syndrome and impaired functional recovery following CT-negative mTBI. They provide further evidence linking axonal injury to persisting brain dysfunction after uncomplicated mTBI. A SNTF blood test, either alone or combined with other markers of axonal injury, may have important utilities for research, prognosis, management and treatment of concussion.

Although neurotechnology careers are on the rise, and neuroscience curriculums have significantly grown at the undergraduate and graduate levels, increasing neuroscience and neurotechnology exposure in high school curricula has been an ongoing challenge. This is due, in part, to difficulties in converting cutting-edge neuroscience research into hands-on activities that are accessible for high school students and affordable for high school educators. Here, we describe and characterize a low-cost, easy-to-construct device to enable students to record rapid Drosophila melanogaster (fruit fly) behaviors during optogenetics experiments. The device is generated from inexpensive Arduino kits and utilizes a smartphone for video capture, making it easy to adopt in a standard biology laboratory. We validate this device is capable of replicating optogenetics experiments performed with more sophisticated setups at leading universities and institutes. We incorporate the device into a high school neuroengineering summer workshop. We find student participation in the workshop significantly enhances their understanding of key neuroscience and neurotechnology concepts, demonstrating how this device can be utilized in high school settings and undergraduate research laboratories seeking low-cost alternatives.

Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.

Traumatic brain injury (TBI) can induce progressive neurodegeneration in association with chronic inflammation. Since chronic treatment with the non-steroidal anti-inflammatory drug (NSAID), ibuprofen, improves functional and histopathologic outcome in a mouse model of Alzheimer's disease (AD), we investigated whether it would also improve long-term outcome following TBI. Anesthetized adult rats were subjected to fluid percussion brain injury. Over the following 4 months the injured animals received ibuprofen per os (formulated in feed) at the approximate doses of 20 mg/kg body wt/day (n=13), 40 mg/kg body wt/day (n=13), or control (feed only, n=12). Sham animals underwent surgery without injury or ibuprofen treatment (n=9). At 4 months post-injury, a Morris water maze task revealed a profound learning dysfunction in all three injured groups compared to the sham group. Surprisingly, the learning ability of injured animals treated with either chronic ibuprofen regimen was significantly worsened compared to non-treated injured animals. However, there was no difference in the extent of progressive atrophy of the cortex or hippocampus between treated and non-treated injured animals. These data may have important implications for TBI patients who are often prescribed NSAIDs for chronic pain.

Tebotelimab, a bispecific PD-1×LAG-3 DART molecule that blocks both PD-1 and LAG-3, was investigated for clinical safety and activity in a phase 1 dose-escalation and cohort-expansion clinical trial in patients with solid tumors or hematologic malignancies and disease progression on previous treatment. Primary endpoints were safety and maximum tolerated dose of tebotelimab when administered as a single agent (n = 269) or in combination with the anti-HER2 antibody margetuximab (n = 84). Secondary endpoints included anti-tumor activity. In patients with advanced cancer treated with tebotelimab monotherapy, 68% (184/269) experienced treatment-related adverse events (TRAEs; 22% were grade ≥3). No maximum tolerated dose was defined; the recommended phase 2 dose (RP2D) was 600 mg once every 2 weeks. There were tumor decreases in 34% (59/172) of response-evaluable patients in the dose-escalation cohorts, with objective responses in multiple solid tumor types, including PD-1-refractory disease, and in LAG-3+ non-Hodgkin lymphomas, including CAR-T refractory disease. To enhance potential anti-tumor responses, we tested margetuximab plus tebotelimab. In patients with HER2+ tumors treated with tebotelimab plus margetuximab, 74% (62/84) had TRAEs (17% were grade ≥3). The RP2D was 600 mg once every 3 weeks. The confirmed objective response rate in these patients was 19% (14/72), including responses in patients typically not responsive to anti-HER2/anti-PD-1 combination therapy. ClinicalTrials.gov identifier: NCT03219268 .

Traumatic axonal injury (TAI) is the most common and important pathology of traumatic brain injury (TBI). However, little is known about potential indirect effects of TAI on dendrites. In this study, we used a well-established in vitro model of axonal stretch injury to investigate TAI-induced changes in dendrite morphology. Axons bridging two separated rat cortical neuron populations plated on a deformable substrate were used to create a zone of isolated stretch injury to axons. Following injury, we observed the formation of dendritic alterations or beading along the dendrite shaft. Dendritic beading formed within minutes after stretch then subsided over time. Pharmacological experiments revealed a sodium-dependent mechanism, while removing extracellular calcium exacerbated TAI's effect on dendrites. In addition, blocking ionotropic glutamate receptors with the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 prevented dendritic beading. These results demonstrate that axon mechanical injury directly affects dendrite morphology, highlighting an important bystander effect of TAI. The data also imply that TAI may alter dendrite structure and plasticity in vivo. An understanding of TAI's effect on dendrites is important since proper dendrite function is crucial for normal brain function and recovery after injury.

Identified neuron classes in vertebrate cortical [1-4] and subcortical [5-8] areas and invertebrate peripheral [9-11] and central [12-14] brain neuropils encode specific visual features of a panorama. How downstream neurons integrate these features to control vital behaviors, like escape, is unclear [15]. In Drosophila, the timing of a single spike in the giant fiber (GF) descending neuron [16-18] determines whether a fly uses a short or long takeoff when escaping a looming predator [13]. We previously proposed that GF spike timing results from summation of two visual features whose detection is highly conserved across animals [19]: an object's subtended angular size and its angular velocity [5-8, 11, 20, 21]. We attributed velocity encoding to input from lobula columnar type 4 (LC4) visual projection neurons, but the size-encoding source remained unknown. Here, we show that lobula plate/lobula columnar, type 2 (LPLC2) visual projection neurons anatomically specialized to detect looming [22] provide the entire GF size component. We find LPLC2 neurons to be necessary for GF-mediated escape and show that LPLC2 and LC4 synapse directly onto the GF via reconstruction in a fly brain electron microscopy (EM) volume [23]. LPLC2 silencing eliminates the size component of the GF looming response in patch-clamp recordings, leaving only the velocity component. A model summing a linear function of angular velocity (provided by LC4) and a Gaussian function of angular size (provided by LPLC2) replicates GF looming response dynamics and predicts the peak response time. We thus present an identified circuit in which information from looming feature-detecting neurons is combined by a common post-synaptic target to determine behavioral output.