Comparison of helicopter and ground transportation in pediatric trauma patients.

BACKGROUND: Decision making regarding transportation mode after a traumatic injury may have a significant impact on outcomes, due to differences in time to definitive care. The objective of this study was to determine if transport mode had an impact on in-hospital mortality and discharge disposition in pediatric trauma patients. METHODS: Data were abstracted from the National Trauma Data Bank from 2007 to 2016 comparing helicopter and ground transportation modes effects on mortality and discharge outcomes. The primary outcome was in-hospital death, while the secondary outcome was discharge home without services (DCHWOS). Analyses included logistic regression modeling and propensity score matching. RESULTS: Significant variables from univariate analysis were included in the multivariate, propensity-matched regression model. Pediatric trauma patients transported by helicopter had lower odds of mortality (OR 0.69 [0.64,0.75]) and higher odds of DCHWOS (1.29 [1.20,1.39]). There were no differences in overall mechanism, but individual injury patterns showed higher odds of mortality. CONCLUSION: Critical decisions regarding triage of patients by different modes of transport occur every day. This study supports the current literature on the topic and shows a potential additional benefit of a meaningful discharge outcome for those transported by helicopter. IMPACT: This study may impact prehospital triage decision making process for pediatric trauma patients on mortality. Prehospital transport mode may contribute to pediatric trauma discharge outcomes. Highlights the need for future research regarding non-clinical data that is unable to be abstracted from national databases (e.g., family dynamics, insurance status, weather, access to post-discharge resources).

Pooled Analysis of Trauma Centers Better Predicts Risk Factors for Firearm Violence Reinjury.

INTRODUCTION: Many trauma centers use the first firearm injury admission as a reachable moment to mitigate reinjury. Understanding repeat firearm violence can be difficult in metropolitan areas with multiple trauma centers and laws that prohibit sharing private health information across health systems. We hypothesized that risk factors for repeat firearm violence could be better understood using pooled data from two major metropolitan trauma centers. METHODS: Two level I trauma center registries were queried (2007-2017) for firearm injury admissions using International Classification of Diseases, Ninth and Tenth Revision (ICD9/10) Ecodes. A pseudo encryption tool allowed sharing of deidentified firearm injury and repeat firearm injury data without disclosing private health information. Factors associated with firearm reinjury admissions including, age, sex, race, payor, injury severity, intent, and discharge, were assessed by multivariable logistic regression. RESULTS: We identified 2145 patients with firearm injury admissions, 89 of whom had a subsequent repeat firearm injury admission. Majority of repeat firearm admissions were assaulted (91%), male (97.8%), and non-Hispanic Black (86.5%). 31.5% of repeat firearm injury admissions were admitted to a different trauma center from their initial admission. Independent predictors of repeat firearm injuries were age (adjusted odds ratio [aOR] 0.94, P < 0.001), male sex (aOR 6.18, P = 0.013), non-Hispanic Black race (aOR 5.14, P = 0.007), or discharge against medical advice (aOR 6.64, P=<0.001). CONCLUSIONS: Nearly a third of repeat firearm injury admissions would have been missed in the current study without pooled metropolitan trauma center data. The incidence of repeat firearm violence is increasing and those at the highest risk for reinjury need to be targeted for mitigating interventions.

Comparing Firearm and Stabbing Injuries in the Pediatric Trauma Population Using Propensity Matching.

OBJECTIVES: The objective of this study was to compare differences in mortality and nonhome discharge in pediatric patients with firearm and stab injuries, while minimizing bias. Our secondary objective was to assess the influence of insurance on these same outcomes. METHODS: Patients aged 0 to 17 years included in the National Trauma Data Bank (2007-2015) with firearm and stabbing injury were matched by propensity score. Logistic regression was used to assess associations of injury type and insurance with long-term care discharge and death. RESULTS: The average age was 14.8 years, 19.2% were female, 48% were African American, 58.4% had an injury severity score ≤8, and assaults accounted for 73.1% of cases. Firearm injuries were associated with a higher risk of discharge to long-term care (adjusted odds ratio [aOR], 2.07) compared with propensity-matched patients who were stabbed. Similarly, we found a higher risk of mortality in those with firearm injuries compared with stabbing injuries (aOR, 1.85). Regardless of mechanism, self-pay insurance status was associated with a higher risk of mortality (aOR, 2.41). When compared with stab wound patients with commercial insurance, self-pay firearm-injured patients were found to have an increased risk of mortality (aOR, 5.25). CONCLUSIONS: Pediatric victims of firearm violence were more likely to die or need additional care outside the home than victims of other types of penetrating injury when accounting for confounding characteristics to minimize bias.

Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions.

UNLABELLED: Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. SIGNIFICANCE STATEMENT: Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and spatially linked to attenuation of RF at sites of forward membrane protrusion. Importantly, clutching of RF is modulated by extracellular matrix (ECM) proteins and soluble axon guidance cues, suggesting that it may regulate axon guidance in vivo. Consistent with this notion, we found that RF rates of spinal neuron growth cones were slower in vivo than what was observed in vitro. Together, our study provides the best evidence that growth cone-ECM adhesions clutch RF locally to guide axons in vivo.

Current state of virtual reality simulation in robotic surgery training: a review.

BACKGROUND: Worldwide, the annual number of robotic surgical procedures continues to increase. Robotic surgical skills are unique from those used in either open or laparoscopic surgery. The acquisition of a basic robotic surgical skill set may be best accomplished in the simulation laboratory. We sought to review the current literature pertaining to the use of virtual reality (VR) simulation in the acquisition of robotic surgical skills on the da Vinci Surgical System. MATERIALS AND METHODS: A PubMed search was conducted between December 2014 and January 2015 utilizing the following keywords: virtual reality, robotic surgery, da Vinci, da Vinci skills simulator, SimSurgery Educational Platform, Mimic dV-Trainer, and Robotic Surgery Simulator. Articles were included if they were published between 2007 and 2015, utilized VR simulation for the da Vinci Surgical System, and utilized a commercially available VR platform. RESULTS: The initial search criteria returned 227 published articles. After all inclusion and exclusion criteria were applied, a total of 47 peer-reviewed manuscripts were included in the final review. CONCLUSIONS: There are many benefits to utilizing VR simulation for robotic skills acquisition. Four commercially available simulators have been demonstrated to be capable of assessing robotic skill. Three of the four simulators demonstrate the ability of a VR training curriculum to improve basic robotic skills, with proficiency-based training being the most effective training style. The skills obtained on a VR training curriculum are comparable with those obtained on dry laboratory simulation. The future of VR simulation includes utilization in assessment for re-credentialing purposes, advanced procedural-based training, and as a warm-up tool prior to surgery.

CIP4 coordinates with phospholipids and actin-associated proteins to localize to the protruding edge and produce actin ribs and veils.

Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production decreases CIP4 at the membrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge. We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons.

Synaptic regulation of microtubule dynamics in dendritic spines by calcium, F-actin, and drebrin.

Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.

Characterizing long-term outcomes following AMA discharges after assault-related penetrating trauma.

BACKGROUND: Patients discharged against medical advice do not receive adequate treatment and have a greater risk of readmission. This study assessed the rate of discharges against medical advice following assault-related penetrating trauma, with secondary aims to evaluate long term pre/post-injury hospitalizations and mortality. METHODS: Adult assault-related penetrating injuries admitted to a Level 1 Trauma Center were identified in the prospectively maintained database. Chart review was conducted for hospitalizations ± 5 years from index injury and statewide mortality data was used to identify deaths outside of hospital care. RESULTS: Out of a total of 1,744 assault-related penetrated injuries, 3.2% (52/1630) of survivors discharged against medical advice. Reasons for discharge against medical advice included: unknown (38%), home/child/family/pets (25%), unhappy with care/restrictions (23%), and work/money/other (13%). Post-discharge mortality did not differ between routine (6.5%) and against medical advice discharge (3.9%). Against medical advice and routine discharge had similar rates of any hospitalization (38.5 v 28.2%) and trauma hospitalization in prior 5-years (35 v 36%). However, significantly more against medical advice discharges had prior hospitalizations involving drug or alcohol abuse (65 v 38%), but not mental health diagnosis (55 v 55%). Significantly more against medical advice discharges have post-injury hospitalizations compared to routine discharges (48 vs 26.5%); however, include similar rates of repeat traumatic injury (36 v 32%). CONCLUSIONS: Those with against medical advice discharges were significantly more likely to have prior hospitalizations involving drug or alcohol abuse and significantly higher rates of post-injury hospitalizations. However, we did not see an increase in repeat traumatic injury or post-discharge mortality in those with against medical advice discharges when compared to those with routine discharges.

Retrospective analysis of the effects of hypocalcemia in severely injured trauma patients.

BACKGROUND: It has been suggested that the Lethal Triad be modified to include hypocalcemia, coined as the Lethal Diamond. Hypocalcemia in trauma has been attributed to multiple mechanisms, but new evidence suggests that traumatic injury may result in the development of hypoCa independent of blood transfusion. We hypothesize that hypocalcemia is associated with increased blood product requirements and mortality. METHODS: A retrospective study of 1,981 severely injured adult trauma patients from 2016 to 2019. Ionized calcium (iCa) levels were obtained on arrival and subjects were categorized by a threshold iCa level of 1.00 mmol/L and compared. Univariable and multivariable logistic regression analysis was performed. RESULTS: The hypocalcemia (iCa <1.00 mmol/L) group had increased rate of overall (p = 0.001), 4-hr (p = 0.007), and 24-hr (p = 0.003) mortality. There was no difference in prehospital transfusion volume between groups (p = 0.25). Hypocalcemia was associated with increased blood product requirements at 4 h (p <0.001), 24 h (p <0.001), and overall hospital length of stay (p <0.001). Logistic regression analysis showed increased odds of 4-hour mortality (OR 0.077 [95 % CI 0.011, 0.523], p = 0.009) and 24-hour mortality (OR 0.121 [95 % CI 0.019, 0.758], p = 0.024) for every mmol/L increase in iCa. CONCLUSIONS: This study shows the association of hypoCa and traumatic injury. Severe hypoCa was associated with increased odds of early and overall mortality and increased blood product requirements. These results support the need for future prospective trials assessing the role of hypocalcemia in trauma.

Firearm injury survival is only the beginning: The impact of socioeconomic factors on unplanned readmission after injury.

BACKGROUND: Firearm trauma remain a national crisis disproportionally impacting minority populations in the United States. Risk factors leading to unplanned readmission after firearm injury remain unclear. We hypothesized that socioeconomic factors have a major impact on unplanned readmission following assault-related firearm injury. METHODS: The 2016-2019 Nationwide Readmission Database of the Healthcare Cost and Utilization Project was used to identify hospital admissions in those aged >14 years with assault-related firearm injury. Multivariable analysis assessed factors associated with unplanned 90-day readmission. RESULTS: Over 4 years, 20,666 assault-related firearm injury admissions were identified that resulted in 2,033 injuries with subsequent 90-day unplanned readmission. Those with readmissions tended to be older (31.9 vs 30.3 years), had a drug or alcohol diagnosis at primary hospitalization (27.1% vs 24.1%), and had longer hospital stays at primary hospitalization (15.5 vs 8.1 days) [all P<0.05]. The mortality rate in the primary hospitalization was 4.5%. Primary readmission diagnoses included: complications (29.6%), infection (14.5%), mental health (4.4%), trauma (15.6%), and chronic disease (30.6%). Over half of the patients readmitted with a trauma diagnosis were coded as new trauma encounters. 10.3% of readmission diagnoses included an additional 'initial' firearm injury diagnosis. Independent predictors of 90-day unplanned readmission were public insurance (aOR 1.21, P = 0.008), lowest income quartile (aOR 1.23, P = 0.048), living in a larger urban region (aOR 1.49, P = 0.01), discharge requiring additional care (aOR 1.61, P < 0.001), and discharge against medical advice (aOR 2.39, P < 0.001). CONCLUSIONS: Here we present socioeconomic risk factors for unplanned readmission after assault-related firearm injury. Better understanding of this population can lead to improved outcomes, decreased readmissions, and decreased financial burden on hospitals and patients. Hospital-based violence intervention programs may use this to target mitigating intervention programs in this population.

The effects of hypocalcemia in severely injured pediatric trauma patients.

INTRODUCTION: The role of calcium is ubiquitous in human physiology. Emerging evidence suggests that the lethal triad be revised to include hypocalcemia (hypoCa) and thus be known as the lethal diamond . There are data showing that traumatic injury may result in hypoCa independent from the mechanism of calcium chelation by citrate-based blood preservatives. Minimal literature exists analyzing the role of hypoCa in pediatric trauma patients. We hypothesize that there is an independent association of hypoCa with increased blood product requirements and mortality. METHODS: A retrospective cohort study of severely injured pediatric trauma patients was conducted. Trauma registry data were collected from January 2016 to August 2021. Ionized calcium (iCa) levels were obtained from arrival blood draws. Subjects were categorized into two groups by a threshold iCa level of 1.00 mmol/L and compared. Shock Index Pediatric Adjusted scores were used to adjust for age-specific differences in vital signs. RESULTS: A total of 142 patients were compared, of which 46.5% were hypocalcemic (iCa <1.00 mmol/L). Patients were well matched in terms of demographics and injury severity. The hypocalcemic group had lower systolic blood pressure and a higher percentage of Shock Index Pediatric Adjusted-positive patients. Weight-adjusted transfusion volumes were significantly higher in the hypocalcemic group at both the 4-hour and 24-hour time points without a difference in prehospital transfusion requirements. There was no observed difference in early or in-hospital mortality. CONCLUSION: This study contributes to the body of literature regarding the association between hypoCa and traumatic injury in the pediatric population. Hypocalcemia was associated with increased blood product requirements without a difference in prehospital transfusion requirements, suggesting a possible independent association. Further prospective studies are needed to better understand this relationship. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Burden of unintentional pediatric firearm injury: An examination of the Nationwide Readmission Database.

BACKGROUND: Significant increases in firearm-related mortality in the US pediatric population drive an urgent need to study these injuries to drive prevention policies. The purpose of this study was (1) to characterize those with and without readmissions, (2) to identify risk factors for 90-day unplanned readmission, and (3) to examine reasons for hospital readmission. METHODS: The 2016-2019 Nationwide Readmission Database of the Healthcare Cost and Utilization Project was used to identify hospital admissions with unintentional firearm injury in patients younger than 18 years. Ninety-day unplanned readmission characteristics were assessed and detailed. Multivariable regression analysis was used to assess factors associated with unplanned 90-day readmission. RESULTS: Over 4 years, 1,264 unintentional firearm injury admissions resulted in 113 subsequent readmissions (8.9%). There were no significant differences in age or payor, but more women (14.7% vs. 23%) and older children (13-17 years [80.5%]) had readmissions. The mortality rate during primary hospitalization was 5.1%. Survivors of initial firearm injury were more frequently readmitted if they had a mental health diagnosis (22.1% vs. 13.8%; p = 0.017). Readmission diagnosis included complications (15%), mental health or drug/alcohol (9.7%), trauma (33.6%), a combination of the prior three (28.3%), and chronic disease (13.3%). More than a third (38.9%) of the trauma readmissions were for new traumatic injury. Female children, those with longer lengths of stay, and those with more severe injuries were more likely to have unplanned 90-day readmissions. Mental health and drug/alcohol abuse diagnoses were not an independent predictor for readmission. CONCLUSION: This study provides insight into the characteristics of and risk factors for unplanned readmission in the pediatric unintentional firearm injury population. In addition to using prevention strategies, the utilization of trauma-informed care must be integrated into all aspects of care for this population to help minimize the long-term psychological impact of surviving firearm injury. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

BDNF-induced increase of PSD-95 in dendritic spines requires dynamic microtubule invasions.

Microtubules (MTs) are capable of entering dendritic spines in mature hippocampal neurons through dynamic polymerization. Although these MT invasions are directly associated with neuronal activity, their function remains unknown. Here we demonstrate in mouse hippocampal neurons that MT entries into spines regulate the increase in postsynaptic density-95 (PSD-95) protein after brain-derived neurotrophic factor (BDNF) treatment. Using multiwavelength total internal reflectance fluorescence microscopy, we show that BDNF prolonged the average MT dwell time in spines and that this effect was dependent on TrkB receptor activation. Further examination revealed that peaks of MT polymerization into spines corresponded to rapid PSD-95 increases in the spine head. Over time, spines targeted by MTs after BDNF application, but not before, showed a robust increase in PSD-95. Conversely, spines completely devoid of MT invasions showed no significant change in the level of PSD-95. Pharmacological inhibition of MT dynamics abolished the BDNF-induced increase in PSD-95. Together, these results support the hypothesis that the well known increase in PSD-95 within spines after BDNF treatment is dependent on MT invasions of dendritic spines. Thus, our study provides a direct link between dynamic MTs and the postsynaptic structure, and provides a functional role for MT invasion of dendritic spines.

Firearm trauma: Race and insurance influence mortality and discharge disposition.

BACKGROUND: Health insurance and race impact mortality and discharge outcomes in the general trauma population. It remains unclear if disparities exist by race and/or insurance in outcomes following firearm injuries. The purpose of this study was to assess differences in mortality and discharge based on race and insurance status following firearm injuries. METHODS: The National Trauma Data Bank (2007-2016) was queried for firearm injuries by International Classification of Diseases, Ninth/Tenth Revision, Ecodes. Patients with known discharge disposition, age (18-64 years), race, and insurance were included in analysis (N = 120,005). To minimize bias due to missing data, we used multiple imputation for variables associated with outcomes following traumatic injury: Injury Severity Score, Glasgow Coma Scale score, respiratory rate, systolic blood pressure, and sex. Multivariable regression analysis was additionally adjusted for age, sex, Injury Severity Score, intent, Glasgow Coma Scale score, systolic blood pressure, heart rate, respiratory rate, year, and clustered by facility to assess differences in mortality and discharge disposition. RESULTS: The average age was 31 years, 88.6% were male, and 50% non-Hispanic Blacks. Overall mortality was 11.5%. Self-pay insurance was associated with a significant increase in mortality rates in all racial groups compared with non-Hispanic Whites with commercial insurance. Hispanic commercial, Medicaid, and self-pay patients were significantly less likely to discharge with posthospital care compared with commercially insured non-Hispanic Whites. When examining racial differences in mortality and discharge by individual insurance types, commercially insured non-Hispanic Black and other race patients were significantly less likely to die compared with similarly insured non-Hispanic White patients. Regardless of race, no significant differences in mortality were observed in Medicaid or self-pay patients compared with non-Hispanic White patients. CONCLUSION: Victims of firearm injuries with a self-pay insurance status have a significantly higher rate of mortality. Hispanic patients regardless of insurance status were significantly less likely to discharge with posthospital care compared with non-Hispanic Whites with commercial insurance. Continued efforts are needed to understand and address the relationship between insurance status, race, and outcomes following firearm violence. LEVEL OF EVIDENCE: Prognostic and epidemiologic, Level IV.

Severe Hypothermia and Frostbite Requiring ECMO and Four Limb Amputations.

Severe hypothermia and frostbite can result in significant morbidity and mortality. We present a case of a patient with severe hypothermia and frostbite due to cold exposure after a snowmobile crash. He presented in cardiac arrest with a core temperature of 19°C requiring prolonged cardiopulmonary resuscitation, active internal rewarming, venoarterial extracorporeal membrane oxygenation, and subsequently amputations of all four extremities. Although severe hypothermia and frostbite can be a fatal condition, the quick action of Emergency Medical Services, emergency physicians, trauma surgeons, cardiothoracic surgeons, intensivists, and the burn team contributed to a successful recovery for this patient including a good neurological outcome. This case highlights the importance of a strong interdisciplinary team in treating this condition.

Adhesive micro-line periodicity determines guidance of axonal outgrowth.

Adhesive micro-lines of various sub-cellular geometries were created using a non-traditional micro stamping technique. This technique employed the use of commercially available diffraction gratings as the molds for the micro stamps, a method which is quick and inexpensive, and which could easily be adopted as a patterning tool in a variety of research efforts. The atypical saw-tooth profile of the micro stamps enabled a unique degree of control and flexibility over patterned line and gap widths. Cortical neurons cultured on patterned poly-lysine micro-lines on PDMS exhibit a startling transition in axonal guidance: From the expected parallel guidance to an unexpected perpendicular guidance that becomes dominant as patterned lines and gaps become sufficiently narrow. This transition is most obvious when the lines are narrow relative to gaps, while the periodicity of the pattern is reduced. Axons growing perpendicular to micro-lines exhibited 'vinculated' growth, a unique morphological phenotype consisting of periodic orthogonal extensions along the axon.

The F-BAR protein CIP4 inhibits neurite formation by producing lamellipodial protrusions.

Neurite formation is a seminal event in the early development of neurons. However, little is known about the mechanisms by which neurons form neurites. F-BAR proteins function in sensing and inducing membrane curvature. Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, regulates endocytosis in a variety of cell types. However, there is little data on how CIP4 functions in neurons. Here we show that CIP4 plays a novel role in neuronal development by inhibiting neurite formation. Remarkably, CIP4 exerts this effect not through endocytosis, but by producing lamellipodial protrusions. In primary cortical neurons CIP4 is concentrated specifically at the tips of extending lamellipodia and filopodia, instead of endosomes as in other cell types. Overexpression of CIP4 results in lamellipodial protrusions around the cell body, subsequently delaying neurite formation and enlarging growth cones. These effects depend on the F-BAR and SH3 domains of CIP4 and on its ability to multimerize. Conversely, cortical neurons from CIP4-null mice initiate neurites twice as fast as controls. This is the first study to demonstrate that an F-BAR protein functions differently in neuronal versus nonneuronal cells and induces lamellipodial protrusions instead of invaginations or filopodia-like structures.

Nucleofection and primary culture of embryonic mouse hippocampal and cortical neurons.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8 hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (>2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

Dynamic microtubules promote synaptic NMDA receptor-dependent spine enlargement.

Most excitatory synaptic terminals in the brain impinge on dendritic spines. We and others have recently shown that dynamic microtubules (MTs) enter spines from the dendritic shaft. However, a direct role for MTs in long-lasting spine plasticity has yet to be demonstrated and it remains unclear whether MT-spine invasions are directly influenced by synaptic activity. Lasting changes in spine morphology and synaptic strength can be triggered by activation of synaptic NMDA receptors (NMDARs) and are associated with learning and memory processes. To determine whether MTs are involved in NMDAR-dependent spine plasticity, we imaged MT dynamics and spine morphology in live mouse hippocampal pyramidal neurons before and after acute activation of synaptic NMDARs. Synaptic NMDAR activation promoted MT-spine invasions and lasting increases in spine size, with invaded spines exhibiting significantly faster and more growth than non-invaded spines. Even individual MT invasions triggered rapid increases in spine size that persisted longer following NMDAR activation. Inhibition of either NMDARs or dynamic MTs blocked NMDAR-dependent spine growth. Together these results demonstrate for the first time that MT-spine invasions are positively regulated by signaling through synaptic NMDARs, and contribute to long-lasting structural changes in targeted spines.