Child Opportunity Index and Pediatric Intensive Care Outcomes: A Multicenter Retrospective Study in the United States.

OBJECTIVES: To evaluate for associations between a child's neighborhood, as categorized by Child Opportunity Index (COI 2.0), and 1) PICU mortality, 2) severity of illness at PICU admission, and 3) PICU length of stay (LOS). DESIGN: Retrospective cohort study. SETTING: Fifteen PICUs in the United States. PATIENTS: Children younger than 18 years admitted from 2019 to 2020, excluding those after cardiac procedures. Nationally-normed COI category (very low, low, moderate, high, very high) was determined for each admission by census tract, and clinical features were obtained from the Virtual Pediatric Systems LLC (Los Angeles, CA) data from each site. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among 33,901 index PICU admissions during the time period, median patient age was 4.9 years and PICU mortality was 2.1%. There was a higher percentage of admissions from the very low COI category (27.3%) than other COI categories (17.2-19.5%, p < 0.0001). Patient admissions from the high and very high COI categories had a lower median Pediatric Index of Mortality 3 risk of mortality (0.70) than those from the very low, low, and moderate COI groups (0.71) ( p < 0.001). PICU mortality was lowest in the very high (1.7%) and high (1.9%) COI groups and highest in the moderate group (2.5%), followed by very low (2.3%) and low (2.2%) ( p = 0.001 across categories). Median PICU LOS was between 1.37 and 1.50 days in all COI categories. Multivariable regression revealed adjusted odds of PICU mortality of 1.30 (95% CI, 0.94-1.79; p = 0.11) for children from a very low versus very high COI neighborhood, with an odds ratio [OR] of 0.996 (95% CI, 0.993-1.00; p = 0.05) for mortality for COI as an ordinal value from 0 to 100. Children without insurance coverage had an OR for mortality of 3.58 (95% CI, 2.46-5.20; p < 0.0001) as compared with those with commercial insurance. CONCLUSIONS: Children admitted to a cohort of U.S. PICUs were often from very low COI neighborhoods. Children from very high COI neighborhoods had the lowest risk of mortality and observed mortality; however, odds of mortality were not statistically different by COI category in a multivariable model. Children without insurance coverage had significantly higher odds of PICU mortality regardless of neighborhood.

A Core Outcome Measurement Set for Pediatric Critical Care.

OBJECTIVES: To identify a PICU Core Outcome Measurement Set (PICU COMS), a set of measures that can be used to evaluate the PICU Core Outcome Set (PICU COS) domains in PICU patients and their families. DESIGN: A modified Delphi consensus process. SETTING: Four webinars attended by PICU physicians and nurses, pediatric surgeons, rehabilitation physicians, and scientists with expertise in PICU clinical care or research ( n = 35). Attendees were from eight countries and convened from the Pediatric Acute Lung Injury and Sepsis Investigators Pediatric Outcomes STudies after PICU Investigators and the Eunice Kennedy Shriver National Institute of Child Health and Human Development Collaborative Pediatric Critical Care Research Network PICU COS Investigators. SUBJECTS: Measures to assess outcome domains of the PICU COS are as follows: cognitive, emotional, overall (including health-related quality of life), physical, and family health. Measures evaluating social health were also considered. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Measures were classified as general or additional based on generalizability across PICU populations, feasibility, and relevance to specific COS domains. Measures with high consensus, defined as 80% agreement for inclusion, were selected for the PICU COMS. Among 140 candidate measures, 24 were delineated as general (broadly applicable) and, of these, 10 achieved consensus for inclusion in the COMS (7 patient-oriented and 3 family-oriented). Six of the seven patient measures were applicable to the broadest range of patients, diagnoses, and developmental abilities. All were validated in pediatric populations and have normative pediatric data. Twenty additional measures focusing on specific populations or in-depth evaluation of a COS subdomain also met consensus for inclusion as COMS additional measures. CONCLUSIONS: The PICU COMS delineates measures to evaluate domains in the PICU COS and facilitates comparability across future research studies to characterize PICU survivorship and enable interventional studies to target long-term outcomes after critical illness.

The Impact of Strict Public Health Restrictions on Pediatric Critical Illness.

OBJECTIVES: To characterize the impact of public health interventions on the volume and characteristics of admissions to the PICU. DESIGN: Multicenter retrospective cohort study. SETTING: Six U.S. referral PICUs during February 15, 2020-May 14, 2020, compared with the same months during 2017-2019 (baseline). PATIENTS: PICU admissions excluding admissions for illnesses due to severe acute respiratory syndrome coronavirus 2 and readmissions during the same hospitalization. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Primary outcome was admission volumes during the period of stay-at-home orders (March 15, 2020-May 14, 2020) compared with baseline. Secondary outcomes were hospitalization characteristics including advanced support (e.g., invasive mechanical ventilation), PICU and hospital lengths of stay, and mortality. We used generalized linear mixed modeling to compare patient and admission characteristics during the stay-at-home orders period to baseline. We evaluated 7,960 admissions including 1,327 during March 15, 2020-May 14, 2020. Daily admissions and patients days were lower during the period of stay-at-home orders compared with baseline: median admissions 21 (interquartile range, 17-25) versus 36 (interquartile range, 30-42) (p < 0.001) and median patient days 93.0 (interquartile range, 55.9-136.7) versus 143.6 (interquartile range, 108.5-189.2) (p < 0.001). Admissions during the period of stay-at-home orders were less common in young children and for respiratory and infectious illnesses and more common for poisonings, endocrinopathies and for children with race/ethnicity categorized as other/unspecified. There were no differences in hospitalization characteristics except fewer patients received noninvasive ventilation during the period of stay-at-home orders. CONCLUSIONS: Reductions in PICU admissions suggest that much of pediatric critical illness in younger children and for respiratory and infectious illnesses may be preventable through targeted public health strategies.

A Framework for Pediatric Intensivists Providing Compassionate Extubation at Home.

For families facing end-of-life decisions for their critically ill children, compassionate extubation at home is a valuable service that pediatric intensivists can provide. Compassionate extubation at home is resource intensive and can be logistically challenging. Discouragingly, guidance on compassionate extubation at home in the literature is limited. We developed an evidence- and experience-based framework for compassionate extubation at home addressing common planning challenges and resource management. Our objective is to share this framework and an accompanying checklist, so that pediatric intensivists in other institutions can adapt these tools for their use, reducing barriers to providing compassionate extubation at home for critically ill children at the end of life.

Characteristics of Burn-Injured Children in 117 U.S. PICUs (2009-2017): A Retrospective Virtual Pediatric Systems Database Study.

OBJECTIVES: To describe characteristics and outcomes of children with burn injury treated in U.S. PICUs. DESIGN: Retrospective study of admissions in the Virtual Pediatric Systems, LLC, database from 2009 to 2017. SETTING: One hundred and seventeen PICUs in the United States. PATIENTS: Patients less than 18 years old admitted with an active diagnosis of burn at admission. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 2,056 patients were included. They were predominantly male (62.6%) and less than 6 years old (66.7%). Cutaneous burns were recorded in 92.1% of patients, mouth/pharynx burns in 5.8%, inhalation injury in 5.1%, and larynx/trachea/lung burns in 4.5%. Among those with an etiology recorded (n = 861), scald was most common (38.6%), particularly in children less than 2 years old (67.8%). Fire/flame burns were most common (46.6%) in children greater than or equal to 2 years. Multiple organ failure was present in 26.2% of patients. Most patients (89%) were at facilities without American Burn Association pediatric verification. PICU mortality occurred in 4.5% of patients. On multivariable analysis using Pediatric Index of Mortality 2, greater than or equal to 30% total body surface area burned was significantly associated with mortality (odds ratio, 5.40; 95% CI, 2.16-13.51; p = 0.0003). When Pediatric Risk of Mortality III was used, greater than or equal to 30% total body surface area burned (odds ratio, 5.45; 95% CI, 1.95-15.26; p = 0.001) and inhalation injury (odds ratio, 5.39; 95% CI, 1.58-18.42; p = 0.007) were significantly associated with mortality. Among 366 survivors (18.6%) with Pediatric Cerebral Performance Category or Pediatric Overall Performance Category data, 190 (51.9%) had a greater than or equal to 1 point increase in Pediatric Cerebral Performance Category or Pediatric Overall Performance Category disability category and 80 (21.9%) had a new designation of moderate or severe disability, or persistent vegetative state. CONCLUSIONS: Burn-injured patients in U.S. PICUs have a substantial burden of organ failure, morbidity, and mortality. Coordination among specialized facilities may be particularly important in this population, especially for those with higher % total body surface area burned or inhalation injury.

Sirtuins and Immuno-Metabolism of Sepsis.

Sepsis and septic shock are the leading causes of death in non-coronary intensive care units worldwide. During sepsis-associated immune dysfunction, the early/hyper-inflammatory phase transitions to a late/hypo-inflammatory phase as sepsis progresses. The majority of sepsis-related deaths occur during the hypo-inflammatory phase. There are no phase-specific therapies currently available for clinical use in sepsis. Metabolic rewiring directs the transition from hyper-inflammatory to hypo-inflammatory immune responses to protect homeostasis during sepsis inflammation, but the mechanisms underlying this immuno-metabolic network are unclear. Here, we review the roles of NAD+ sensing Sirtuin (SIRT) family members in controlling immunometabolic rewiring during the acute systemic inflammatory response associated with sepsis. We discuss individual contributions among family members SIRT 1, 2, 3, 4 and 6 in regulating the metabolic switch between carbohydrate-fueled hyper-inflammation to lipid-fueled hypo-inflammation. We further highlight the role of SIRT1 and SIRT2 as potential "druggable" targets for promoting immunometabolic homeostasis and increasing sepsis survival.

Alkaline Phosphatase in Infant Cardiopulmonary Bypass: Kinetics and Relationship to Organ Injury and Major Cardiovascular Events.

OBJECTIVES: To determine the kinetics of alkaline phosphatase (AP) activity and concentration after infant cardiopulmonary bypass, including isoform-specific changes, and to measure the association between postoperative AP activity and major postoperative cardiovascular events, organ injury/dysfunction, and postoperative support requirements STUDY DESIGN: Prospective cohort study of 120 infants ≤120 days of age undergoing cardiopulmonary bypass. AP total and isoform-specific activity was assessed at 6 time points (preoperation, rewarming, 6, 24, 48, and 72 hours postoperation). Low AP activity was defined as ≤80 U/L. AP concentrations and biomarkers of organ injury/dysfunction were collected through 24 hours postoperation. Major cardiovascular events were defined as cardiac arrest, mechanical circulatory support, or death. RESULTS: AP activity loss occurred primarily during the operation (median decrease 89 U/L; P < .0001) secondary to decreased bone and liver 2 isoforms. Activity declined through 24 hours in 27% of patients. AP activity strongly correlated with serum concentration (r = 0.87-0.91; P < .0001). Persistent low AP activity at 72 hours was associated independently with occurrence of a major cardiac event (OR 5.6; P < .05). Early AP activity was associated independently with subsequent vasoactive-inotropic score (P < .001), peak lactate (P < .0001), peak creatinine (P < .0005), N-terminal pro-brain natriuretic peptide (P < .05), and intestinal fatty acid binding protein (P < .005). CONCLUSIONS: AP activity decreases during infant cardiopulmonary bypass and may continue to decrease for 24 hours. Activity loss is secondary to decreased bone and liver 2 isoform concentrations. Early low AP activity is associated independently with subsequent postoperative support and organ injury/dysfunction, and persistence of AP activity ≤80 U/L at 72 hours is associated independently with increased odds of major cardiovascular events.

Drug delivery systems for prolonged duration local anesthesia.

Numerous drug delivery systems have been applied to the problem of providing prolonged duration local anesthesia (PDLA). Here we review the rationale for PDLA, the desirable features for and important attributes of such systems, and specific examples that have been developed.

State-dependent function of neocortical chandelier cells.

Chandelier (axoaxonic) cells (ChCs) are a distinct group of GABAergic interneurons that innervate the axon initial segments of pyramidal cells. However, their circuit role and the function of their clearly defined anatomical specificity remain unclear. Recent work has demonstrated that chandelier cells can produce depolarizing GABAergic PSPs, occasionally driving postsynaptic targets to spike. On the other hand, other work suggests that ChCs are hyperpolarizing and may have an inhibitory role. These disparate functional effects may reflect heterogeneity among ChCs. Here, using brain slices from transgenic mouse strains, we first demonstrate that, across different neocortical areas and genetic backgrounds, upper Layer 2/3 ChCs belong to a single electrophysiologically and morphologically defined population, extensively sampling Layer 1 inputs with asymmetric dendrites. Consistent with being a single cell type, we find electrical coupling between ChCs. We then investigate the effect of chandelier cell activation on pyramidal neuron spiking in several conditions, ranging from the resting membrane potential to stimuli designed to approximate in vivo membrane potential dynamics. We find that under quiescent conditions, chandelier cells are capable of both promoting and inhibiting spike generation, depending on the postsynaptic membrane potential. However, during in vivo-like membrane potential fluctuations, the dominant postsynaptic effect was a strong inhibition. Thus, neocortical chandelier cells, even from within a homogeneous population, appear to play a dual role in the circuit, helping to activate quiescent pyramidal neurons, while at the same time inhibiting active ones.

Nonadherence to appropriate tidal volume and PEEP in children with pARDS at a single center.

BACKGROUND: Low tidal volume and adequate positive end-expiratory pressure (PEEP) are evidence-based approaches for pediatric acute respiratory distress syndrome (pARDS), however, data are limited regarding their use since pARDS guidelines were revised in 2015. OBJECTIVE: To identify prevalence of, and factors associated with, nonadherence to appropriate tidal volume and PEEP in children with pARDS. METHODS: Retrospective cohort study of children 1 month to <18 years with pARDS who received invasive mechanical ventilation from 2016 to 2018 in a single pediatric intensive care unit (PICU). RESULTS: At 24 h after meeting pARDS criteria, 48/86 (56%) patients received tidal volume ≤8 ml/kg of ideal body weight and 45/86 (52%) received appropriate PEEP, with 22/86 (26%) receiving both. Among patients ≥2 years of age, a lower proportion of patients with overweight/obesity (9/25, 36%) had appropriate tidal volume versus those in the normal or underweight category (16/22, 73%, p = 0.02). When FIO2 was ≥50%, PEEP was appropriate in 19/60 (32%) cases versus 26/26 (100%) with FIO2 < 50% (p < 0.0001). pARDS was documented in the progress note in 7/86 (8%) patients at 24 h. Severity of pARDS, documentation in the progress note, and other clinical factors were not significantly associated with use of appropriate tidal volume and PEEP, however pARDS was documented more commonly in patients with severe pARDS. CONCLUSIONS: In a single PICU in the United States, children with pARDS did not receive appropriate tidal volume for ideal body weight nor PEEP. Targets for improving tidal volume and PEEP adherence may include overweight patients and those receiving FIO2 ≥ 50%, respectively.

Continuing Care For Critically Ill Children Beyond Hospital Discharge: Current State of Follow-up.

OBJECTIVES: Survivors of the PICU face long-term morbidities across health domains. In this study, we detail active PICU follow-up programs (PFUPs) and identify perceptions and barriers about development and maintenance of PFUPs. METHODS: A web link to an adaptive survey was distributed through organizational listservs. Descriptive statistics characterized the sample and details of existing PFUPs. Likert responses regarding benefits and barriers were summarized. RESULTS: One hundred eleven respondents represented 60 institutions located in the United States (n = 55), Canada (n = 3), Australia (n = 1), and the United Kingdom (n = 1). Details for 17 active programs were provided. Five programs included broad PICU populations, while the majority were neurocritical care (53%) focused. Despite strong agreement on the need to assess and treat morbidity across multiple health domains, 29% were physician only programs, and considerable variation existed in services provided by programs across settings. More than 80% of all respondents agreed PFUPs provide direct benefits and are essential to advancing knowledge on long-term PICU outcomes. Respondents identified "lack of support" as the most important barrier, particularly funding for providers and staff, and lack of clinical space, though successful programs overcome this challenge using a variety of funding resources. CONCLUSIONS: Few systematic multidisciplinary PFUPs exist despite strong agreement about importance of this care and direct benefit to patients and families. We recommend stakeholders use our description of successful programs as a framework to develop multidisciplinary models to elevate continuity across inpatient and outpatient settings, improve patient care, and foster collaboration to advance knowledge.

Of mice and men, and chandeliers.

How does the human neocortex reliably propagate information through neural circuits? One mechanism appears to involve relying on strong connections from pyramidal neurons to interneurons and a depolarizing action of cortical chandelier cells.

Long-Term Outcomes and the Post-Intensive Care Syndrome in Critically Ill Children: A North American Perspective.

Advances in medical and surgical care for children in the pediatric intensive care unit (PICU) have led to vast reductions in mortality, but survivors often leave with newly acquired or worsened morbidity. Emerging evidence reveals that survivors of pediatric critical illness may experience a constellation of physical, emotional, cognitive, and social impairments, collectively known as the "post-intensive care syndrome in pediatrics" (PICs-P). The spectrum of PICs-P manifestations within each domain are heterogeneous. This is attributed to the wide age and developmental diversity of children admitted to PICUs and the high prevalence of chronic complex conditions. PICs-P recovery follows variable trajectories based on numerous patient, family, and environmental factors. Those who improve tend to do so within less than a year of discharge. A small proportion, however, may actually worsen over time. There are many gaps in our current understanding of PICs-P. A unified approach to screening, preventing, and treating PICs-P-related morbidity has been hindered by disparate research methodology. Initiatives are underway to harmonize clinical and research priorities, validate new and existing epidemiologic and patient-specific tools for the prediction or monitoring of outcomes, and define research priorities for investigators interested in long-term outcomes.

Prolonged Duration Topical Corneal Anesthesia With the Cationic Lidocaine Derivative QX-314.

PURPOSE: Topical corneal local anesthetics are short acting and may impair corneal healing. In this study we compared corneal anesthesia and toxicity of topically applied N-ethyl lidocaine (QX-314) versus the conventional local anesthetic, proparacaine (PPC). METHODS: Various concentrations of QX-314 and 15 mM (0.5%) PPC were topically applied to rat corneas. Corneal anesthesia was assessed with a Cochet-Bonnet esthesiometer at predetermined time points. PC12 cells were exposed to the same solutions to assess cytotoxicity. Repeated topical corneal administration in rats was then used to assess for histologic evidence of toxicity. Finally, we created uniform corneal epithelial defects in rats and assessed the effect of repeated administration of these compounds on the defect healing rate. RESULTS: QX-314 (15 mM) and PPC (15 mM) caused similar total duration (114 ± 17 and 87 ± 16 minutes, respectively; P = 0.06) of anesthesia. The depth of anesthesia was similar between these low-dose groups at 15 minutes after application (1.8 ± 0.3- and 2.0 ± 0.8-cm filament lengths). QX-314 (100 mM) provided more prolonged corneal anesthesia (174 ± 13 minutes; P < 0.0001), with improved depth at 15 minutes (0.7 ± 0.3-cm filament length; P = 0.007). All tested concentrations of QX-314 demonstrated similar or less toxicity than 0.5% PPC. CONCLUSIONS: Topical administration of QX-314 is effective for corneal anesthesia and demonstrates no histologic signs of local toxicity in a rodent model. In higher concentrations, QX-314 provides more than twofold the duration of anesthetic effect than does 0.5% PPC. TRANSLATIONAL RELEVANCE: Our study reveals a clinically relevant compound providing prolonged duration topical corneal anesthesia.

Networks of parvalbumin-positive interneurons in the basolateral amygdala.

The amygdala is a temporal lobe structure that is required for processing emotional information. Polymodal sensory information enters the amygdala at the level of the basolateral amygdala (BLA) and undergoes local processing, after which the behavioral and autonomic responses that accompany emotions are initiated. Two main neuron types are present in the BLA, pyramidal-like principal neurons that use glutamate as their transmitter, and local circuit interneurons that use GABA as their transmitter. Although the properties of principal neurons are known in some detail, very little is known about the properties of BLA interneurons or the local circuits in which they are involved. Using mice in which EGFP (enhanced green fluorescent protein) is expressed under the control of the parvalbumin promoter, we characterized the properties of parvalbumin-positive interneurons in the BLA. By making recordings from interneuron-interneuron and interneuron-principal neuron pairs, we analyzed the intrinsic circuitry of the BLA. We show that parvalbumin-positive interneurons can be divided into four subtypes as defined by their firing properties. Interneurons are electrically coupled in subtype-specific networks and exhibit subtype-specific heterogeneities in their synaptic dynamics and patterns of connectivity. We propose that these properties allow networks of parvalbumin-expressing neurons to perform an array of information-processing tasks within the BLA.

GABAergic excitation in the basolateral amygdala.

GABA-containing interneurons are a diverse population of cells whose primary mode of action in the mature nervous system is inhibition of postsynaptic target neurons. Using paired recordings from parvalbumin-positive interneurons in the basolateral amygdala, we show that, in a subpopulation of interneurons, single action potentials in one interneuron evoke in the postsynaptic interneuron a monosynaptic inhibitory synaptic current, followed by a disynaptic excitatory glutamatergic synaptic current. Interneuron-evoked glutamatergic events were blocked by antagonists of either AMPA/kainate or GABA(A) receptors, and could be seen concurrently in both presynaptic and postsynaptic interneurons. These results show that single action potentials in a GABAergic interneuron can drive glutamatergic principal neurons to threshold, resulting in both feedforward and feedback excitation. In interneuron pairs that both receive glutamatergic inputs after an interneuron spike, electrical coupling and bidirectional GABAergic connections occur with a higher probability relative to other interneuron pairs. We propose that this form of GABAergic excitation provides a means for the reliable and specific recruitment of homogeneous interneuron networks in the basal amygdala.

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass.

RATIONALE: Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. OBJECTIVES: 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. METHODS AND RESEARCH: Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4µmol/L vs 12.9µmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9µmol/L vs 2.7µmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). CONCLUSIONS: Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to impaired capacity to clear adenosine monophosphate. AP supplementation improves serum clearance of adenosine monophosphate to adenosine. These findings represent a potential therapeutic mechanism for alkaline phosphatase infusion during cardiac surgery. NEW AND NOTEWORTHY: We identify alkaline phosphatase (AP) as the primary soluble ectonucleotidase in infants undergoing cardiopulmonary bypass and show decreased capacity to clear AMP when AP activity decreases post-bypass. Supplementation of AP ex vivo improves this capacity and may represent the beneficial therapeutic mechanism of AP infusion seen in phase 2 studies.

Spatial distribution of neurons innervated by chandelier cells.

Chandelier (or axo-axonic) cells are a distinct group of GABAergic interneurons that innervate the axon initial segments of pyramidal cells and are thus thought to have an important role in controlling the activity of cortical circuits. To examine the circuit connectivity of chandelier cells (ChCs), we made use of a genetic targeting strategy to label neocortical ChCs in upper layers of juvenile mouse neocortex. We filled individual ChCs with biocytin in living brain slices and reconstructed their axonal arbors from serial semi-thin sections. We also reconstructed the cell somata of pyramidal neurons that were located inside the ChC axonal trees and determined the percentage of pyramidal neurons whose axon initial segments were innervated by ChC terminals. We found that the total percentage of pyramidal neurons that were innervated by a single labeled ChC was 18-22 %. Sholl analysis showed that this percentage peaked at 22-35 % for distances between 30 and 60 µm from the ChC soma, decreasing to lower percentages with increasing distances. We also studied the three-dimensional spatial distribution of the innervated neurons inside the ChC axonal arbor using spatial statistical analysis tools. We found that innervated pyramidal neurons are not distributed at random, but show a clustered distribution, with pockets where almost all cells are innervated and other regions within the ChC axonal tree that receive little or no innervation. Thus, individual ChCs may exert a strong, widespread influence on their local pyramidal neighbors in a spatially heterogeneous fashion.

Spatial light modulator microscopy.

The use of spatial light modulators (SLMs) for two-photon laser microscopy is described. SLM phase modulation can be used to generate nearly any spatiotemporal pattern of light, enabling simultaneous illumination of any number of selected regions of interest. We take advantage of this flexibility to perform fast two-photon imaging or uncaging experiments on dendritic spines and neocortical neurons. By operating in the spatial Fourier plane, an SLM can effectively mimic any arbitrary optical transfer function and thus replace, in software, many of the functions provided by hardware in standard microscopes, such as focusing, magnification, and aberration correction.