Design strategies for dynamic closed-loop optogenetic neurocontrol in vivo.

OBJECTIVE: Controlling neural activity enables the possibility of manipulating sensory perception, cognitive processes, and body movement, in addition to providing a powerful framework for functionally disentangling the neural circuits that underlie these complex phenomena. Over the last decade, optogenetic stimulation has become an increasingly important and powerful tool for understanding neural circuit function, owing to the ability to target specific cell types and bidirectionally modulate neural activity. To date, most stimulation has been provided in open-loop or in an on/off closed-loop fashion, where previously-determined stimulation is triggered by an event. Here, we describe and demonstrate a design approach for precise optogenetic control of neuronal firing rate modulation using feedback to guide stimulation continuously. APPROACH: Using the rodent somatosensory thalamus as an experimental testbed for realizing desired time-varying patterns of firing rate modulation, we utilized a moving average exponential filter to estimate firing rate online from single-unit spiking measured extracellularly. This estimate of instantaneous rate served as feedback for a proportional integral (PI) controller, which was designed during the experiment based on a linear-nonlinear Poisson (LNP) model of the neuronal response to light. MAIN RESULTS: The LNP model fit during the experiment enabled robust closed-loop control, resulting in good tracking of sinusoidal and non-sinusoidal targets, and rejection of unmeasured disturbances. Closed-loop control also enabled manipulation of trial-to-trial variability. SIGNIFICANCE: Because neuroscientists are faced with the challenge of dissecting the functions of circuit components, the ability to maintain control of a region of interest in spite of changes in ongoing neural activity will be important for disambiguating function within networks. Closed-loop stimulation strategies are ideal for control that is robust to such changes, and the employment of continuous feedback to adjust stimulation in real-time can improve the quality of data collected using optogenetic manipulation.

Bacteraemia during direct laryngoscopy and endotracheal intubation: a study using a multiple culture, large volume technique.

Bacteraemia secondary to orotracheal intubation has been reported to occur in 0-5.3% of patients. Bacteraemia detection is dependent upon several factors including the volume of blood per culture and the number of cultures. Prior studies used small volumes of blood and one or two cultures, and may therefore have underestimated the incidence of bacteraemia. Sixty-two adult patients who underwent direct laryngoscopy and endotracheal intubation were studied. Baseline blood cultures were sterile in all patients. After intubation, four blood cultures were obtained in ten minutes, with 10 ml being evenly divided between aerobic and anaerobic media. Two patients (3.2%) became bacteraemic. This is a lower incidence than occurs in association with other procedures for which The American Heart Association does not recommend administration of prophylactic antibiotics. Therefore, prophylactic antibiotics are not recommended prior to direct laryngoscopy. However, when a prophylactic antibiotic is administered prior to surgery, it would be best to administer the antibiotic prior to direct laryngoscopy and intubation.

Lung cancer model system using 3-methylcholanthrene in inbred strains of mice.

A model system has been established for studying lung carcinogenesis using intratracheal instillation of 3-methylcholanthrene in C3H/AnfCum and C57BL/Cum X C3H/AnfCum F1 (hereafter called BC3F1/Cum) mice. The animals in these studies were screened for adventitious agents and were free throughout their lifetime of two important lung viruses, Sendai virus and pneumonia virus of mice. Under these conditions, the occurrence of spontaneous and chemically indiced lung cancers was determined over the lifetime of the animals. Data were analyzed by the actuarial method for lung tumor probability. Probability was found to be dose and time dependent. Over 95% of the 3-methylcholanthrene-treated BC3F1/Cum and over 88% of the C3H/AnfCum mice were found at death to have pulmonary carcinomas. Tumors observed in animals which died up to 40 weeks on test were almost always squamous cell carcinomas (approximately 85%), while tumors which were observed in animals which died after 50 weeks were mainly alveolar adenocarcinomas (approximately 80%). Both tumors types metastasized widely. Spontaneous lung cancers (only alveolar adenocarcinomas were observed) occurred in these two strains at low frequency and were expressed late in life. Thus, the system described affords a suitable model to study the induction, expression, and progression of lung tumors under conditions where a vast majority of animals develop neoplasia.

Correlation of inducibility of aryl hydrocarbon hydroxylase with susceptibility to 3-methylcholanthrene-induced lung cancers.

C57BL/6Cum, DBA/2Cum, first filia (F1), and backcross progeny from these 2 parental strains of mice were evaluated for their susceptibility to 3-methylcholanthrene-induced lung cancers. In the crosses among these mice, aryl hydrocarbon hydroxylase (AHH) responsiveness segregated as a single autosomal dominant gene (the Ah locus). AHH responsive mice (Ahb allele) expressed 40-60 units AHH activity/g wet wt liver following intraperitoneal treatment with 3-methylcholanthrene (MCA) compared to AHH non-responsive mice (Ahd allele) which expressed 7-11 units AHH activity/g wet wt liver after MCA treatment. Intratracheal administration of 500 microgram MCA for a total of 4 times at weekly intervals yielded a variety of pulmonary cancers, including squamous cell carcinomas, alveolar adenocarcinomas, and adeno-squamous cell carcinomas among mice that survived 1 year after the carcinogen treatment. The AHH responsive C57BL/6Cum, F1, and C57BL/6Cum X F1 animals were much more susceptible to MCA-induced lung cancers than the AHH non-responsive DBA/2Cum mice. The lung cancers were also not randomly distributed in DBA/2Cum X F1 backcross progeny since significantly more lung cancers were found in AHH-responsive progeny than in AHH non-responsive mice. Data support genetic linkage between susceptibility to MCA-induced lung carcinomas and the Ahb allele.

Effects of subcutaneous administration of 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene on spontaneous reticulum cell neoplasms in SJL/J mice.

Inbred 4-week-old and 16-week-old SJL/J mice were treated sc with either 150 or 450 microgram 3-methylcholanthrene (MCA). No difference was noted in their subcutaneous tumor response related to age. Although the incidences of reticulum cell neoplasms (RCN's) were the same in the controls for the 2 age groups, more 16-week-old animals developed RCN's after carcinogen treatment than did 4-week-old SJL/J mice. More 16-week-old mice developed both subcutaneous tumors at the site of MCA injection and systemic RCN's than did mice treated at 4 weeks of age. This incidence was related to the coinciding of the chronologic age of the mice for RCN development with the latency for chemical carcinogen-induced subcutaneous tumor development. Mice treated with 7,12-dimethylbenz[a]anthracene at 16 weeks of age responded in the same way as did those treated with MCA.

Effects of subcutaneous administration of chemical carcinogens on leukemia in C58 mice.

The effects of sc administration of 3-methylcholanthrene (MCA), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (BP) on spontaneous viral leukemia and subcutaneous sarcoma induction have been studied in weanling C58/J mice. MCA produced significantly more sarcomas at the inoculation site than did DMBA or BP; moreover, it interfered with leukemia development. DMBA produced fewer sarcomas, and the incidence of leukemia was comparable to that found in the controls. BP accelerated the incidence of leukemia, although no sarcomas were produced. When the effect of the age of the mice at the time of MCA treatment on the incidence of leukemia and sarcomas was studied, newborn and weanling mice were found to develop primarily sarcomas, whereas no sarcomas were produced in the 16-week-old mice, and 52-64% of the 16-week-old mice developed leukemia. The reason no sarcomas were found in the C58 mice was apparently different from the reason no sarcomas were found in AKR mice, inasmuch as the AKR mice did not live long enough for sarcomas to develop. Immunologic surveillance may have played a part in the sarcoma suppression in the C58 mouse.

Comparison of the effects of beeswax: trioctanoin and trioctanoin vehicles on 3-methylcholanthrene, benzo[a]pyrene, and 7,12-dimethylbenz[a]anthracene subcutaneous carcinogenesis in three strains of mice and one hybrid.

Subcutaneous tumor induction with three dose levels of 3-methylcholanthrene (MCA), benzo[a]pyrene (BP), and 7,12-dimethylbenz[a]anthracene (DMBA) in two vehicles was studied in C3H/Anf Cum, C57BL/6 Cum, DBA/2J, and (C57BLXC3H/Anf)F1 (BC3F1/Cum) mice. Median tumor dose levels were significantly lower when the three carcinogens were suspended in trioctanoin. When beeswax: trioctanoin (B:T) was used as a vehicle, the three carcinogens differed in their abilities to be absorbed or solubilized from the vehicle by the three strains of mice and the hybrid. In C3H/Anf mice, BP in B:T failed to produce tumors. In BC3F1 mice, no tumors were produced by MCA, BP, or DMBA in B:T. In C57BL/6 mice, no tumors were produced with DMBA or MCA in B:T. In DBA/2 mice, no tumors were produced by BP or MCA in B:T. These results indicated that the interpretation of tumor induction results obtained with B:T vehicle may be related to the conditions of bioassay rather than to the carcinogenic potential of a compound.

Immunosuppressive effects of 3-methylcholanthrene given intratracheally in various inbred strains of mice.

The immunosuppressive effects of 3-methylcholanthrene (MCA) given intratracheally have been shown to correlate with susceptibility of some inbred strains of mice to MCA-induced carcinogenesis. In susceptible strains of mice, C3Hf and C57BL/6, intratracheal administration of MCA resulted in profound systemic immunodepression. This effect was not observed in resistant DBA/2 mice. This immunodepressive effect correlated with the inducibility of the aryl hydrocarbon hydroxylase enzyme complex and the inducibility of pulmonary tumors by MCA.

Studies on pulmonary aryl hydrocarbon hydroxylase activity in inbred strains of mice.

Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.