Layer 6b controls brain state via apical dendrites and the higher-order thalamocortical system.

The deepest layer of the cortex (layer 6b [L6b]) contains relatively few neurons, but it is the only cortical layer responsive to the potent wake-promoting neuropeptide orexin/hypocretin. Can these few neurons significantly influence brain state? Here, we show that L6b-photoactivation causes a surprisingly robust enhancement of attention-associated high-gamma oscillations and population spiking while abolishing slow waves in sleep-deprived mice. To explain this powerful impact on brain state, we investigated L6b's synaptic output using optogenetics, electrophysiology, and monoCaTChR ex vivo. We found powerful output in the higher-order thalamus and apical dendrites of L5 pyramidal neurons, via L1a and L5a, as well as in superior colliculus and L6 interneurons. L6b subpopulations with distinct morphologies and short- and long-term plasticities project to these diverse targets. The L1a-targeting subpopulation triggered powerful NMDA-receptor-dependent spikes that elicited burst firing in L5. We conclude that orexin/hypocretin-activated cortical neurons form a multifaceted, fine-tuned circuit for the sustained control of the higher-order thalamocortical system.

Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig-Tick-Rickettsia System.

The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.

Multiplex TaqMan® Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick-Rickettsia System.

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.

Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis.

Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host-pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.

Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group Rickettsia Exposure.

Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group Rickettsia (SFGR) species. The immunofluorescence antibody assay (IFA) has been the gold standard for the serodiagnosis of rickettsial infections such as spotted fever rickettsiosis (SFR). However, the IFA is semi-quantitative and subjective, requiring a high level of expertise to interpret it correctly. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Rickettsia parkeri infection in the guinea pig. Our ELISA is an objective, quantitative, and high-throughput assay that shows greater sensitivity and resolution in observed titers than the IFA. We methodically optimized relevant parameters in sequence for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR species after tick transmission. Guinea pigs are a useful model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species.

An optimized five-color/seven-parameter flow cytometry panel for immunophenotyping guinea pig peripheral blood lymphocytes.

Guinea pigs are an ideal animal model for the study of several infectious diseases, including tuberculosis, legionellosis, brucellosis, and spotted fever rickettsiosis. In comparison to the murine model, clinical signs in guinea pigs are more representative of disease in humans, the guinea pig immune system is more similar to that of the human, and their large size offers logistic advantages for sample collection while following disease progression. Unfortunately, the advantage of using guinea pigs in biomedical research, particularly in understanding the immune response to infectious agents, is limited in large part by the paucity of available reagents and lack of genetically manipulated strains. Here, we expand the utility of guinea pigs in biomedical research by establishing an optimized five-color/seven-parameter polychromatic flow cytometric assay for immunophenotyping lymphocytes. This assay fills a need for immunophenotyping peripheral blood lymphocytes and is an improvement over current published flow cytometry assays for guinea pigs. We anticipate that our approach will be an important starting point for developing new assays to evaluate the cellular immune response to infectious diseases in the guinea pig model. Importantly, we are currently using this assay for evaluating immunity to spotted fever rickettsiosis in a guinea pig-tick-Rickettsia system, where CD8+ T cells are a critical contributor to the immune response. Developing resources to utilize the guinea pig more effectively will enhance our ability to understand infectious diseases where the guinea pig would otherwise be the ideal model.

Multidimensional fluorescence studies of the phenolic content of dissolved organic carbon in humic substances.

Indicators suggest that the amount of dissolved organic carbon (DOC) in natural waters may be increasing. Climate change has been proposed as a potential contributor to the trend, and under such a mechanism, the phenolic content of DOC may also be increasing. This study explores the assessment of the phenolic character of DOC using multidimensional fluorescence spectroscopy as a more convenient alternative to traditional wet chemistry methods. Parallel factor analysis (PARAFAC) is applied to fluorescence excitation emission matrices (EEMs) of humic samples to analyze inherent phenolic content. The PARAFAC results are correlated with phenol concentrations derived from the Folin-Ciocalteau reagent-based method. The reagent-based method reveals that the phenolic content of five International Humic Substance Society (IHSS) samples varies from approximately 5.2 to 22 ppm Tannic Acid Equivalents (TAE). A four-component PARAFAC fit is applied to the EEMs of the IHSS sample dataset and it is determined by PARAFAC score correlations with phenol concentrations from the reagent-based method that components C2, C3, and C4 have the highest probability of containing phenolic groups. The results show the potential for PARAFAC analysis of multidimensional fluorescence data for monitoring the phenolic content of DOC.