Cholinergic stimulation stabilizes TRPM4 in the plasma membrane of cortical pyramidal neurons.

TRPM4 is a calcium activated non-selective cation channel, impermeable to Ca2+, in neurons it has been implicated in the regulation of the excitability and in the persistent firing. Cholinergic stimulation is also implicated in changes in excitability that leads neurons to an increased firing frequency, however it is not clear whether TRPM4 is involved in the cholinergic-induced increase in firing frequency. Here using a combination of patch clamp electrophysiology, Ca2+ imaging, immunofluorescence, fluorescence recovery after photobleaching (FRAP) and pharmacological approach, we demonstrate that carbachol (Cch) increases firing frequency, intracellular Ca2+ and that TRPM4 inhibition using 9-Ph and CBA reduces firing frequency and decreases the peak in intracellular Ca2+ induced by Cch in cortical pyramidal neurons in culture. Moreover, we determined that cholinergic stimulation reduces TRPM4 recycling and stabilizes TRPM4 in the plasma membrane. Together our results indicate that cholinergic stimulation increases firing in a TRPM4 dependent manner, and also increases the TRPM4 stability in the membrane, suggesting that TRPM4 is locked in microdomains in the membrane, possibly signaling or cytoskeleton proteins complexes.

The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

BACKGROUND AND PURPOSE: The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels. EXPERIMENTAL APPROACH: Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice. KEY RESULTS: Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism. CONCLUSION AND IMPLICATIONS: These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors.

Untangling Macropore Formation and Current Facilitation in P2X7.

Macropore formation and current facilitation are intriguing phenomena associated with ATP-gated P2X7 receptors (P2X7). Macropores are large pores formed in the cell membrane that allow the passage of large molecules. The precise mechanisms underlying macropore formation remain poorly understood, but recent evidence suggests two alternative pathways: a direct entry through the P2X7 pore itself, and an indirect pathway triggered by P2X7 activation involving additional proteins, such as TMEM16F channel/scramblase. On the other hand, current facilitation refers to the progressive increase in current amplitude and activation kinetics observed with prolonged or repetitive exposure to ATP. Various mechanisms, including the activation of chloride channels and intrinsic properties of P2X7, have been proposed to explain this phenomenon. In this comprehensive review, we present an in-depth overview of P2X7 current facilitation and macropore formation, highlighting new findings and proposing mechanistic models that may offer fresh insights into these untangled processes.

Optical control of PIEZO1 channels.

PIEZO proteins are unusually large, mechanically-activated trimeric ion channels. The central pore features structural similarities with the pore of other trimeric ion channels, including purinergic P2X receptors, for which optical control of channel gating has been previously achieved with photoswitchable azobenzenes. Extension of these chemical optogenetics methods to mechanically-activated ion channels would provide tools for specific manipulation of pore activity alternative to non-specific mechanical stimulations. Here we report a light-gated mouse PIEZO1 channel, in which an azobenzene-based photoswitch covalently tethered to an engineered cysteine, Y2464C, localized at the extracellular apex of the transmembrane helix 38, rapidly triggers channel gating upon 365-nm-light irradiation. We provide evidence that this light-gated channel recapitulates mechanically-activated PIEZO1 functional properties, and show that light-induced molecular motions are similar to those evoked mechanically. These results push the limits of azobenzene-based methods to unusually large ion channels and provide a simple stimulation means to specifically interrogate PIEZO1 function.

Photo-isomerizable tweezers to probe ionotropic receptor mechanisms.

Ligand-gated ion channels (LGIC, also referred to as ionotropic receptors) are important transmembrane proteins that open to allow ions to flow across the membrane and locally modify the membrane potential in response to the binding of a ligand. For more than a decade, a tremendous effort has been carried out in the determination of many LGIC structures in high resolution, leading to an unprecedented molecular description of channel gating. However, it is sometimes difficult to classify experimentally derived structures to their corresponding functional states, and alternative methods may help resolve or refine this issue. In this review, we focus on the application of photo-isomerizable tweezers (PIT) as a powerful strategy to interrogate molecular mechanisms of LGIC while assessing their functionality by electrophysiology.

P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation.

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

Hybrid QM/MM Simulations Confirm Zn(II) Coordination Sphere That Includes Four Cysteines from the P2 × 4R Head Domain.

To ascertain the role of Zn(II) as an allosteric modulator on P2X4R, QM/MM molecular dynamic simulations were performed on the WT and two P2X4R mutants suggested by previous electrophysiological data to affect Zn(II) binding. The Gibbs free energy for the reduction of the putative P2X4R Zn(II) binding site by glutathione was estimated at -22 kcal/mol. Simulations of the WT P2X4R head domain revealed a flexible coordination sphere dominated by an octahedral geometry encompassing C126, N127, C132, C149, C159 and a water molecule. The C132A mutation disrupted the metal binding site, leading to a coordination sphere with a majority of water ligands, and a displacement of the metal ion towards the solvent. The C132A/C159A mutant exhibited a tendency towards WT-like stability by incorporating the R148 backbone to the coordination sphere. Thus, the computational findings agree with previous experimental data showing Zn(II) modulation for the WT and C132A/C159A variants, but not for the C132A mutant. The results provide molecular insights into the nature of the Zn(II) modulation in P2X4R, and the effect of the C132A and C132A/C159A mutations, accounting for an elusive modulation mechanism possibly occurring in other extracellular or membrane protein.

ICAN (TRPM4) Contributes to the Intrinsic Excitability of Prefrontal Cortex Layer 2/3 Pyramidal Neurons.

Pyramidal neurons in the medial prefrontal cortical layer 2/3 are an essential contributor to the cellular basis of working memory; thus, changes in their intrinsic excitability critically affect medial prefrontal cortex (mPFC) functional properties. Transient Receptor Potential Melastatin 4 (TRPM4), a calcium-activated nonselective cation channel (CAN), regulates the membrane potential in a calcium-dependent manner. In this study, we uncovered the role of TRPM4 in regulating the intrinsic excitability plasticity of pyramidal neurons in the mouse mPFC layer of 2/3 using a combination of conventional and nystatin perforated whole-cell recordings. Interestingly, we found that TRPM4 is open at resting membrane potential, and its inhibition increases input resistance and hyperpolarizes membrane potential. After high-frequency stimulation, pyramidal neurons increase a calcium-activated non-selective cation current, increase the action potential firing, and the amplitude of the afterdepolarization, these effects depend on intracellular calcium. Furthermore, pharmacological inhibition or genetic silencing of TRPM4 reduces the firing rate and the afterdepolarization after high frequency stimulation. Together, these results show that TRPM4 plays a significant role in the excitability of mPFC layer 2/3 pyramidal neurons by modulating neuronal excitability in a calcium-dependent manner.

Evaluation of trypanocidal properties of ferrocenyl and cyrhetrenyl N-acylhydrazones with pendant 5-nitrofuryl group.

Four N-acylhydrazones of general formulae [R1-C(O)-NH-N=C(R2)(5-nitrofuryl)] with (R1 = ferrocenyl or cyrhetrenyl and R2 = H or Me) are synthesized and characterized in solution and in the solid-state. Comparative studies of their stability in solution under different experimental conditions and their electrochemical properties are reported. NMR studies reveal that the four compounds are stable in DMSO­d6 and complementary UV-Vis studies confirm that they also exhibit high stability in mixtures DMSO:H2O at 37 °C. Electrochemical studies show that the half-wave potential of the nitro group of the N-acylhydrazones is smaller than that of the standard drug nifurtimox and the reduction process follows a self-protonation mechanism. In vitro studies on the antiparasitic activities of the four complexes and the nifurtimox against Trypanosoma cruzi and Trypanosoma brucei reveal that: i) the N-acylhydrazones have a potent inhibitory growth activity against both parasites [EC50 in the low micromolar (in T. cruzi) or even in the nanomolar (in T. brucei) range] and ii) cyrhetrenyl derivatives are more effective than their ferrocenyl analogs. Parallel studies on the L6 rat skeletal myoblast cell line have also been conducted, and the selectivity indexes determined. Three of the four N-acylhydrazones showed higher selectivity towards T. brucei than the standard drug nifurtimox. Additional studies suggest that the organometallic compounds are bioactivated by type I nitroreductase enzymes.

New Insights of the Zn(II)-Induced P2 × 4R Positive Allosteric Modulation: Role of Head Receptor Domain SS2/SS3, E160 and D170.

P2 × 4R is allosterically modulated by Zn(II), and despite the efforts to understand the mechanism, there is not a consensus proposal; C132 is a critical amino acid for the Zn(II) modulation, and this residue is located in the receptor head domain, forming disulfide SS3. To ascertain the role of the SS2/SS3 microenvironment on the rP2 × 4R Zn(II)-induced allosteric modulation, we investigated the contribution of each individual SS2/SS3 cysteine plus carboxylic acid residues E118, E160, and D170, located in the immediate vicinity of the SS2/SS3 disulfide bonds. To this aim, we combined electrophysiological recordings with protein chemical alkylation using thiol reagents such as N-ethylmaleimide or iodoacetamide, and a mutation of key amino acid residues together with P2 × 4 receptor bioinformatics. P2 × 4R alkylation in the presence of the metal obliterated the allosteric modulation, a finding supported by the site-directed mutagenesis of C132 and C149 by a corresponding alanine. In addition, while E118Q was sensitive to Zn(II) modulation, the wild type receptor, mutants E160Q and D170N, were not, suggesting that these acid residues participate in the modulatory mechanism. Poisson-Boltzmann analysis indicated that the E160Q and D170N mutants showed a shift towards more positive electrostatic potential in the SS2/SS3 microenvironment. Present results highlight the role of C132 and C149 as putative Zn(II) ligands; in addition, we infer that acid residues E160 and D170 play a role attracting Zn(II) to the head receptor domain.

Loss of Social/Non-social Context Discrimination by Movement Acceleration in the Valproate Model of Autism.

Autism spectrum disorder (ASD) is a neurodevelopmental alteration characterized by social/communicative deficits, repetitive/stereotyped movements, and restricted/obsessive interests. However, there is not much information about whether movement alterations in ASD comprise modifications at the basic kinematic level, such as trajectory and velocity, which may contribute to the higher level of processing that allows the perception and interpretation of actions performed by others, and hence, impact social interaction. In order to further explore possible motor alterations in ASD, we analyzed movement parameters in the Valproate (VPA) animal model of autism. We found that VPA-treated rats displayed greater movement acceleration, reduced distance between stops, spent more time in the corner of the open-field arena, and executed a number of particular behaviors; for example, supported rearing and circling, with no major changes in distance and velocity. However, in the social interaction test, we found other alterations in the movement parameters. In addition to increased acceleration, VPA-rats displayed reduced velocity, increased stops, reduced distance/stop and lost the social/non-social area discrimination that is characteristic of control rats in acceleration and stops variables. Hence, even if prenatal VPA-treatment could have a minor effect in motor variables in a non-social context, it has a crucial effect in the capacity of the animals to adjust their kinematic variables when social/non-social context alternation is required.

Region-Specific Reduction of BDNF Protein and Transcripts in the Hippocampus of Juvenile Rats Prenatally Treated With Sodium Valproate.

Autism is a neurodevelopmental disorder characterized by a deep deficit in language and social interaction, accompanied by restricted, stereotyped and repetitive behaviors. The use of genetic autism animal models has revealed that the alteration of the mechanisms controlling the formation and maturation of neural circuits are points of convergence for the physiopathological pathways in several types of autism. Brain Derived Neurotrophic Factor (BDNF), a key multifunctional regulator of brain development, has been related to autism in several ways. However, its precise role is still elusive, in part, due to its extremely complex posttranscriptional regulation. In order to contribute to this topic, we treated prenatal rats with Valproate, a well-validated model of autism, to analyze BDNF levels in the hippocampus of juvenile rats. Valproate-treated rats exhibited an autism-like behavioral profile, characterized by a deficit in social interaction, anxiety-like behavior and repetitive behavior. In situ hybridization (ISH) experiments revealed that Valproate reduced BDNF mRNA, especially long-3'UTR-containing transcripts, in specific areas of the dentate gyrus (DG) and CA3 regions. At the same time, Valproate reduced BDNF immunoreactivity in the suprapyramidal and lucidum layers of CA3, but improved hippocampus-dependent spatial learning. The molecular changes reported here may help to explain the cognitive and behavioral signs of autism and reinforce BDNF as a potential molecular target for this neurodevelopmental disorder.

Characteristics, Structure, and Effects of an On-Line Tool for Improvement in Adolescents' Competency for Interaction With Alcohol: The e-ALADOTM Utility.

This research report aims to present the characteristics, structure and effects of a psychoeducational technological innovation (called the e-ALADO Program) for the prevention of alcohol intake in adolescents. Based on the Competency model for interaction with alcohol, this program consists of a total of 24 lessons that promote conceptual, procedural, and attitudinal learning, in ICT format (www.alado.es). The hypothesis of this validation study established that adolescents treated with the program would improve their levels of competence and their interaction behavior with alcohol, depending of their personal level of self-regulation. A total of 148 adolescents from 12 to 16 years of age from three Spanish educational centers with different sociocultural contexts participated. A quasi-experimental methodology with repeated measures and use of inferential analysis was used (ANOVAs and MANOVAs). The results show a main principal effect of the Treatment variable, of the Self-Regulation Level variable, and an interaction effect of Treatment × Self-regulation in the conceptual and attitudinal subcompetence for interaction with alcohol. The results are discussed in the face of new technological developments that allow the evaluation and intervention in the prevention of alcohol intake with adolescents. An important implication of this work is related to the importance of self-regulation as a psychological variable. Also, the suitability of psychoeducational interventions with new technological formats in the prevention of adolescents' alcohol intake as entrepreneurial activity.

A Review on the General Stability of Mood Disorder Diagnoses Along the Lifetime.

PURPOSE OF REVIEW: The purpose of this review is to review the most recent literature regarding diagnostic stability of mood disorders, focusing on epidemiological, clinical-psychopathological, and neurobiological data for unipolar and bipolar affective disorders. RECENT FINDINGS: Unipolar depression follows a chronic course in at least half of all cases and presents a considerable diagnostic stability across all age ranges. Studies using latent class analysis are allowing improved profiling of depressive subtypes and assessment of their prevalence. Advances have been made in our understanding of the neurobiological underpinnings of depression, with data highlighting the roles of amyloid deposits, the ApoE4 allele, and atrophy of the anterior hippocampus or frontal cortex. The diagnostic instability of bipolar disorder is manifest in the early years, seen in both the extent of diagnostic delay and the high rate of diagnostic conversion from unipolar depression. Regarding disruptive mood dysregulation disorder, we have little data to date, but those which exist indicate a high rate of comorbidity and minimal diagnostic stability for this disorder. Diagnostic stability varies substantially among mood disorders, which would be related to the validity of current diagnostic categories and our diagnostic accuracy.

The Adolescent's Competency for Interacting with Alcohol as a Determinant of Intake: The Role of Self-Regulation.

The competency for interacting with alcohol is a highly useful Educational Psychology model for preventing and for understanding the different behavioral levels of this interaction. Knowledge of facts, concepts and principles about alcohol use, self-regulated behavior, and attitudes toward alcohol are predictive of adequate interaction with alcohol. The objective of this study was to empirically evaluate this postulated relationship. A total of 328 Spanish adolescents participated, between the ages of 12 and 17. All were enrolled in 1st-4th year of compulsory secondary education, in the context of the ALADO Program for prevention of alcohol intake in adolescents. An ex post facto design was used, with inferential analyses and SEM analyses. Results show an interdependence relationship, with significant structural prediction between the behavioral levels defined and the level of alcohol intake, with principles, self-regulating control and attitudes carrying more weight. Analyses are presented, as are implications for psychoeducational intervention using preventive programs based on this competency model.

Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators.

Prenatal valproate treatment produces autistic-like behavior and increases metabotropic glutamate receptor 1A-immunoreactivity in the hippocampus of juvenile rats.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by deficits in social communication and social interaction, and repetitive and stereotypical patterns of behavior. Previously, a common physiopathological pathway, involving the control of synaptic protein synthesis, was proposed as a convergence point in ASD. In particular, a role for local mRNA translation activated by class I metabotropic glutamate receptor type 5 (mGluR5) was suggested in genetic syndromes with autistic signs and in the prenatal exposition to the valproate model of autism. However, the role of the other members of class I metabotropic glutamate receptors, including mGluR1, has been poorly studied. The present study analyzed the immunoreactivity for mGluR1a in the hippocampus of rats prenatally treated with valproate. Pregnant dams (embryonic day 12.5) were injected with valproate (450 mg/kg) and subsequently, the behavior and mGluR1a were evaluated at postnatal day 30. Experimental rats exhibited social deficit, repetitive conduct and anxious behaviors compared with that of the control animals. Additionally, the present study observed an increased level of mGluR1a-immunoreactivity in the hilus of dentate gyrus and in the CA1 alveus region of the hippocampus. These results suggested an over­functioning of mGluR1a signaling in the hippocampus, induced in the valproate model of autism, which may serve a role in cognitive and behavioral signs of ASD.

Aspectos da esfingofauna (lepidoptera, sphingidae), em área de terra-firme, no estado do Amazonas, Brasil

Abundance, richness, flight hour, sex ratio of Sphingidae were recorded at Itacoatiara, State of Amazonas, on a disturbed area of upland rainforest brazilian Amazon during thirten months (40 nights) between july/1990 - july/1991. 61 species including all collected material were obtained with predominance of Dilophonotini (29 species) followed by Sphingini (14), Philampelini (7), Smerinthini (6) and Macroglossini (5). The community of Sphingidae is compared with others localities data of Neotropical region (South and Central America).

Abundância, riqueza, horário de vôo, razão sexual de Sphingidae são estudadas no Município de Itacoatiara, Estado do Amazonas, em área perturbada de terra-firme na Amazônia brasileira, durante treze meses consecutivos (40 noites), entre julho/1990 - julho/1991. Obteve-se 61 espécies, incluindo todo material coletado, com a predominância de Dilophonotini (29 espécies), seguida por Sphingini (14), Philampelini (7), Smerinthini (6) e Macroglossini (5). A comunidade de Sphingidae é comparada com dados de outras localidades da região Neotropical (Américas do Sul e Central).

Antimicrobianos en diarrea aguda / Antimicrobial agents in acute diarrhea

Las enfermedades diarreicas son una de las causas más frecuentes de morbilidad y mortalidad en niños, siendo responsables de alrededor de 3.3 millones de muertes al año en países en vías de desarrollo, la mayoría por deshidratación. Los virus son la causa principal de las diarreas deshidratantes en niños menores de 2 años; no se conoce tratamiento antimicrobiano para ellos, pero la diarrea que producen es de carácter autolimitado si se previenen correctamente la deshidratación y la desnutrición, que son las complicaciones más frecuentes. Se analizan los mecanismos de producción de diarrea por distintas etiologías, esquemas de tratamiento apropiados y efectos indeseables de los distintos antimicrobianos. Se concluye que no es conveniente usar antimicrobianos de rutina en diarrea aguda y que sólo están indicados en casos de diarrea con sangre (disentería), cólera, giardiasis o en pacientes inmunocomprometidos. El resto de los pacientes la diarrea aguda es autolimitada

Thermodynamics in nests of two melipona species in brasil

SUMMARY Thermodynamics within 10 nests of Melipona rufiventris and M. seminigra were recorded during 48 horurs with thermocouple probes. Strikingly similar patterns were found for both species. Homeostasis did not occur; temperatures within the brood area, honey and pollen stored in pots and nest cavity space all followed ambient temperature fluctuations. Nest temperatures were consistently higher than ambient in all portions of the nest except the upper extremith of vertically elongate hives. Near the brood, temperature fluctuations were damped and displayed a time lag of one to two hours in following ambient temperature. The thoracic temperature of resting worker bees was near 34°C, and the average brood temperature was 31 32°. The involucrum surrounding the brood retained a portion of radiated heat from immatures and workers resting between combs, and brood temperature was two to three degrees higher than internal nest temperature immediately outside the involucrum. The brood chamber, the largest nest structure, contained from 2000 to 6000 immatures, and adult bee populations were less than 1000. The brood nest acts as a heat source at the base of the nest and dissipates heat upwards, creating a thermal gradient. Immature bees appear to supply most of the heat for the nest, and excess heat is shunted by fanning workers through the nest entrance, usually connected to the brood area. There is no evidence of evaporative cooling from water brought into the nest in these or other species of Meliponinae.

Resumo A termodinâmica em 10 ninhos de Molipona rufiventris paraensis e Melipona seminigra merrillae foi registrada através de sensores termoelétricos. Padrões similares foram encontrados para ambas as espécies. Aparentemente não ocorre homeostasis, já que as temperaturas internas registradas da câmara de cría, dos potes de armazenamento de mel e de pólen e a do espaço da cavidade do ninho todos acompanhavam as flutuações da temperatura do meio ambiente. No entanto, as temperaturas registradas no ninho foram consideravelmente mais altas que as do meio ambiente em todas as porções do mesmo fazendo exceção a extremidade superior das colmeias verticais. Perto da cría as flutuações de temperatura acompanhavam as flutuações extranidais (do meio ambiente) com uma defasagem de 1 a 2 horas. A temperatura torácica das operárias pousadas nos favos foi perto de 34°C e a média da cría entre 31° e 32°C. O invólucro ao redor da cría retínha uma porção da radiação térmica dos imaturos e das operárias pousadas entre os favos e a temperatura da cría foi de 2 a 3°C mais alta do que a temperatura do ninho imediatamente fora do invólucro. A câmara de cría continha de 2.000 a 6.000 imaturos e a população de adultos foi menor do que 1.000. A cría do ninho atua como uma fonte térmica na base do ninho dissipando calor para a câmara criando um gradiente térmico. As abelhas imaturas parecem acrescentar mais energia térmica para o ninho.