A perspective on astrocyte regulation of neural circuit function and animal behavior

Studies over the past two decades have demonstrated that astrocytes are tightly associated with neurons and play pivotal roles in neural circuit development, operation, and adaptation in health and disease. Nevertheless, precisely how astrocytes integrate diverse neuronal signals, modulate neural circuit structure and function at multiple temporal and spatial scales, and influence animal behavior or disease through aberrant excitation and molecular output remains unclear. This Perspective discusses how new and state-of-the-art approaches, including fluorescence indicators, opto- and chemogenetic actuators, genetic targeting tools, quantitative behavioral assays, and computational methods, might help resolve these longstanding questions. It also addresses complicating factors in interpreting astrocytes' role in neural circuit regulation and animal behavior, such as their heterogeneity, metabolism, and inter-glial communication. Research on these questions should provide a deeper mechanistic understanding of astrocyte-neuron assemblies' role in neural circuit function, complex behaviors, and disease

Inspiratory off-switch mediated by optogenetic activation of inhibitory neurons in the preBötzinger complex in vivo

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 µm multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4–5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex

GABA-glycine cotransmitting neurons in the ventrolateral medulla: development and functional relevance for breathing

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

Adenosine triphosphate (ATP) is the central energy carrier of all cells and knowledge on the dynamics of the concentration of ATP ([ATP]) provides important insights into the energetic state of a cell. Several genetically encoded fluorescent nanosensors for ATP were developed, which allow following the cytosolic [ATP] at high spatial and temporal resolution using fluorescence microscopy. However, to calibrate the fluorescent signal to [ATP] has remained challenging. To estimate basal cytosolic [ATP] ([ATP] ([ATP0) in astrocytes, we here took advantage of two ATP nanosensors of the ATeam-family (ATeam1.03; ATeam1.03YEMK) with different affinities for ATP. Altering [ATP] by external stimuli resulted in characteristic pairs of signal changes of both nanosensors, which depend on [ATP]0. Using this dual nanosensor strategy and epifluorescence microscopy, [ATP]0 was estimated to be around 1.5 mM in primary cultures of cortical astrocytes from mice. Furthermore, in astrocytes in acutely isolated cortical slices from mice expressing both nanosensors after stereotactic injection of AAV-vectors, 2-photon microscopy revealed [ATP]0 of 0.7 mM to 1.3 mM. Finally, the change in [ATP] induced in the cytosol of cultured cortical astrocytes by application of azide, glutamate, and an increased extracellular concentration of KC were calculated as 0.50 mM, 0.16 mM, and 0.07 mM, respectively. In summary, the dual nanosensor approach adds another option for determining the concentration of [ATP] to the increasing toolbox of fluorescent nanosensors for metabolites. This approach can also be applied to other metabolites when two sensors with different binding properties are available

Extinction of cue-evoked food seeking recruits a GABAergic interneuron ensemble in the dorsal medial prefrontal cortex of mice

Animals must quickly adapt food-seeking strategies to locate nutrient sources in dynamically changing environments. Learned associations between food and environmental cues that predict its availability promote food-seeking behaviors. However, when such cues cease to predict food availability, animals undergo 'extinction' learning, resulting in the inhibition of food-seeking responses. Repeatedly activated sets of neurons, or 'neuronal ensembles', in the dorsal medial prefrontal cortex (dmPFC) are recruited following appetitive conditioning and undergo physiological adaptations thought to encode cue-reward associations. However, little is known about how the recruitment and intrinsic excitability of such dmPFC ensembles are modulated by extinction learning. Here, we used in vivo 2-Photon imaging in male Fos-GFP mice that express green fluorescent protein (GFP) in recently behaviorally-activated neurons to determine the recruitment of activated pyramidal and GABAergic interneuron mPFC ensembles during extinction. During extinction, we revealed a persistent activation of a subset of interneurons which emerged from a wider population of interneurons activated during the initial extinction session. This activation pattern was not observed in pyramidal cells, and extinction learning did not modulate the excitability properties of activated neurons. Moreover, extinction learning reduced the likelihood of reactivation of pyramidal cells activated during the initial extinction session. Our findings illuminate novel neuronal activation patterns in the dmPFC underlying extinction of food-seeking, and in particular, highlight an important role for interneuron ensembles in this inhibitory form of learning

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD+ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD+ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K+, typical correlates of neuronal activity, induced a more reduced NADH/NAD+ redox ratio. While application of glutamate decreased [ATP], K+ as well as the combination of glutamate and K+ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K+ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg2+ caused a shift of the NADH/NAD+ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions

Genetic impairment of folate metabolism regulates cortical interneurons and social behavior

IntroductionThe implications of folate deficiency in neuropsychiatric disorders were demonstrated in numerous studies. Genetic deficiency in a key folate metabolism enzyme, MTHFR, is an example of the interaction between genetic and environmental risk factors: the maternal MTHFR deficiency governs in-utero nutrient availability, and the embryo’s Mthfr genotype influences its ability to metabolize folates. Here, we explore how the maternal and offspring Mthfr genotypes affect cortical interneuron densities and distributions, mouse social outcome, and the relation of the different interneuron patterns to cortical excitability.MethodsTwo experiments were conducted to examine the effects of maternal and offspring Mthfr-KO heterozygosity. Mice were tested for direct social interactions (DSIs), repetitive behavior and cortical laminar distribution of interneuron populations expressing glutamate-decarboxylase-65, parvalbumin and somatostatin. Susceptibility to seizure was tested by exposure to pentylenetetrazole (PTZ).ResultsMaternal Mthfr+/− genotype was associated with suppressed social activities and reduced interneuron densities in all layers of the retrosplenial cortex (RSC). Somatostatin density and the somatostatin/parvalbumin ratio in the RSC and frontal cortex positively correlated with social behavior in the mice. An interaction between maternal and offspring Mthfr genotypes resulted in higher susceptibility of wild-type offspring to PTZ induced seizure.DiscussionMaternal folate metabolism was shown to be critical to interneuron ontogenesis. Our results demonstrate that interneurons have a specific susceptibility to folate deficiency that may mediate folate’s involvement in neuropsychiatric disease. The relations between cortical somatostatin interneuron patterns and social behavior highlight this subpopulation of interneurons as a target for further research

Cell Type-Dependent Activation Sequence During Rhythmic Bursting in the PreBötzinger Complex in Respiratory Rhythmic Slices From Mice

Spontaneous respiratory rhythmic burst activity can be preserved in the preBötzinger Complex (preBötC) of rodent medullary transverse slices. It is known, that the activation sequence of inspiratory neurons in the preBötC stochastically varies from cycle to cycle. To test whether the activation timing of an inspiratory neuron depends on its neurotransmitter, we performed calcium imaging of preBötC neurons using double-transgenic mice expressing EGFP in GlyT2+ neurons and tdTomato in GAD65+ neurons. Five types of inspiratory neurons were identified using the fluorescence protein expression and the maximum cross-correlation coefficient between neuronal calcium fluctuation and field potential. Regarding the activation sequence, irregular type putative excitatory (GlyT2-/GAD65-) neurons and irregular type glycinergic (GlyT2+/GAD65-) neurons tended to be activated early, while regular type putative excitatory neurons, regular type glycinergic neurons tended to be activated later. In conclusion, the different cell types define a general framework for the stochastically changing activation sequence of inspiratory neurons in the preBötC

Relation between activity‐induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons

Excitatory neuronal activity results in the influx of Na+ through voltage- and ligand-gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+]i) is mainly mediated by the Na+/K+-ATPase (NKA) and is one of the major energy-consuming processes in the brain. Here, we analysed the relation between different patterns of activity-induced [Na+]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium-binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+]i oscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3–0.4 mm in both compartments. Global [Na+]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i. Our results suggest that ATP consumption by the NKA following global [Na+]i transients temporarily overrides its availability, causing a decrease in [ATP]i. Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i, suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions