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Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

New York University Langone Medical Center

In addition to being essential supporters of neuronal function, astrocytes are now recognized as active elements in the brain. Astrocytes sense and integrate synaptic activity and, depending on intracellular Ca(2+) levels, release gliotransmitters (e.g. glutamate, d-serine and ATP) that have feedback actions on neurons. Recent experimental results have raised the possibility that quantitative variations in gliotransmission might contribute to disorders of the nervous system. Here, we discuss targeted molecular genetic approaches that have demonstrated that alterations in protein expression in astrocytes can lead to serious changes in neuronal function. We also introduce the concept of 'astrocyte activation spectrum' in which enhanced and reduced gliotransmission might contribute to epilepsy and schizophrenia, respectively. The results of future experimental tests of the astrocyte activation spectrum, which relates gliotransmission to neurological and psychiatric disorders, might point to a new therapeutic target in the brain.

The tripartite synapse: roles for gliotransmission in health and disease.

In the mammalian brain, astrocytes modulate neuronal function, in part, by synchronizing neuronal firing and coordinating synaptic networks. Little, however, is known about how this is accomplished from a structural standpoint. To investigate the structural basis of astrocyte-mediated neuronal synchrony and synaptic coordination, the three-dimensional relationships between cortical astrocytes and neurons was investigated. Using a transgenic and viral approach to label astrocytes with enhanced green fluorescent protein, we performed a three-dimensional reconstruction of astrocytes from tissue sections or live animals in vivo. We found that cortical astrocytes occupy nonoverlapping territories similar to those described in the hippocampus. Using immunofluorescence labeling of neuronal somata, a single astrocyte enwraps on average four neuronal somata with an upper limit of eight. Single-neuron dye-fills allowed us to estimate that one astrocyte contacts 300-600 neuronal dendrites. Together with the recent findings showing that glial Ca2+ signaling is restricted to individual astrocytes in vivo, and that Ca2+ signaling leads to gliotransmission, we propose the concept of functional islands of synapses in which groups of synapses confined within the boundaries of an individual astrocyte are modulated by the gliotransmitter environment controlled by that astrocyte. Our description offers a new structurally based conceptual framework to evaluate functional data involving interactions between neurons and astrocytes in the mammalian brain.

Synaptic islands defined by the territory of a single astrocyte.

Astrocytes modulate neuronal activity by releasing chemical transmitters via a process termed gliotransmission. The role of this process in the control of behavior is unknown. Since one outcome of SNARE-dependent gliotransmission is the regulation of extracellular adenosine and because adenosine promotes sleep, we genetically inhibited the release of gliotransmitters and asked if astrocytes play an unsuspected role in sleep regulation. Inhibiting gliotransmission attenuated the accumulation of sleep pressure, assessed by measuring the slow wave activity of the EEG during NREM sleep, and prevented cognitive deficits associated with sleep loss. Since the sleep-suppressing effects of the A1 receptor antagonist CPT were prevented following inhibition of gliotransmission and because intracerebroventricular delivery of CPT to wild-type mice mimicked the transgenic phenotype, we conclude that astrocytes modulate the accumulation of sleep pressure and its cognitive consequences through a pathway involving A1 receptors.

Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss.

Astrocytes are known to release several transmitters to impact neuronal activity. Cell-specific molecular genetic attenuation of vesicular release has shown that ATP is a primary astrocytic transmitter in situ and in vivo. In this review, we discuss the biology of astrocytic ATP release highlighting the exciting discovery that lysosomes might be primary stores for the release of this gliotransmitter. In addition, we discuss the role of ATP and its metabolite adenosine on synaptic transmission and the coordination of synaptic networks. Finally, we discuss the recent elucidation of the involvement of this form of glial signaling in the modulation of mammalian behavior. By controlling neuronal A1-receptor signaling, astrocytes modulate mammalian sleep homeostasis and are essential for mediating the cognitive consequences of sleep deprivation. These discoveries begin to paint a new picture of brain function in which slow-signaling glia modulate fast synaptic transmission and neuronal firing to impact behavioral output. Because these cells have privileged access to synapses, they may be valuable targets for the development of novel therapies for many neurological and psychiatric conditions.

Tripartite synapses: roles for astrocytic purines in the control of synaptic physiology and behavior.

Gliotransmission, the release of molecules from astrocytes, regulates neuronal excitability and synaptic transmission in situ. Whether this process affects neuronal network activity in vivo is not known. Using a combination of astrocyte-specific molecular genetics, with in vivo electrophysiology and pharmacology, we determined that gliotransmission modulates cortical slow oscillations, a rhythm characterizing nonrapid eye movement sleep. Inhibition of gliotransmission by the expression of a dominant negative SNARE domain in astrocytes affected cortical slow oscillations, reducing the duration of neuronal depolarizations and causing prolonged hyperpolarizations. These network effects result from the astrocytic modulation of intracortical synaptic transmission at two sites: a hypofunction of postsynaptic NMDA receptors, and by reducing extracellular adenosine, a loss of tonic A1 receptor-mediated inhibition. These results demonstrate that rhythmic brain activity is generated by the coordinated action of the neuronal and glial networks.

Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo.

The past decade has seen an explosion of research on roles of neuron-astrocyte interactions in the control of brain function. We highlight recent studies performed on the tripartite synapse, the structure consisting of pre- and postsynaptic elements of the synapse and an associated astrocytic process. Astrocytes respond to neuronal activity and neurotransmitters, through the activation of metabotropic receptors, and can release the gliotransmitters ATP, d-serine, and glutamate, which act on neurons. Astrocyte-derived ATP modulates synaptic transmission, either directly or through its metabolic product adenosine. d-serine modulates NMDA receptor function, whereas glia-derived glutamate can play important roles in relapse following withdrawal from drugs of abuse. Cell type-specific molecular genetics has allowed a new level of examination of the function of astrocytes in brain function and has revealed an important role of these glial cells that is mediated by adenosine accumulation in the control of sleep and in cognitive impairments that follow sleep deprivation.

Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior.

Although astrocytes are increasingly recognized as important modulators of neuronal excitability and information transfer at the synapse, whether these cells regulate neuronal network activity has only recently started to be investigated. In this article, we highlight the role of astrocytes in the modulation of circuit function with particular focus on sleep-related rhythmogenesis. We discuss recent data showing that these glial cells regulate slow oscillations, a specific thalamocortical activity that characterizes non-REM sleep, and sleep-associated behaviors. Based on these findings, we predict that our understanding of the genesis and tuning of thalamocortical rhythms will necessarily go through an integrated view of brain circuits in which non-neuronal cells can play important neuromodulatory roles.

Integrated brain circuits: neuron-astrocyte interaction in sleep-related rhythmogenesis.

Thalamocortical dynamics, the millisecond to second changes in activity of thalamocortical circuits, are central to perception, action and cognition. Generated by local circuitry and sculpted by neuromodulatory systems, these dynamics reflect the expression of vigilance states. In sleep, thalamocortical dynamics are thought to mediate "offline" functions including memory consolidation and synaptic scaling. Here, I discuss thalamocortical sleep dynamics and their modulation by the ascending arousal system and locally released neurochemicals. I focus on modulation of these dynamics by electrically silent astrocytes, highlighting the role of purinergic signaling in this glial form of communication. Astrocytes modulate cortical slow oscillations, sleep behavior, and sleep-dependent cognitive function. The discovery that astrocytes can modulate sleep dynamics and sleep-related behaviors suggests a new way of thinking about the brain, in which integrated circuits of neurons and glia control information processing and behavioral output.

Thalamocortical dynamics of sleep: roles of purinergic neuromodulation.

Sleep deprivation (SD) can have a negative impact on cognitive function, but the mechanism(s) by which SD modulates memory remains unclear. We have previously shown that astrocyte-derived adenosine is a candidate molecule involved in the cognitive deficits following a brief period of SD (Halassa et al., 2009). In this study, we examined whether genetic disruption of soluble N-ethylmaleimide-sensitive factor attached protein (SNARE)-dependent exocytosis in astrocytes (dnSNARE mice) or pharmacological blockade of A1 receptor signaling using an adenosine A1 receptor (A1R) antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), could prevent the negative effects of 6 h of SD on hippocampal late-phase long-term potentiation (L-LTP) and hippocampus-dependent spatial object recognition memory. We found that SD impaired L-LTP in wild-type mice but not in dnSNARE mice. Similarly, this deficit in L-LTP resulting from SD was prevented by a chronic infusion of CPT. Consistent with these results, we found that hippocampus-dependent memory deficits produced by SD were rescued in dnSNARE mice and CPT-treated mice. These data provide the first evidence that astrocytic ATP and adenosine A1R activity contribute to the effects of SD on hippocampal synaptic plasticity and hippocampus-dependent memory, and suggest a new therapeutic target to reverse the hippocampus-related cognitive deficits induced by sleep loss.

Astrocyte-derived adenosine and A1 receptor activity contribute to sleep loss-induced deficits in hippocampal synaptic plasticity and memory in mice.

The thalamic reticular nucleus (TRN) is hypothesized to regulate neocortical rhythms and behavioral states. Using optogenetics and multi-electrode recording in behaving mice, we found that brief selective drive of TRN switched the thalamocortical firing mode from tonic to bursting and generated state-dependent neocortical spindles. These findings provide causal support for the involvement of the TRN in state regulation in vivo and introduce a new model for addressing the role of this structure in behavior.

Selective optical drive of thalamic reticular nucleus generates thalamic bursts and cortical spindles.

Integrated within neural circuits, astrocytes have recently been shown to modulate brain rhythms thought to mediate sleep function. Experimental evidence suggests that local impact of astrocytes on single synapses translates into global modulation of neuronal networks and behavior. We discuss these findings in the context of current conceptual models of sleep generation and function, each of which have historically focused on neural mechanisms. We highlight the implications and the challenges introduced by these results from a conceptual and computational perspective. We further provide modeling directions on how these data might extend our knowledge of astrocytic properties and sleep function. Given our evolving understanding of how local cellular activities during sleep lead to functional outcomes for the brain, further mechanistic and theoretical understanding of astrocytic contribution to these dynamics will undoubtedly be of great basic and translational benefit.

Astrocyte regulation of sleep circuits: experimental and modeling perspectives.

Sleep impairments are comorbid with a variety of neurological and psychiatric disorders including depression, epilepsy, and alcohol abuse. Despite the prevalence of these disorders, the cellular mechanisms underlying the interaction between sleep disruption and behavior remain poorly understood. In this study, the impact of chronic sleep loss on sleep homeostasis was examined in C57BL/6J mice following 3 d of sleep restriction. The electroencephalographic power of slow-wave activity (SWA; 0.5-4 Hz) in nonrapid eye movement (NREM) sleep and adenosine tone were measured during and after sleep restriction, and following subsequent acute sleep deprivation. During the first day of sleep restriction, SWA and adenosine tone increased, indicating a homeostatic response to sleep loss. On subsequent days, SWA declined, and this was accompanied by a corresponding reduction in adenosine tone caused by a loss of one source of extracellular adenosine. Furthermore, the response to acute sleep deprivation (6 h) was significantly attenuated in sleep-restricted mice. These effects were long-lasting with reduced SWA and adenosine tone persisting for at least 2 weeks. To investigate the behavioral consequences of chronic sleep restriction, sensitivity to the motor-impairing effects of alcohol was also examined. Sleep-restricted mice were significantly less sensitive to alcohol when tested 24 h after sleep restriction, an effect that persisted for 4 weeks. Intracerebroventricular infusion of an adenosine A1 receptor antagonist produced a similar decrease in sensitivity to alcohol. These results suggest that chronic sleep restriction induces a sustained impairment in adenosine-regulated sleep homeostasis and consequentially impacts the response to alcohol.

Chronic sleep restriction disrupts sleep homeostasis and behavioral sensitivity to alcohol by reducing the extracellular accumulation of adenosine.

Behavioral state is known to influence interactions between thalamus and cortex, which are important for sensation, action, and cognition. The thalamic reticular nucleus (TRN) is hypothesized to regulate thalamo-cortical interactions, but the underlying functional architecture of this process and its state dependence are unknown. By combining the first TRN ensemble recording with psychophysics and connectivity-based optogenetic tagging, we found reticular circuits to be composed of distinct subnetworks. While activity of limbic-projecting TRN neurons positively correlates with arousal, sensory-projecting neurons participate in spindles and show elevated synchrony by slow waves during sleep. Sensory-projecting neurons are suppressed by attentional states, demonstrating that their gating of thalamo-cortical interactions is matched to behavioral state. Bidirectional manipulation of attentional performance was achieved through subnetwork-specific optogenetic stimulation. Together, our findings provide evidence for differential inhibition of thalamic nuclei across brain states, where the TRN separately controls external sensory and internal limbic processing facilitating normal cognitive function. PAPERFLICK:

Michael M. Halassa

The Neuroscience Institute

New York University Langone Medical Center

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

Michael M. Halassa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Neuroscience Institute

New York University Langone Medical Center

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The Neuroscience Institute