Imaging Intracellular Ca<sup>2+</sup> Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

University of California Los Angeles

We assessed the potential of using multielectrode arrays (MEAs) to investigate several physiological properties of the calyx of Held synapse in the medial nucleus of the trapezoid body of gerbil. Due to the large size of the synapse, it became widely employed in studies on synaptic mechanisms. Electrical stimulation at the midline evoked a characteristic compound signal consisting of a presynaptic volley (C(1)) and a postsynaptic response (C(2)). The C(1) was blocked by tetrodotoxin, whilst the C(2) was blocked by perfusion of low Ca(2+) external solution, or the AMPA-R antagonists CNQX, and GYKI52466. NMDA-R blocker D-AP5, partially inhibited the postsynaptic response at P12, but showed no effect in P30 animals. The inhibitory effects of GABA or glycine on postsynaptic responses were reciprocal with regard to animal's maturity: GABA caused a pronounced reduction of C(2) amplitude in P20-22 animals, while glycine showed a stronger inhibition in P27-28 animals. Low-frequency super-threshold stimulation of the afferents induced facilitation of the postsynaptic C(2) amplitudes and only minor changes in temporal characteristics of the signals. At stimulation frequencies >200 Hz, however, significant depression occurs accompanied by increases in transmission delay and in the width of the postsynaptic response. This study suggests MEAs as a useful tool to study calyx of Held synapse by simultaneous recordings of pre- and postsynaptic elements of synaptically interconnected neurons in the auditory brainstem. Moreover, MEAs enable convenient analysis of activity-dependent depression and modulation of neuronal activity by glycine and GABA at later developmental stages not accessible to patch recordings.

Synaptic transmission and short-term plasticity at the calyx of Held synapse revealed by multielectrode array recordings.

Neuronal nitric oxide synthase (nNOS) is broadly expressed in the brain and associated with synaptic plasticity through NMDAR-mediated calcium influx. However, its physiological activation and the mechanisms by which nitric oxide (NO) influences synaptic transmission have proved elusive. Here, we exploit the unique input-specificity of the calyx of Held to characterize NO modulation at this glutamatergic synapse in the auditory pathway. NO is generated in an activity-dependent manner by MNTB principal neurons receiving a calyceal synaptic input. It acts in the target neuron and adjacent inactive neurons to modulate excitability and synaptic efficacy, inhibiting postsynaptic Kv3 potassium currents (via phosphorylation), reducing EPSCs and so increasing action potential duration and reducing transmission fidelity. We conclude that NO serves as a volume transmitter and slow dynamic modulator, integrating spontaneous and evoked neuronal firing, thereby providing an index of global activity and regulating information transmission across a population of active and inactive neurons.

Nitric oxide is a volume transmitter regulating postsynaptic excitability at a glutamatergic synapse.

Extracellular signals from the endbulb of Held-spherical bushy cell (SBC) synapse exhibit up to three component waves ('P', 'A' and 'B'). Signals lacking the third component (B) are frequently observed but as the origin of each of the components is uncertain, interpretation of this lack of B has been controversial: is it a failure to release transmitter or a failure to generate or propagate an action potential? Our aim was to determine the origin of each component. We combined single- and multiunit in vitro methods in Mongolian gerbils and Wistar rats and used pharmacological tools to modulate glutamate receptors or voltage-gated sodium channels. Simultaneous extra- and intracellular recordings from single SBCs demonstrated a presynaptic origin of the P-component, consistent with data obtained with multielectrode array recordings of local field potentials. The later components (A and B) correspond to the excitatory postsynaptic potential (EPSP) and action potential of the SBC, respectively. These results allow a clear interpretation of in vivo extracellular signals. We conclude that action potential failures occurring at the endbulb-SBC synaptic junction largely reflect failures of the EPSP to trigger an action potential and not failures of synaptic transmission. The data provide the basis for future investigation of convergence of excitatory and inhibitory inputs in modulating transmission at a fully functional neuronal system using physiological stimulation.

Presynaptic and postsynaptic origin of multicomponent extracellular waveforms at the endbulb of Held-spherical bushy cell synapse.

There is a well-established link between hyperbilirubinaemia and hearing loss in paediatrics, but the cellular mechanisms have not been elucidated. Here we used the Gunn rat model of hyperbilirubinaemia to investigate bilirubin-induced hearing loss. In vivo auditory brainstem responses revealed that Gunn rats have severe auditory deficits within 18 h of exposure to high bilirubin levels. Using an in vitro preparation of the auditory brainstem from these rats, extracellular multi-electrode array recording from the medial nucleus of the trapezoid body (MNTB) showed longer latency and decreased amplitude of evoked field potentials following bilirubin exposure, suggestive of transmission failure at this synaptic relay. Whole-cell patch-clamp recordings confirmed that the electrophysiological properties of the postsynaptic MNTB neurons were unaffected by bilirubin, with no change in action potential waveforms or current-voltage relationships. However, stimulation of the trapezoid body was unable to elicit large calyceal EPSCs in MNTB neurons of hyperbilirubinaemic rats, indicative of damage at a presynaptic site. Multi-photon imaging of anterograde-labelled calyceal projections revealed axonal staining and presynaptic profiles around MNTB principal neuron somata. Following induction of hyperbilirubinaemia the giant synapses were largely destroyed. Electron microscopy confirmed loss of presynaptic calyceal terminals and supported the electrophysiological evidence for healthy postsynaptic neurons. MNTB neurons express high levels of neuronal nitric oxide synthase (nNOS). Nitric oxide has been implicated in mechanisms of bilirubin toxicity elsewhere in the brain, and antagonism of nNOS by 7-nitroindazole protected hearing during bilirubin exposure. We conclude that bilirubin-induced deafness is caused by degeneration of excitatory synaptic terminals in the auditory brainstem.

Acute hyperbilirubinaemia induces presynaptic neurodegeneration at a central glutamatergic synapse.

Activity-dependent changes in synaptic strength are well established as mediating long-term plasticity underlying learning and memory, but modulation of target neuron excitability could complement changes in synaptic strength and regulate network activity. It is thought that homeostatic mechanisms match intrinsic excitability to the incoming synaptic drive, but evidence for involvement of voltage-gated conductances is sparse. Here, we show that glutamatergic synaptic activity modulates target neuron excitability and switches the basis of action potential repolarization from Kv3 to Kv2 potassium channel dominance, thereby adjusting neuronal signaling between low and high activity states, respectively. This nitric oxide-mediated signaling dramatically increases Kv2 currents in both the auditory brain stem and hippocampus (>3-fold) transforming synaptic integration and information transmission but with only modest changes in action potential waveform. We conclude that nitric oxide is a homeostatic regulator, tuning neuronal excitability to the recent history of excitatory synaptic inputs over intervals of minutes to hours.

Nitric oxide is an activity-dependent regulator of target neuron intrinsic excitability.

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged âˆ¼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

Intracellulaires Ca(2+) transitoires sont consid&eacute;r&eacute;es comme un signal primaire par lequel les astrocytes interagissent avec les neurones et les vaisseaux sanguins. Avec les m&eacute;thodes couramment utilis&eacute;es, Ca(2+) a &eacute;t&eacute; &eacute;tudi&eacute;e que dans les corps cellulaires astrocyte et branches &eacute;paisses, laissant les rameaux fine distale et endfeet qui sont plus proches de synapses neuronales et des vaisseaux sanguins en grande partie inexplor&eacute;s. Ici, &agrave; l&#39;aide de formes cytosoliques et attach&eacute; &agrave; la membrane de cod&eacute; g&eacute;n&eacute;tiquement Ca(2+) indicateurs (GECIs ; cyto-GCaMP3 et Lck-GCaMP3), nous rapportons des approches bien caract&eacute;ris&eacute;s que surmonter ces limites. Nous avons utilis&eacute; des microinjections in vivo de virus ad&eacute;no-associ&eacute;s &agrave; exprimer GECIs dans les astrocytes et &eacute;tudi&eacute; Ca(2+) signaux dans des tranches d&#39;hippocampe aigu&euml;s in vitro de souris adultes (&sim;P80 ans) deux semaines apr&egrave;s l&#39;infection. Nos donn&eacute;es r&eacute;v&egrave;lent un panorama mousseux de fa&ccedil;on inattendue nombreuses, fr&eacute;quentes, de mani&egrave;re &eacute;quivalente &agrave; l&#39;&eacute;chelle et tr&egrave;s localis&eacute;es Ca(2+) microdomaines territoire entier astrocyte in situ dans des tranches d&#39;hippocampe aigu&euml;s, conform&eacute;ment &agrave; la r&eacute;partition des rameaux p&eacute;risynaptique d&eacute;crits &agrave; l&#39;aide de la microscopie &eacute;lectronique. Signaux d&#39;endfeet ont &eacute;t&eacute; r&eacute;v&eacute;l&eacute;es avec une clart&eacute; particuli&egrave;re. Les outils et les approches exp&eacute;rimentales, que nous d&eacute;crivons en d&eacute;tail permettent l&#39;&eacute;tude syst&eacute;matique des signaux Ca(2+) int&eacute;rieur des astrocytes enti&egrave;res, y compris au sein des rameaux fine p&eacute;risynaptique et endfeet associ&eacute;s &agrave; bateau, permettant une &eacute;valuation rigoureuse de comment les astrocytes contribuent au fonctionnement du cerveau.

Imagerie calcique microdomaines dans territoires astrocyte ensemble et endfeet avec GCaMPs exprim&eacute;es &agrave; l&#39;aide de virus ad&eacute;no-associ&eacute;s.

Intracellulare Ca(2+) transitori sono considerati un segnale primario da cui astrociti interagiscono con i neuroni e vasi sanguigni. Con i metodi comunemente utilizzati esistenti, Ca(2+) &egrave; stato studiato solo all&#39;interno di somata astrociti e folti rami, lasciando i ramoscelli sottili distale ed endfeet che sono pi&ugrave; prossima alla sinapsi neuronali e dei vasi sanguigni in gran parte inesplorati. Qui, utilizzando forme citosoliche e membrana-legato di geneticamente codificati Ca(2+) indicatori (GECIs; cyto-GCaMP3 e Lck-GCaMP3), relazione approcci ben caratterizzati che superano queste limitazioni. Abbiamo utilizzato in vivo microiniezioni di virus adeno-associati ad per esprimere GECIs negli astrociti e studiato Ca(2+) segnali a fette hippocampal acute in vitro da topi adulti (et&agrave; &sim;P80) due settimane dopo l&#39;infezione. I nostri dati rivelano un panorama scintillante di inaspettatamente numerose, frequenti, in modo equivalente in scala e altamente localizzato Ca(2+) microdomains all&#39;interno dei territori di astrociti intero in situ entro fette hippocampal acute, coerente con la distribuzione di ramoscelli di perisynaptic descritto utilizzando la microscopia elettronica. Segnali da endfeet sono state rivelate con particolare chiarezza. Gli strumenti e approcci sperimentali che descriviamo in dettaglio consentono lo studio sistematico dei Ca(2+) segnali all&#39;interno di interi astrociti, compreso all&#39;interno di ramoscelli sottili perisynaptic e vaso-associated endfeet, permettendo la valutazione rigorosa di quanto gli astrociti contribuiscono alla funzione del cervello.

Imaging microdomains calcio all&#39;interno di territori Astrocita intera ed endfeet con GCaMPs espresso utilizzando virus adeno-associati.

Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K(+) channel functional expression, leading to elevated in vivo striatal extracellular K(+), which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K(+), ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K(+) homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.

Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.

The spatiotemporal activities of astrocyte Ca²⁺ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²⁺ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²⁺ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²⁺ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²⁺ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

Martin D. Haustein

Department of Physiology

University of California Los Angeles

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Martin D. Haustein

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

University of California Los Angeles

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Department of Physiology

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators