A major goal in neuroscience is the development of optical reporters of membrane potential that are easy to use, have limited phototoxicity, and achieve the speed and sensitivity necessary for detection of individual action potentials in single neurons. Here we present a novel, two-component optical approach that attains these goals. By combining DiO, a fluorescent neuronal tracer dye, with dipicrylamine (DPA), a molecule whose membrane partitioning is voltage-sensitive, optical signals related to changes in membrane potential based on FRET (Förster resonance energy transfer) are reported. Using DiO/DPA in HEK-293 cells with diffraction-limited laser spot illumination, depolarization-induced fluorescence changes of 56% per 100 mV ( ϳ 0.1 ms) were obtained, while in neuronal cultures and brain slices, action potentials (APs) generated a ⌬F/F per 100 mV of Ͼ25%. The high sensitivity provided by DiO/DPA enabled the detection of subthreshold activity and high-frequency APs in single trials from somatic, axonal, or dendritic membrane compartments. Recognizing that DPA can depress excitability, we assayed the amplitude and duration of single APs, burst properties, and spontaneous firing in neurons of primary cultures and brain slices and found that they are undetectably altered by up to 2 M DPA and only slightly perturbed by 5 M DPA. These findings substantiate a simple, noninvasive method that relies on a neuronal tracer dye for monitoring electrical signal flow, and offers unique flexibility for the study of signaling within intact neuronal circuits.

Key points• Pharmacological activation of nicotinic ACh receptors (nAChRs) excites striatal interneurones and induces a GABA-mediated current in medium spiny projecting neurones (MSNs) via feedforward inhibition. Abstract Choline acetyltransferase-expressing interneurones (ChAT)+ of the striatum influence the activity of medium spiny projecting neurones (MSNs) and striatal output via a disynaptic mechanism that involves GABAergic neurotransmission. Using transgenic mice that allow visual identification of MSNs and distinct populations of GABAergic interneurones expressing neuropeptide Y (NPY) + , parvalbumin (PV) + and tyrosine hydroxylase (TH) + , we further elucidate this mechanism by studying nicotinic ACh receptor (nAChR)-mediated responses. First, we determined whether striatal neurones exhibit pharmacologically induced nicotinic responses by performing patch-clamp recordings. With high [Cl − ] i , our results showed increased spontaneous IPSC frequency and amplitude in MSNs as well as in the majority of interneurones. However, direct nAChR-mediated activity was observed in interneurones but not MSNs. In recordings with physiological [Cl − ] i , these responses manifested as inward currents in the presence of tetrodotoxin and bicuculline methobromide. Nicotinic responses in MSNs were primarily mediated through GABA A receptors in feedforward inhibition. To identify the GABAergic interneurones that mediate the response, we performed dual recordings from GABAergic interneurones and MSNs. Both TH + and neurogliaform subtypes of NPY + (NPY + NGF) interneurones form synaptic connections with MSNs, although the strength of connectivity, response kinetics and pharmacology differ between and within the two populations. Importantly, both cell types appear to contribute to nAChR-mediated GABAergic responses in MSNs. Our data offer insight into the striatal network activity under cholinergic control, and suggest that subclasses of recently identified TH + and R. Luo and M. J. Janssen have contributed equally to this work.

Corticotrophin Releasing Factor (CRF) is a critical stress-related neuropeptide in major output pathways of the amygdala, including the central nucleus (CeA), and in a key projection target of the CeA, the bed nucleus of the stria terminalis (BnST). While progress has been made in understanding the contributions and characteristics of CRF as a neuropeptide in rodent behavior, little attention has been committed to determine the properties and synaptic physiology of specific populations of CRF-expressing (CRF(+)) and non-expressing (CRF(-)) neurons in the CeA and BnST. Here, we fill this gap by electrophysiologically characterizing distinct neuronal subtypes in CeA and BnST. Crossing tdTomato or channelrhodopsin-2 (ChR2-YFP) reporter mice to those expressing Cre-recombinase under the CRF promoter allowed us to identify and manipulate CRF(+) and CRF(-) neurons in CeA and BnST, the two largest areas with fluorescently labeled neurons in these mice. We optogenetically activated CRF(+) neurons to elicit action potentials or synaptic responses in CRF(+) and CRF(-) neurons. We found that GABA is the predominant co-transmitter in CRF(+) neurons within the CeA and BnST. CRF(+) neurons are highly interconnected with CRF(-) neurons and to a lesser extent with CRF(+) neurons. CRF(+) and CRF(-) neurons differentially express tonic GABA currents. Chronic, unpredictable stress increase the amplitude of evoked IPSCs and connectivity between CRF(+) neurons, but not between CRF(+) and CRF(-) neurons in both regions. We propose that reciprocal inhibition of interconnected neurons controls CRF(+) output in these nuclei.

Dellal SS, Luo R, Otis TS. GABA A receptors increase excitability and conduction velocity of cerebellar parallel fiber axons. J Neurophysiol 107: 2958 -2970. First published February 29, 2012 doi:10.1152/jn.01028.2011In the adult mammalian brain, GABA A receptors (GABA A Rs) are responsible for the predominant forms of synaptic inhibition, but these receptors can excite neurons when the chloride equilibrium potential (E Cl ) is depolarized. In many mature neurons, GABA A Rs are found on presynaptic terminals where they exert depolarizing effects. To understand whether excitatory GABA action affects axonal function, we used transverse cerebellar slices to measure the effects of photolysis of caged GABA on the initiation and propagation of compound parallel fiber (PF) action potentials (APs). Photolysis of caged GABA increased the amplitude and conduction velocity of PF APs; GABA reuptake blockers and a positive modulator of GABA A Rs enhanced these effects. In contrast, a modulator selective for ␦-subunit-containing GABA A Rs did not enhance these effects and responsiveness remained in ␦ Ϫ/Ϫ mice, arguing that ␦-subunit-containing GABA A Rs are not required. Synaptically released GABA also increased PF excitability, indicating that the mechanism is engaged by physiological signals. A Hodgkin-Huxleystyle compartmental model of the PF axon and granule cell body was constructed, and this model recapitulated the GABA-dependent decrease in AP threshold and the increase in conduction velocity, features that were sensitive to E Cl and to the voltage dependence of sodium channel inactivation. The model also predicts that axonal GABA A Rs could affect orthodromic spike initiation. We conclude that GABA acting on cerebellar PFs facilitates both spike generation and propagation, allowing axons of granule cells to passively integrate signals from inhibitory interneurons and influence information flow in the input layer to the cerebellar cortex.␥-aminobutyric acid; presynaptic modulation; axonal excitability; ␦-subunit; fiber volley; caged ␥-aminobutyric acid THE CLASSICAL STUDIES OF MUSCLE afferent inputs to spinal motor neurons that gave rise to the concept of presynaptic inhibition also eventually led to the first identification of presynaptic GABA A receptors (GABA A Rs) (Eccles et al. 1963). Subsequent work by many laboratories demonstrated that this GABA A R-mediated presynaptic inhibition results from a depolarizing action of GABA on presynaptic afferent terminals (Rudomin and Schmidt 1999). Since those initial findings, GABA A Rs have been observed on presynaptic terminals in a variety of brain regions, including auditory brain stem, ventral tegmental area, hypothalamus, amygdala, hippocampus, cerebellum, and cortex, and in most of these locations GABA has been found to be depolarizing ( Although there have been many studies on the consequences of presynaptic GABA A Rs on synaptic transmission, relatively little is known about whether these receptors affect axonal excitability. To directly test whether transient acti...

Striatonigral and striatopallidal projecting medium spiny neurons (MSNs) express dopamine D1 (D1+) and D2 receptors (D2+), respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic, and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic, and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2, and a β subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory post-synaptic current (IPSC). Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the β3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2 μM), a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the β3 subunit knock out mice. This suggests that the β3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the β3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its role in mediating synaptic input from striatal neurogliaform interneurons.