P2X receptors are ATP-gated ion channels made up of three similar or identical subunits. It is unknown whether ligand binding is intersubunit or intrasubunit, either for agonists or for allosteric modulators. Zinc binds to rat P2X 2 receptors and acts as an allosteric modulator, potentiating channel opening. To probe the location of this zinc binding site, P2X 2 receptors bearing mutations of the histidines at positions 120 and 213 were expressed in Xenopus oocytes. Studies of H120C and H213C mutants produced five lines of evidence consistent with the hypothesis that the residues in these positions bind zinc. Mixing of subunits containing the H120A or H213A mutation generated receptors that showed zinc potentiation, even though neither of these mutant receptors showed zinc potentiation on its own. Furthermore, expression of trimeric concatamers with His 3 Ala mutations at some but not all six positions showed that zinc potentiation correlated with the number of intersubunit histidine pairs. These results indicate that zinc potentiation requires an interaction across a subunit interface. Expression of the H120C/H213C double mutant resulted in the formation of ectopic disulfide bonds that could be detected by changes in the physiological properties of the receptors after treatment with reducing and oxidizing agents. Immunoblot analysis of H120C/H213C protein separated under nonreducing conditions demonstrated that the ectopic bonds were between adjacent subunits. Taken together, these data indicate that His 120 and His 213 sit close to each other across the interface between subunits and are likely to be key components of the zinc binding site in P2X 2 receptors.P2X receptors are oligomeric, ATP-gated cation channels that are distributed widely throughout the central and peripheral nervous systems of mammals and are known to be involved in fast excitatory synaptic transmission (1). P2X receptors are structurally distinct from the nicotinic receptor and ionotropic glutamate receptor channel superfamilies. In mammals, the P2X family has seven members encoded by different genes (P2X 1-7 ) that can associate as homo-and heteromeric channel assemblies (1). P2X receptors are thought to be trimers (2) with subunits that have intracellular N and C termini, two transmembrane domains, and a large extracellular loop (1).Colocalization of zinc with P2X 2 receptors in the nervous system suggests a physiological role for this divalent cation in modulating ATP-evoked currents (3, 4). Extracellular zinc potentiates P2X receptor currents in rat sensory and sympathetic neurons as well as in rat PC12 cells (1). The potentiating effect of zinc on P2X 2 receptors results from a decrease in the EC 50 for ATP without a concomitant change in the maximum response to ATP (5, 6).We have previously identified two molecular determinants of zinc potentiation for the rat P2X 2 subunit (6); mutation of either His 120 or His 213 to alanine eliminated potentiation by zinc. Direct participation of these residues in zinc binding is consistent with t...

Many of our daily activities, such as riding a bike to work or reading a book in a noisy cafe, and highly skilled activities, such as a professional playing a tennis match or a violin concerto, depend upon the ability of the brain to quickly make moment-to-moment adjustments to our behavior in response to the results of our actions. Particularly, they depend upon the ability of the neocortex to integrate the information provided by the sensory organs (bottom-up information) with internally generated signals such as expectations or attentional signals (top-down information). This integration occurs in pyramidal cells (PCs) and their long apical dendrite, which branches extensively into a dendritic tuft in layer 1 (L1). The outermost layer of the neocortex, L1 is highly conserved across cortical areas and species. Importantly, L1 is the predominant input layer for top-down information, relayed by a rich, dense mesh of long-range projections that provide signals to the tuft branches of the PCs. Here, we discuss recent progress in our understanding of the composition of L1 and review evidence that L1 processing contributes to functions such as sensory perception, cross-modal integration, controlling states of consciousness, attention, and learning. Expected final online publication date for the Annual Review of Neuroscience, Volume 44 is July 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Photochemical switches represent a powerful method for improving pharmacological therapies and controlling cellular physiology. Here we report the photo-regulation of GABAA receptors (GABAARs) by a derivative of propofol (2,6-diisopropylphenol), a GABAAR allosteric modulator, that we have modified to contain photo-isomerizable azobenzene. Using α1β2γ2 GABAARs expressed in Xenopus laevis oocytes and native GABAARs of isolated retinal ganglion cells, we show that the trans-azobenzene isomer of the new compound (trans-MPC088), generated by visible light (wavelengths ~440 nm), potentiates the GABA-elicited response and at higher concentrations directly activates the receptors. cis-MPC088, generated from trans-MPC088 by UV light (~365 nm), produces little if any receptor potentiation/activation. In cerebellar slices, MPC088 co-applied with GABA affords bidirectional photo-modulation of Purkinje cell membrane current and spike-firing rate. The findings demonstrate photo-control of GABAARs by an allosteric ligand and open new avenues for fundamental and clinically oriented research on GABAARs, a major class of neurotransmitter receptors in the central nervous system.

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...

We describe a fast activity-dependent homeostatic regulation of intrinsic excitability of identified neurons in mouse dorsal striatum, the striatal output neurons. It can be induced by brief bursts of activity, is expressed on a time scale of seconds, limits repetitive firing, and can convert regular firing patterns to irregular ones. We show it is due to progressive recruitment of the KCNQ2/3 channels that generate the M current. This homeostatic mechanism is significantly reduced in striatal output neurons of the R6/2 transgenic mouse model of Huntington's disease, at an age when the neurons are hyperactive in vivo and the mice begin to exhibit locomotor impairment. Furthermore, it can be rescued by bath perfusion with retigabine, a KCNQ channel activator, and chronic treatment improves locomotor performance. Thus, M-current dysfunction may contribute to the hyperactivity and network dysregulation characteristic of this neurodegenerative disease, and KCNQ2/3 channel regulation may be a target for therapeutic intervention.untington's disease (HD) is a fatal inherited autosomal neurodegenerative disorder, with its primary symptoms being progressive development of motor and cognitive dysfunction (1). The mutated gene, huntingtin (HTT), and its mutation, an expansion of the number of CAG repeats, were identified 20 y ago. However, the mechanism(s) underlying the pathological changes that culminate in the degeneration of striatal output neurons (SONs) remain unknown. Early animal models (2) generated a number of testable hypotheses, most notable being that the neurons degenerate because of a hyperactivity that leads to a build-up of excitotoxic molecules. However, more recent studies implicate alternative pathologies, such as altered transcriptional activity, calcium regulation and mitochondrial function, or disruptions in normal neuronal patterns of activity (3) and show that neuronal dysfunction and behavioral and motor symptoms of HD precede neurodegeneration (2). These studies have been facilitated by access to transgenic mice models, including R6/1 and R6/2 mice, which express a truncated region of the mutant human HTT gene with expanded CAG repeats (4). In vitro recordings in both lines revealed that SONs are depolarized and have higher input resistances than do wild-type (WT) controls, at a stage where deficits in locomotor activity begin to be manifest (5-7). Furthermore, in vivo recordings indicate that at 5-9 wk of age, when the mice exhibit overt motor deficits, R6/2 SONs have higher firing rates and more regular discharge patterns compared with WT (8, 9). In contrast, neurodegeneration and death occur later (2). Hence, we asked whether cellular mechanisms that influence excitability might be altered in the early stages of HD and might serve as targets for alleviating associated behavioral symptoms.Hyperactivity and related changes in neuronal firing patterns could reflect alterations in synaptic transmission and its activitydependent modifications or in intrinsic membrane properties governing neuronal e...

Cortical GABAergic interneurons (INs) represent a diverse population of locally projecting cells that provide specialized forms of inhibition to pyramidal neurons and other INs. Most recent work on INs has focused on subtypes distinguished by expression of Parvalbumin (PV), Somatostatin (SST), or Vasoactive Intestinal Peptide (VIP), but a fourth group that includes neurogliaform cells (NGFCs) has remained largely enigmatic due to a lack of genetic tools. Here, we show that these INs can be accessed experimentally using intersectional genetics with the gene Id2. We find that outside of layer 1 (L1), the vast majority of Id2 INs are NGFCs that express high levels of neuropeptide Y (NPY) and exhibit a late-spiking firing pattern, with extensive local connectivity. While much sparser, non-NGFC Id2 IN had more variable properties, with most corresponding to a diverse group of INs that strongly express the neuropeptide CCK. In vivo, using silicon probe recordings, we observed several intriguing aspects of NGFC activity, including a strong rebound in activity immediately following the cortical down state during NREM sleep. Our study provides insights into IN diversity and NGFC distribution and properties, and outlines an intersectional genetics approach for further study of this neglected group of INs.

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C5-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.