Neural-network oscillations at distinct frequencies have been implicated in the encoding, consolidation and retrieval of information in the hippocampus. Some GABA (gamma-aminobutyric acid)-containing interneurons fire phase-locked to theta oscillations (4-8 Hz) or to sharp-wave-associated ripple oscillations (120-200 Hz), which represent different behavioural states. Interneurons also entrain pyramidal cells in vitro. The large diversity of interneurons poses the question of whether they have specific roles in shaping distinct network activities in vivo. Here we report that three distinct interneuron types--basket, axo-axonic and oriens-lacunosum-moleculare cells--visualized and defined by synaptic connectivity as well as by neurochemical markers, contribute differentially to theta and ripple oscillations in anaesthetized rats. The firing patterns of individual cells of the same class are remarkably stereotyped and provide unique signatures for each class. We conclude that the diversity of interneurons, innervating distinct domains of pyramidal cells, emerged to coordinate the activity of pyramidal cells in a temporally distinct and brain-state-dependent manner.

CA9 is a membrane-tethered, carbonic anhydrase (CA) enzyme, expressed mainly at the external surface of cells, that catalyzes reversible CO 2 hydration. Expression is greatly enhanced in many tumors, particularly in aggressive carcinomas. The functional role of CA9 in tumors is not well established. Here we show that CA9, when expressed heterologously in cultured spheroids (0.5-mm diameter, ϳ25,000 cells) of RT112 cells (derived from bladder carcinoma), induces a nearuniform intracellular pH (pH i ) throughout the structure. Dynamic pH i changes during displacements of superfusate CO 2 concentration are also spatially coincident (within 2 s). In contrast, spheroids of wild-type RT112 cells lacking CA9 exhibit an acidic core (ϳ0.25 pH i reduction) and significant time delays (ϳ9 s) for pH i changes in core versus peripheral regions. pH i non-uniformity also occurs in CA9-expressing spheroids after selective pharmacological inhibition of the enzyme. In isolated RT112 cells, pH i regulation is unaffected by CA9 expression. The influence of CA9 on pH i is thus only evident in multicellular tissue. Diffusion-reaction modeling indicates that CA9 coordinates pH i spatially by facilitating CO 2 diffusion in the unstirred extracellular space of the spheroid. We suggest that pH i coordination may favor survival and growth of a tumor. By disrupting spatial pH i control, inhibition of CA9 activity may offer a novel strategy for the clinical treatment of CA9-associated tumors.Intracellular H ϩ ions are powerful modulators of physiological and biochemical processes, including cell growth, division, and development (1-3). Many metabolic reactions within the cell generate H ϩ ions as end-products (4). Because of their high chemical reactivity, particularly with intracellular proteins, these ions must be removed if normal cell function is to continue. Tumors display a notably high rate of H ϩ ion production, partly because their growth demands a ready supply of energy, but also because much of the metabolism that supports this is anaerobic (5). Energy production via extramitochondrial routes is associated with a larger metabolic flux of acid per ATP molecule (4). Indeed, growing tumors are typically hypoxic, resulting in stabilization of hypoxia-inducible factor 1␣, which upregulates a variety of proteins, including enzymes that control glycolytic lactic acid production (6). If intracellular H ϩ ions accumulate, intracellular pH (pH i ) 3 falls and cellular activity becomes compromised. One solution is for cells to express plasmalemmal ion transport proteins that extrude excess H ϩ equivalents (7). Extrusion is achieved either by expelling H ϩ ions directly (e.g. via Na ϩ -H ϩ exchangers, H ϩ -lactate co transporters, and H ϩ -ATPases), or by importing HCO 3 Ϫ anions (e.g. via Na ϩ -dependent HCO 3 Ϫ transporters), to neutralize intracellular H ϩ ions. The pH i of tumor cells, like that of most other eukaryotic cells, appears to be relatively alkaline (pH i ϳ 7.2), indicating that they possess an efficient system for eliminating...

Presynaptic metabotropic glutamate receptors (mGluRs) show a highly selective expression and subcellular location in nerve terminals modulating neurotransmitter release. We have demonstrated that alternatively spliced variants of mGluR8, mGluR8a and mGluR8b, have an overlapping distribution in the hippocampus, and besides perforant path terminals, they are expressed in the presynaptic active zone of boutons making synapses selectively with several types of GABAergic interneurons, primarily in the stratum oriens. Boutons labeled for mGluR8 formed either type I or type II synapses, and the latter were GABAergic. Some mGluR8-positive boutons also expressed mGluR7 or vasoactive intestinal polypeptide. Interneurons strongly immunopositive for the muscarinic M2 or the mGlu1 receptors were the primary targets of mGluR8-containing terminals in the stratum oriens, but only neurochemically distinct subsets were innervated by mGluR8-enriched terminals. The majority of M2-positive neurons were mGluR8 innervated, but a minority, which expresses somatostatin, was not. Rare neurons coexpressing calretinin and M2 were consistently targeted by mGluR8-positive boutons. In vivo recording and labeling of an mGluR8-decorated and strongly M2-positive interneuron revealed a trilaminar cell with complex spike bursts during theta oscillations and strong discharge during sharp wave/ripple events. The trilaminar cell had a large projection from the CA1 area to the subiculum and a preferential innervation of interneurons in the CA1 area in addition to pyramidal cell somata and dendrites. The postsynaptic interneuron type-specific expression of the high-efficacy presynaptic mGluR8 in both putative glutamatergic and in identified GABAergic terminals predicts a role in adjusting the activity of interneurons depending on the level of network activity.

No abstract

In the hippocampal CA1 region, metabotropic glutamate subtype 1 (mGluR1) receptors have been implicated in a variety of physiological responses to glutamate, which include modulation of synaptic transmission and plasticity, as well as neuronal excitability and synchronization. The mGluR1alpha isoform is characteristically expressed only by nonprincipal cells, and it is particularly enriched in somatostatin (SS)-containing interneurons in stratum oriensalveus. Anatomical and physiological data have indicated the presence of mGluR1alpha in several distinct classes of interneurons with their somata located also in strata pyramidale, radiatum, and lacunosum moleculare. Each different interneuron subtype, as defined by functionally relevant criteria, including input/ output characteristics and expression of selective molecular markers, subserves distinct functions in local hippocampal circuits. We have investigated which of the different CA1 interneuron classes express mGluR1alpha by immunofluorescent labeling, combining antibodies to mGluR1alpha, calcium-binding proteins, and neuropeptides, and by intracellular labeling in vitro. Several types of interneuron that are immunopositive for mGluR1alpha each targeted different domains of pyramidal cells and included (1) O-LM inter-neurons, found to coexpress both SS and parvalbumin (PV); (2) interneurons with target selectivity for other interneurons, expressing vasoactive intestinal polypeptide (VIP) and/or the calcium-binding protein calretinin; (3) procholecystokinin-immunopositive interneurons probably non-basket and dendrite-targeting; and (4) an as-yet unidentified SS-immunoreactive but PV-immunonegative interneuron class, possibly corresponding to oriens-bistratified cells. Estimation of the relative proportion of mGluR1alpha-positive interneurons showed 43%, 46%, and 30% co-labeling with SS, VIP, or PV, respectively. The identification of the specific subclasses of CA1 interneurons expressing mGluR1alpha provides the network basis for assessing the contribution of this receptor to the excitability of the hippocampus.

Background and purpose: Intracellular pH (pH i ) in heart is regulated by sarcolemmal H þ -equivalent transporters such as Na þ -H þ exchange (NHE) and Na þ -HCO 3 À cotransport (NBC). Inhibition of NBC influences pH i and can be cardioprotective in animal models of post-ischaemic reperfusion. Apart from a rabbit polyclonal NBC-antibody, a selective NBC inhibitor compound has not been studied. Compound S0859 (C 29 H 24 ClN 3 O 3 S) is a putative NBC inhibitor. Here, we provide the drug's chemical structure, test its potency and selectivity in ventricular cells and assess its suitability for experiments on cardiac contraction. Experimental approach: pH i recovery from intracellular acidosis was monitored using pH-epifluorescence (SNARFfluorophore) in guinea pig, rat and rabbit isolated ventricular myocytes. Electrically evoked cell shortening (contraction) was measured optically. With CO 2 /HCO 3 À -buffered superfusates containing 30 mM cariporide (to inhibit NHE), pH i recovery is mediated by NBC. Key results: S0859, an N-cyanosulphonamide compound, reversibly inhibited NBC-mediated pH i recovery (K i ¼ 1.7 mM, full inhibition at B30 mM). In HEPES-buffered superfusates, NHE-mediated pH i recovery was unaffected by 30 mM S0859. With CO 2 /HCO 3 À buffer, pH i recovery from intracellular alkalosis (mediated by Cl À /HCO 3 À and Cl À /OH À exchange) was also unaffected. Selective NBC-inhibition was not due to action on carbonic anhydrase (CA) enzymes, as 100 mM acetazolamide (a membrane-permeant CA-inhibitor) had no significant effect on NBC activity. pH i recovery from acidosis was associated with increased contractile-amplitude. The time course of recovery of pH i and contraction was slowed by S0859, confirming that NBC is a significant controller of contractility during acidosis. Conclusions and implications: Compound S0859 is a selective, high-affinity generic NBC inhibitor, potentially important for probing the transporter's functional role in heart and other tissues.

Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3− ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein–protein binding (a ‘transport metabolon’) or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3− or H+ transport via the native acid-extruding proteins, Na+–HCO3− cotransport (NBC) and Na+/H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and —impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3− ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3− buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3− transport, and hence pHi regulation in the heart.

The protocol for penetratin-coupling of siRNA-like molecules used in this Article incorporated suggestions by Carol M. Troy that were based on the method described in ref. 1.