Ruth M. Empson

The perforant path projection to hippocampal area CA1 in the rat hippocampal-entorhinal cortex combined slice.

1. The perforant path projection from layer III of the entorhinal cortex to CA1 of the hippocampus was studied within a hippocampal‐entorhinal combined slice preparation. We prevented contamination from the other main hippocampal pathways by removal of CA3 and the dentate gyrus. 2. Initially the projection was mapped using field potential recordings that suggested an excitatory sink in stratum lacunosum moleculare with an associated source in stratum pyramidale. 3. However, recording intracellularly from CA1 cells, stimulation of the perforant path produced prominent fast GABAA and slow GABAB IPSPs often preceded by small EPSPs. In a small number of cells we observed EPSPs only. 4. CNQX blocked excitatory and inhibitory responses. This indicated the presence of an intervening excitatory synapse between the inhibitory interneurone and the pyramidal cell. 5. Focal bicuculline applications revealed that the major site of GABAA inhibitory input was to stratum radiatum of CA1. 6. The inhibition activated by the perforant path was very effective at reducing simultaneously activated Schaffer collateral mediated EPSPs and suprathreshold‐stimulated action potentials. 7. Blockade of fast inhibition increased excitability and enhanced slow inhibition. Both increases relied upon the activation of NMDA receptors. 8. Perforant path inputs activated prominent and effective disynaptic inhibition of CA1 cells. This has significance for the output of hippocampal processing during normal behaviour and also under pathological conditions.

UNIQUE INACTIVATION PROPERTIES OF NAADP-SENSITIVE CA2+ RELEASE

Ca mobilization from intracellular stores constitutes an important mechanism for generating cytoplasmic Ca signals. Inositol trisphosphate (InsP) and ryanodine receptors are the two families of intracellular Ca release channels that have been identified, which may be regulated by separate intracellular messengers, InsP and cyclic adenosine 5′-diphosphate ribose, respectively. A third molecule, nicotinic acid adenine dinucleotide phosphate (NAADP), has recently been recognized as a potent Ca releasing agent in sea urchin eggs and microsomes. We now report that non-releasing concentrations of NAADP fully and irreversibly inactivate the NAADP-sensitive Ca release mechanism. This phenomenon occurred both in intact sea urchin eggs and in homogenates and is not shared by either InsP or cyclic adenosine 5′-diphosphate ribose. The novel properties of this Ca release mechanism, giving a one-shot Ca release, may be suited to irreversible cellular events.

Morphological and electrophysiological characterization of layer III cells of the medial entorhinal cortex of the rat.

Entorhinal cortex layer III cells send their axons into hippocampal area CA1, forming the less well studied branch of the perforant path. Using electrophysiological and morphological techniques within a slice preparation, we can classify medial entorhinal cortex layer III cells into four different types. Type 1 and 2 cells were projection cells. Type 1 cells fired regularly and possessed high input resistances and long membrane time constants. Electrical stimulation of the lateral entorhinal cortex revealed a strong excitation by both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potentials. Type 2 cells accommodated strongly, had lower input resistances, faster time constants and featured prominent synaptic inhibition. Type 1 and 2 cells responded to repetitive synaptic stimulation with a prolonged hyperpolarization. We identified the two other, presumed local circuit, cell types whose axons remained within the entorhinal cortex. Type 3 cells were regular firing, had high input resistances and slow membrane time constants, while type 4 cells fired at higher frequencies and possessed a faster time constant and lower input resistance than type 3 neurons. Type 3 cells presented long-lasting excitatory synaptic potentials. Type 4 neurons were the only ones with different responses to stimulation from different sites. Upon lateral entorhinal cortex stimulation they responded with an excitatory postsynaptic potential, while a monosynaptic inhibitory postsynaptic potential was evoked from deep layer stimulation. In contrast to type 1 and 2 neurons, none of the local circuit cells could be antidromically activated from deep layers, and prolonged hyperpolarizations following synaptic repetitive stimulation were also absent in these cells. Together, the complementing morphology and the electrophysiological characteristics of all the cells can provide the controlled flexibility required during the transfer of cortical information to the hippocampus.

7-Deaza-8-bromo-cyclic ADP-ribose, the first membrane-permeant, hydrolysis-resistant cyclic ADP-ribose antagonist.

Cyclic ADP-ribose (cADPR) is a putative second messenger that has been demonstrated to mobilize Ca2+in many cell types. Its postulated role as the endogenous regulator of ryanodine-sensitive Ca2+ release channels has been greatly supported by the advent and use of specific cADPR receptor antagonists such as 8-NH2-cADPR (Walseth, T. F., and Lee, H. C. (1993)Biochim. Biophys. Acta 1178, 235–242). However, investigations of the role of cADPR in physiological responses, such as fertilization, stimulus-secretion coupling, and excitation-contraction coupling, have been hindered by the susceptibility of cADPR receptor antagonists to hydrolysis and the need to introduce these molecules into cells by microinjection or patch clamp techniques. We have recently reported on the discovery of a poorly hydrolyzable analogue of cADPR, 7-deaza-cADPR (Bailey, V. C., Sethi, J. K., Fortt, S. M., Galione, A., and Potter, B. V. L. (1997) Chem. Biol. 4, 41–51) but this, like cADPR, is an agonist of ryanodine-sensitive Ca2+ release channels. We therefore explored the possibility of combining antagonistic activity with that of hydrolytic resistance and now report on the biological properties of the first hydrolysis-resistant cADPR receptor antagonist, 7-deaza-8-bromo-cADPR. In addition this compound has the advantage of being membrane-permeable. Together these properties make this hybrid molecule the most powerful tool to date for studying cADPR-mediated Ca2+ signaling in intact cells.

Serotonin reduces synaptic excitation in the superficial medial entorhinal cortex of the rat via a presynaptic mechanism

1 The superficial layers II and III of the entorhinal cortex, which form the main cortical input to the hippocampus, receive a large serotonergic projection from the raphe nuclei and express 5‐HT receptors at high density. Here, we studied the effects of serotonin on the intrinsic properties and excitatory synaptic transmission of the superficial medial entorhinal cortex. 2 Intracellular and patch clamp recordings revealed that serotonin hyperpolarized only one‐third of the cells, approximately, through a potassium conductance via a GTP‐dependent process. 3 Serotonin depressed mixed as well as isolated α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐ propionic acid receptor (AMPAR)‐ and N‐methyl‐D‐aspartic acid receptor (NMDAR)‐mediated excitatory postsynaptic potentials/currents (EPSPs/EPSCsapproximately 40 % reduction with 1 μM serotonin). 4 The effect of serotonin on EPSPs/EPSCs was similar in whole‐cell versus intracellular recordings; it did not require intracellular GTP and was not visible in glutamate applications to excised patches. Miniature EPSCs recorded in the presence of tetrodotoxin and bicuculline were reduced in frequency, but not altered in amplitude. 5 The effects of serotonin on intrinsic properties and EPSPs were partially mimicked by 5‐HT1A receptor agonists (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin hydrobromide (8‐OH‐DPAT) and 5‐carboxamido‐tryptamine maleate (5‐CT), and reduced by 5‐HT1A receptor antagonists S‐(‐)‐5‐fluoro‐8‐hydroxy‐DPAT hydrochloride (S‐UH‐301), 1‐(2‐methoxyphenyl)‐4‐[4‐(2‐phthalimido)butyl]piperazine hydrobromide (NAN‐190) and spiperone. 6 We conclude that serotonin potently suppresses excitatory synaptic transmission via 5‐HT1A receptors in layers II and III of the medial entorhinal cortex by a presynaptic mechanism.

CYCLIC ADP-RIBOSE ENHANCES COUPLING BETWEEN VOLTAGE-GATED CA2+ ENTRY AND INTRACELLULAR CA2+ RELEASE

Ca2+ release from intracellular stores can be activated in neurons by influx of Ca2+through voltage-gated Ca2+ channels. This process, called Ca2+-induced Ca2+ release, relies on the properties of the ryanodine receptor and represents a mechanism by which Ca2+ influx during neuronal activity can be amplified into large intracellular Ca2+ signals. In a differentiated neuroblastoma cell line, we show that caffeine, a pharmacological activator of the ryanodine receptor, released Ca2+ from intracellular stores in a Ca2+-dependent and ryanodine-sensitive manner. The pyridine nucleotide, cyclic ADP-ribose, thought to be an endogenous modulator of ryanodine receptors also amplified Ca2+-induced Ca2+ release in these neurons. Cyclic ADP-ribose enhanced the total cytoplasmic Ca2+ levels during controlled Ca2+ influx through voltage gated channels, in a concentration-dependent and ryanodine-sensitive manner and also increased the sensitivity with which a small amount of Ca2+ influx could trigger additional release from the ryanodine-sensitive intracellular Ca2+ stores. Single cell imaging showed that following the Ca2+ influx, cyclic ADP-ribose enhanced the spatial spread of the Ca2+ signal from the edge of the cell into its center. These powerful actions suggest a role for cyclic ADP-ribose in the functional coupling of neuronal depolarization, Ca2+entry, and global intracellular Ca2+ signaling.

Serotonin blocks different patterns of low Mg2+-induced epileptiform activity in rat entorhinal cortex, but not hippocampus

Low Mg2+-induced epileptiform activity in the entorhinal cortex is characterized by an initial expression of seizure-like events followed by late recurrent discharges. Both these forms of activity as well as the transition between them were blocked by serotonin. In contrast, serotonin had little effect upon the epileptiform activity in areas CA3 and CA1 of the hippocampus. Both forms of epileptiform activity in the entorhinal cortex are sensitive to N-methyl-D-aspartate receptor antagonists and it is shown here that serotonin blocked both types of epileptiform activity through an effective concentration-dependent reduction of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potentials in deep layer entorhinal cortex cells. Serotonin also prolonged or even prevented the transition between the two types of epileptiform activity and we suggest that this may be through activation of the Na+/K+-ATPase. The resistance of epileptiform activity in CA1 and CA3 to serotonin was most likely related to the inability of serotonin to reduce Schaffer collateral-evoked excitatory postsynaptic potentials. Given the strong serotonergic inputs to both the hippocampus and entorhinal cortex, the differential sensitivity of the two regions to serotonin suggests functional differences. In addition since the late recurrent discharges in the entorhinal cortex are resistant to all clinically used anticonvulsants, serotonin may open new avenues for the development of novel anticonvulsant compounds.

Serotonin reduces synaptic excitation of principal cells in the superficial layers of rat hippocampal-entorhinal cortex combined slices

The cells of the entorhinal cortex receive a dense innervation of serotonergic fibres from the Raphe nuclei and express a high density of 5-hydroxytryptamine 1A (5-HT1A) receptors. We investigated the effects of serotonin on excitatory synaptic transmission in principal cells from entorhinal cortex layers II and III within hippocampal-entorhinal cortex combined slices. Although serotonin had an effect upon the membrane conductance of some, but not all cells, its most pronounced action was to reduce stimulus evoked excitatory synaptic potentials and currents (EPSP/Cs). Both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl-D-aspartate receptor-mediated EPSPs were reduced to similar extents over a range of concentrations. Since the principal cells in layer II and layer III are the main projection cells of the entorhinal cortex, these inhibitory effects of serotonin may have implications for the transfer of information to the hippocampus.

Electrophysiological properties of rat subicular neurons in vitro

The electrophysiological properties of 46 bursting cells and 39 regular firing cells were studied in the subiculum of rat combined hippocampal-entorhinal cortex slices. In bursting cells we found a significantly higher resting membrane potential than in regular firing cells. Upon hyperpolarization both cell types expressed a delayed inward rectification with a subsequent afterdepolarization. While in regular firing cells longer lasting depolarizing current injection caused a train of action potentials with a rather marked decline of discharge frequency, bursting cells displayed only little frequency accommodation. Regular firing cells usually displayed a fast and a slow afterhyperpolarization following a train of action potentials, while bursting neurons present only a slow afterhyperpolarization.

Potent depression of stimulus evoked field potential responses in the medial entorhinal cortex by serotonin

The entorhinal cortex (EC), main input structure to the hippocampus, gets innervated by serotonergic terminals from the raphe nuclei and expresses 5‐HT‐receptors at high density. Using extra‐ and intracellular recording techniques we here investigated the effects of serotonin on population and cellular responses within the EC. Stimulation in the lateral entorhinal cortex resulted in complex field potential responses in the superficial EC. The potentials are composed of an early antidromic and a late orthodromic component reflecting the efferent and afferent circuitry. Serotonin (5‐HT) reduced synaptic potentials of the stimulus evoked extracellular field potential at all concentrations tested (0.1–100 μM; 59%‐depression by 10 μM serotonin), while the antidromic response was not significantly changed by up to 50 μM 5‐HT. Depression of field potential responses by serotonin was associated with a significant increase in paired‐pulse facilitation from 1.15 to 1.88. The effects of serotonin on field potential responses were mimicked by 5‐HT1A‐receptor agonists (8‐OH‐DPAT, 5‐CT) and partially prevented by the 5‐HT1A‐receptor antagonist (S‐UH‐301). Moreover, the 5‐HT1A‐receptor antagonist WAY100635 reduced the effect of 5‐CT. Fenfluramine, a serotonin releaser, mimics the effects of serotonin on stimulus‐evoked field potential responses, indicating that synaptically released serotonin can produce the changes in reactivity to afferent stimulation. Depression of isolated AMPA‐receptor mediated EPSCs by serotonin as well as fenfluramine was associated with an increase in paired pulse facilitation, indicating a presynaptic locus of action. We conclude that physiological concentrations of serotonin potently suppresses excitatory synaptic transmission in the superficial entorhinal cortex by a presynaptic mechanism.