Ionic basis of ON and OFF persistent activity in layer III lateral entorhinal cortical principal neurons.

Principal neurons in layer III of the rat lateral entorhinal cortex (LEC) generate a self-sustained plateau potential and persistent spiking following the application of a brief suprathreshold excitatory stimulus delivered in the presence of the muscarinic receptor agonist carbachol. This persistent activity can be terminated by application of a second excitatory stimulus, and these cells can be repeatedly toggled between on and off states by consecutive excitatory stimuli. However, the ionic mechanisms that underlie the production of on and off states in layer III LEC neurons are unknown but seem to involve activity-dependent conductances, since they can be initiated by trains of action potentials evoked by either depolarizing current pulses applied to the cell or by repetitive spiking induced by activation of excitatory synaptic inputs. In this study, we obtained intracellular recordings from rat layer III LEC neurons in vitro, and a series of pharmacological and ionic substitution experiments were performed to identify mechanisms involved in the induction and termination of persistent spiking. Our data indicate that initiation of the on state depends on spike-evoked calcium influx and subsequent activation of calcium-activated nonselective cationic current. Moreover, we show that termination of persistent firing in response to an excitatory stimulus can be blocked by tetraethylammonium or iberiotoxin, suggesting that the activation of calcium-activated potassium current mediated by large conductance calcium-activated K(+) (i.e., BK) channels is required to induce the off state.

Ca(2+)-dependent K(+) currents and spike-frequency adaptation in medial entorhinal cortex layer II stellate cells.

The entorhinal cortex (EC), located in the medial temporal lobe (MTL) of the brain, plays an important functional role in the MTL memory circuit. Medial EC (MEC) Layer II stellate cells (SCs) serve as one of the most prominent target cell types within the EC for inputs arising from higher cortical areas, and these same cells provide most of the output from the EC to the hippocampal region. We used the whole-cell patch clamp technique in a rat in vitro slice preparation to test whether SCs express afterhyperpolarization (AHP) currents and if these currents can be modulated. Our results revealed that SCs contain medium (mI(K(Ca))) and slow (sI(AHP)) Ca(2+)-dependent K(+) currents. Furthermore, we determined that an apamin-sensitive current does not underlie the mAHP in SCs. Our studies also showed that a cAMP-dependent modulation process significantly reduces mI(K(Ca)), sI(AHP), and spike-frequency adaptation in MEC Layer II SCs. Modulation of the firing pattern of SCs resulting from this effect may play an important role in the encoding of information related to memory processes.

Switching between "On" and "Off" states of persistent activity in lateral entorhinal layer III neurons.

Persistent neural spiking maintains information during a working memory task when a stimulus is no longer present. During retention, this activity needs to be stable to distractors. More importantly, when retention is no longer relevant, cessation of the activity is necessary to enable processing and retention of subsequent information. Here, by means of intracellular recording with sharp microelectrode in in vitro rat brain slices, we demonstrate that single principal layer III neurons of the lateral entorhinal cortex (EC) generate persistent spiking activity with a novel ability to reliably toggle between spiking activity and a silent state. Our data indicates that in the presence of muscarinic receptor activation, persistent activity following an excitatory input may be induced and that a subsequent excitatory input can terminate this activity and cause the neuron to return to a silent state. Moreover, application of inhibitory hyperpolarizing stimuli is neither able to decrease the frequency of the persistent activity nor terminate it. The persistent activity can also be initiated and terminated by synchronized synaptic stimuli of layer II/III of the perirhinal cortex. The neuronal ability to switch "On" and "Off" persistent activity may facilitate the concurrent representation of temporally segregated information arriving in the EC and being directed toward the hippocampus.

Mechanism of graded persistent cellular activity of entorhinal cortex layer v neurons.

Working memory is an emergent property of neuronal networks, but its cellular basis remains elusive. Recent data show that principal neurons of the entorhinal cortex display persistent firing at graded firing rates that can be shifted up or down in response to brief excitatory or inhibitory stimuli. Here, we present a model of a potential mechanism for graded firing. Our multicompartmental model provides stable plateau firing generated by a nonspecific calcium-sensitive cationic (CAN) current. Sustained firing is insensitive to small variations in Ca2+ concentration in a neutral zone. However, both high and low Ca2+ levels alter firing rates. Specifically, increases in persistent firing rate are triggered only during high levels of calcium, while decreases in rate occur in the presence of low levels of calcium. The model is consistent with detailed experimental observations and provides a mechanism for maintenance of memory-related activity in individual neurons.

Ionic mechanisms in the generation of subthreshold oscillations and action potential clustering in entorhinal layer II stellate neurons.

A multicompartmental biophysical model of entorhinal cortex layer II stellate cells was developed to analyze the ionic basis of physiological properties, such as subthreshold membrane potential oscillations, action potential clustering, and the medium afterhyperpolarization. In particular, the simulation illustrates the interaction of the persistent sodium current (I(Nap)) and the hyperpolarization activated inward current (Ih) in the generation of subthreshold membrane potential oscillations. The potential role of Ih in contributing to the medium hyperpolarization (mAHP) and rebound spiking was studied. The role of Ih and the slow calcium-activated potassium current Ikappa(AHP) in action potential clustering was also studied. Representations of Ih and I(Nap) were developed with parameters based on voltage-clamp data from whole-cell patch and single channel recordings of stellate cells (Dickson et al., J Neurophysiol 83:2562-2579, 2000; Magistretti and Alonso, J Gen Physiol 114:491-509, 1999; Magistretti et al., J Physiol 521:629-636, 1999a; J Neurosci 19:7334-7341, 1999b). These currents interacted to generate robust subthreshold membrane potentials with amplitude and frequency corresponding to data observed in the whole cell patch recordings. The model was also able to account for effects of pharmacological manipulations, including blockade of Ih with ZD7288, partial blockade with cesium, and the influence of barium on oscillations. In a model with a wider range of currents, the transition from oscillations to single spiking, to spike clustering, and finally tonic firing could be replicated. In agreement with experiment, blockade of calcium channels in the model strongly reduced clustering. In the voltage interval during which no data are available, the model predicts that the slow component of Ih does not follow the fast component down to very short time constants. The model also predicts that the fast component of Ih is responsible for the involvement in the generation of subthreshold oscillations, and the slow component dominates in the generation of spike clusters.

Graded persistent activity in entorhinal cortex neurons.

Working memory represents the ability of the brain to hold externally or internally driven information for relatively short periods of time. Persistent neuronal activity is the elementary process underlying working memory but its cellular basis remains unknown. The most widely accepted hypothesis is that persistent activity is based on synaptic reverberations in recurrent circuits. The entorhinal cortex in the parahippocampal region is crucially involved in the acquisition, consolidation and retrieval of long-term memory traces for which working memory operations are essential. Here we show that individual neurons from layer V of the entorhinal cortex-which link the hippocampus to extensive cortical regions-respond to consecutive stimuli with graded changes in firing frequency that remain stable after each stimulus presentation. In addition, the sustained levels of firing frequency can be either increased or decreased in an input-specific manner. This firing behaviour displays robustness to distractors; it is linked to cholinergic muscarinic receptor activation, and relies on activity-dependent changes of a Ca2+-sensitive cationic current. Such an intrinsic neuronal ability to generate graded persistent activity constitutes an elementary mechanism for working memory.

Muscarinic activation of a cation current and associated current noise in entorhinal-cortex layer-II neurons.

The effects of muscarinic stimulation on the membrane potential and current of in situ rat entorhinal-cortex layer-II principal neurons were analyzed using the whole cell, patch-clamp technique. In current-clamp experiments, application of carbachol (CCh) induced a slowly developing, prolonged depolarization initially accompanied by a slight decrease or no significant change in input resistance. By contrast, in a later phase of the depolarization input resistance appeared consistently increased. To elucidate the ionic bases of these effects, voltage-clamp experiments were then carried out. In recordings performed in nearly physiological ionic conditions at the holding potential of -60 mV, CCh application promoted the slow development of an inward current deflection consistently associated with a prominent increase in current noise. Similarly to voltage responses to CCh, this inward-current induction was abolished by the muscarinic antagonist, atropine. Current-voltage relationships derived by applying ramp voltage protocols during the different phases of the CCh-induced inward-current deflection revealed the early induction of an inward current that manifested a linear current/voltage relationship in the subthreshold range and the longer-lasting block of an outward K(+) current. The latter current could be blocked by 1 mM extracellular Ba(2+), which allowed us to study the CCh-induced inward current (I(CCh)) in isolation. The extrapolated reversal potential of the isolated I(CCh) was approximately 0 mV and was not modified by complete substitution of intrapipette K(+) with Cs(+). Moreover, the extrapolated I(CCh) reversal shifted to approximately -20 mV on removal of 50% extracellular Na(+). These results are consistent with I(CCh) being a nonspecific cation current. Finally, noise analysis of I(CCh) returned an estimated conductance of the underlying channels of approximately 13.5 pS. We conclude that the depolarizing effect of muscarinic stimuli on entorhinal-cortex layer-II principal neurons depends on both the block of a K(+) conductance and the activation of a "noisy" nonspecific cation current. We suggest that the membrane current fluctuations brought about by I(CCh) channel noise may facilitate the "theta" oscillatory dynamics of these neurons and enhance firing reliability and synchronization.

Morphological and electrophysiological characteristics of layer V neurons of the rat lateral entorhinal cortex.

The intrinsic electrophysiological and morphological properties of lateral entorhinal area (LEA) layer V neurons were investigated by sharp electrode intracellular recording and biocytin labeling in vitro. The morphological analysis revealed that layer V of the LEA contains three distinct subtypes of principal neurons, which were classified as pyramidal, horizontal, and polymorphic neurons. Pyramidal cells were the most abundant subtype (57%) and could be further subdivided into neurons with large, small, and star-like somas. Similarly to pyramidal cells, horizontal neurons (11%) had a prominent apical dendrite. However, their distinctive basal dendritic plexus extended primarily in the horizontal plane. Polymorphic neurons (32%) were characterized by a multipolar dendritic organization. Electrophysiological analysis of neurons in the three categories demonstrated a diversity of electrophysiological profiles within each category and no significant differences between groups. Neurons in the three subgroups could display instantaneous and/or time-dependent inward rectification and different degrees of spike frequency adaptation. None of the recorded cells displayed an intrinsic oscillatory bursting discharge. Many neurons in the three subgroups, however, displayed slow (3.5-14 Hz), sustained, subthreshold membrane potential oscillations. The morphological and electrophysiological diversity of principal neurons in the LEA parallels that previously reported for the medial entorhinal area and suggests that, with respect to the deep layers, similar information processing is performed across the mediolateral extent of the entorhinal cortex. Layer V of the entorhinal cortex may undertake very complex operations beyond acting as a relay station of hippocampal processed information to the neocortex.

Simulations of the role of the muscarinic-activated calcium-sensitive nonspecific cation current INCM in entorhinal neuronal activity during delayed matching tasks.

Entorhinal lesions impair performance in delayed matching tasks, and blockade of muscarinic cholinergic receptors also impairs performance in these tasks. Physiological data demonstrate that muscarinic cholinergic receptor stimulation activates intrinsic cellular currents in entorhinal neurons that could underlie the role of entorhinal cortex in performance of these tasks. Here we use a network biophysical simulation of the entorhinal cortex to demonstrate the potential role of this cellular mechanism in the behavioral tasks. Simulations demonstrate how the muscarinic-activated calcium-sensitive nonspecific cation current I(NCM) could provide a cellular mechanism for features of the neuronal activity observed during performance of delayed matching tasks. In particular, I(NCM) could underlie (1) the maintenance of sustained spiking activity during the delay period, (2) the enhancement of spiking activity during the matching period relative to the sample period, and (3) the resistance of sustained activity to distractors. Simulation of a larger entorhinal network with connectivity chosen randomly within constraints on number, distribution, and weight demonstrates appearance of other phenomena observed in unit recordings from awake animals, including match suppression, non-match enhancement, and non-match suppression.