Superficial layers of the medial entorhinal cortex replay independently of the hippocampus.

The hippocampus is thought to initiate systems-wide mnemonic processes through the reactivation of previously acquired spatial and episodic memory traces, which can recruit the entorhinal cortex as a first stage of memory redistribution to other brain areas. Hippocampal reactivation occurs during sharp wave-ripples, in which synchronous network firing encodes sequences of places. We investigated the coordination of this replay by recording assembly activity simultaneously in the CA1 region of the hippocampus and superficial layers of the medial entorhinal cortex. We found that entorhinal cell assemblies can replay trajectories independently of the hippocampus and sharp wave-ripples. This suggests that the hippocampus is not the sole initiator of spatial and episodic memory trace reactivation. Memory systems involved in these processes may include nonhierarchical, parallel components.

Hippocampal network patterns of activity in the mouse.

Genetic engineering of the mouse brain allows investigators to address novel hypotheses in vivo. Because of the paucity of information on the network patterns of the mouse hippocampus, we investigated the electrical patterns in the behaving animal using multisite silicon probes and wire tetrodes. Theta (6-9 Hz) and gamma (40-100 Hz) oscillations were present during exploration and rapid eye movement sleep. Gamma power and theta power were comodulated and gamma power varied as a function of the theta cycle. Pyramidal cells and putative interneurons were phase-locked to theta oscillations. During immobility, consummatory behaviors and slow-wave sleep, sharp waves were present in cornu ammonis region CA1 of the hippocampus stratum radiatum associated with 140-200-Hz "ripples" in the pyramidal cell layer and population burst of CA1 neurons. In the hilus, large-amplitude "dentate spikes" occurred in association with increased discharge of hilar neurons. The amplitude of field patterns was larger in the mouse than in the rat, likely reflecting the higher neuron density in a smaller brain. We suggest that the main hippocampal network patterns are mediated by similar pathways and mechanisms in mouse and rat.

Firing rates of hippocampal neurons are preserved during subsequent sleep episodes and modified by novel awake experience.

What determines the firing rate of cortical neurons in the absence of external sensory input or motor behavior, such as during sleep? Here we report that, in a familiar environment, the discharge frequency of simultaneously recorded individual CA1 pyramidal neurons and the coactivation of cell pairs remain highly correlated across sleep-wake-sleep sequences. However, both measures were affected when new sets of neurons were activated in a novel environment. Nevertheless, the grand mean firing rate of the whole pyramidal cell population remained constant across behavioral states and testing conditions. The findings suggest that long-term firing patterns of single cells can be modified by experience. We hypothesize that increased firing rates of recently used neurons are associated with a concomitant decrease in the discharge activity of the remaining population, leaving the mean excitability of the hippocampal network unaltered.

Behavior-dependent states of the hippocampal network affect functional clustering of neurons.

Local versus distant coherence of hippocampal CA1 pyramidal cells was investigated in the behaving rat. Temporal cross-correlation of pyramidal cells revealed a significantly stronger relationship among local (<140 microm) pyramidal neurons compared with distant (>300 microm) neurons during non-theta-associated immobility and sleep but not during theta-associated running and walking. In contrast, cross-correlation between local pyramidal cell-interneuron pairs was significantly stronger than between distant pairs during theta oscillations but were similar during non-theta-associated behaviors. We suggest that network state-dependent functional clustering of neuronal activity emerges because of the differential contribution of the main excitatory inputs, the perforant path, and Schaffer collaterals during theta and non-theta behaviors.

The application of printed circuit board technology for fabrication of multi-channel micro-drives.

A modular multichannel microdrive ('hyperdrive') is described. The microdrive uses printed circuit board technology and flexible fused silica capillaries. The modular design allows for the fabrication of 4-32 independently movable electrodes or 'tetrodes'. The drives are re-usable and re-loading the drive with electrodes is simple.

Intracellular features predicted by extracellular recordings in the hippocampus in vivo.

Multichannel tetrode array recording in awake behaving animals provides a powerful method to record the activity of large numbers of neurons. The power of this method could be extended if further information concerning the intracellular state of the neurons could be extracted from the extracellularly recorded signals. Toward this end, we have simultaneously recorded intracellular and extracellular signals from hippocampal CA1 pyramidal cells and interneurons in the anesthetized rat. We found that several intracellular parameters can be deduced from extracellular spike waveforms. The width of the intracellular action potential is defined precisely by distinct points on the extracellular spike. Amplitude changes of the intracellular action potential are reflected by changes in the amplitude of the initial negative phase of the extracellular spike, and these amplitude changes are dependent on the state of the network. In addition, intracellular recordings from dendrites with simultaneous extracellular recordings from the soma indicate that, on average, action potentials are initiated in the perisomatic region and propagate to the dendrites at 1.68 m/s. Finally we determined that a tetrode in hippocampal area CA1 theoretically should be able to record electrical signals from approximately 1, 000 neurons. Of these, 60-100 neurons should generate spikes of sufficient amplitude to be detectable from the noise and to allow for their separation using current spatial clustering methods. This theoretical maximum is in contrast to the approximately six units that are usually detected per tetrode. From this, we conclude that a large percentage of hippocampal CA1 pyramidal cells are silent in any given behavioral condition.

Accuracy of tetrode spike separation as determined by simultaneous intracellular and extracellular measurements.

Simultaneous recording from large numbers of neurons is a prerequisite for understanding their cooperative behavior. Various recording techniques and spike separation methods are being used toward this goal. However, the error rates involved in spike separation have not yet been quantified. We studied the separation reliability of "tetrode" (4-wire electrode)-recorded spikes by monitoring simultaneously from the same cell intracellularly with a glass pipette and extracellularly with a tetrode. With manual spike sorting, we found a trade-off between Type I and Type II errors, with errors typically ranging from 0 to 30% depending on the amplitude and firing pattern of the cell, the similarity of the waveshapes of neighboring neurons, and the experience of the operator. Performance using only a single wire was markedly lower, indicating the advantages of multiple-site monitoring techniques over single-wire recordings. For tetrode recordings, error rates were increased by burst activity and during periods of cellular synchrony. The lowest possible separation error rates were estimated by a search for the best ellipsoidal cluster shape. Human operator performance was significantly below the estimated optimum. Investigation of error distributions indicated that suboptimal performance was caused by inability of the operators to mark cluster boundaries accurately in a high-dimensional feature space. We therefore hypothesized that automatic spike-sorting algorithms have the potential to significantly lower error rates. Implementation of a semi-automatic classification system confirms this suggestion, reducing errors close to the estimated optimum, in the range 0-8%.

Ensemble patterns of hippocampal CA3-CA1 neurons during sharp wave-associated population events.

Transfer of neuronal patterns from the CA3 to CA1 region was studied by simultaneous recording of neuronal ensembles in the behaving rat. A nonlinear interaction among pyramidal neurons was observed during sharp wave (SPW)-related population bursts, with stronger synchrony associated with more widespread spatial coherence. SPW bursts emerged in the CA3a-b subregions and spread to CA3c before invading the CA1 area. Synchronous discharge of >10% of the CA3 within a 100 ms window was required to exert a detectable influence on CA1 pyramidal cells. Activity of some CA3 pyramidal neurons differentially predicted the ripple-related discharge of circumscribed groups of CA1 pyramidal cells. We suggest that, in SPW behavioral state, the coherent discharge of a small group of CA3 cells is the primary cause of spiking activity in CA1 pyramidal neurons.

Firing rate and theta-phase coding by hippocampal pyramidal neurons during 'space clamping'.

In the hippocampus, spatial representation of the environment has been suggested to be coded by either the firing rate of pyramidal cell assemblies or the relative timing of the action potentials during the theta EEG cycle. Here, we used a behavioural 'space clamp' method, which involved the confinement of the actively running animal in a defined position in space (running wheel) to examine how 'spatial' and other inputs affect firing rate and timing of hippocampal CA1 pyramidal cells and interneurons. Nineteen per cent of the recorded CA1 pyramidal cells were selectively active while the rat was running in the wheel in a given direction ('wheel' cells). Spatial rotation of the apparatus showed that selective discharge of pyramidal cells in the wheel was under the combined influence of distal and apparatus cues. During steady running, both discharge rate and theta phase were constant. Rotation of the wheel apparatus resulted in a shift of both firing rate and preferred theta phase. The discharge frequency of 'wheel' cells increased threefold (on average) with increasing running velocity. In contrast, change in running speed had relatively little effect on the theta phase-related discharge of 'wheel' cells. Our findings indicate that mechanisms that regulate rate and phase of spikes are overlapping but not necessarily identical.

Ultra-slow oscillation (0.025 Hz) triggers hippocampal afterdischarges in Wistar rats.

Oscillations in neuronal networks are assumed to serve various physiological functions, from coordination of motor patterns to perceptual binding of sensory information. Here, we describe an ultra-slow oscillation (0.025 Hz) in the hippocampus. Extracellular and intracellular activity was recorded from the CA1 and subicular regions in rats of the Wistar and Sprague-Dawley strains, anesthetized with urethane. In a subgroup of Wistar rats (23%), spontaneous afterdischarges (4.7+/-1.6 s) occurred regularly at 40.8+/-15.7 s. The afterdischarge was initiated by a fast increase of population synchrony (100-250 Hz oscillation; "tonic" phase), followed by large-amplitude rhythmic waves and associated action potentials at gamma and beta frequency (15-50 Hz; "clonic" phase). The afterdischarges were bilaterally synchronous and terminated relatively abruptly without post-ictal depression. Single-pulse stimulation of the commissural input could trigger afterdischarges, but only at times when they were about to occur. Commissural stimulation evoked inhibitory postsynaptic potentials in pyramidal cells. However, when the stimulus triggered an afterdischarge, the inhibitory postsynaptic potential was absent and the cells remained depolarized during most of the afterdischarge. Afterdischarges were not observed in the Sprague-Dawley rats. Long-term analysis of interneuronal activity in intact, drug-free rats also revealed periodic excitability changes in the hippocampal network at 0.025 Hz. These findings indicate the presence of an ultra-slow oscillation in the hippocampal formation. The ultra-slow clock induced afterdischarges in susceptible animals. We hypothesize that a transient failure of GABAergic inhibition in a subset of Wistar rats is responsible for the emergence of epileptiform patterns.

Replay and time compression of recurring spike sequences in the hippocampus.

Information in neuronal networks may be represented by the spatiotemporal patterns of spikes. Here we examined the temporal coordination of pyramidal cell spikes in the rat hippocampus during slow-wave sleep. In addition, rats were trained to run in a defined position in space (running wheel) to activate a selected group of pyramidal cells. A template-matching method and a joint probability map method were used for sequence search. Repeating spike sequences in excess of chance occurrence were examined by comparing the number of repeating sequences in the original spike trains and in surrogate trains after Monte Carlo shuffling of the spikes. Four different shuffling procedures were used to control for the population dynamics of hippocampal neurons. Repeating spike sequences in the recorded cell assemblies were present in both the awake and sleeping animal in excess of what might be predicted by random variations. Spike sequences observed during wheel running were "replayed" at a faster timescale during single sharp-wave bursts of slow-wave sleep. We hypothesize that the endogenously expressed spike sequences during sleep reflect reactivation of the circuitry modified by previous experience. Reactivation of acquired sequences may serve to consolidate information.

Fast network oscillations in the hippocampal CA1 region of the behaving rat.

This study examined intermittent, high-frequency (100-200 Hz) oscillatory patterns in the CA1 region of the hippocampus in the absence of theta activity, i.e., during and in between sharp wave (SPW) bursts. Pyramidal and interneuronal activity was phase-locked not only to large amplitude (>7 SD from baseline) oscillatory events, which are present mainly during SPWs, but to smaller amplitude (<4 SD) patterns, as well. Large-amplitude events were in the 140-200 Hz, "ripple" frequency range. Lower-amplitude events, however, contained slower, 100-130 Hz ("slow") oscillatory patterns. Fast ripple waves reversed just below the CA1 pyramidal layer, whereas slow oscillatory potentials reversed in the stratum radiatum and/or in the stratum oriens. Parallel CA1-CA3 recordings revealed correlated CA3 field and unit activity to the slow CA1 waves but not to fast ripple waves. These findings suggest that fast ripples emerge in the CA1 region, whereas slow (100-130 Hz) oscillatory patterns are generated in the CA3 region and transferred to the CA1 field.

Interactions between hippocampus and medial septum during sharp waves and theta oscillation in the behaving rat.

The medial septal region and the hippocampus are connected reciprocally via GABAergic neurons, but the physiological role of this loop is still not well understood. In an attempt to reveal the physiological effects of the hippocamposeptal GABAergic projection, we cross-correlated hippocampal sharp wave (SPW) ripples or theta activity and extracellular units recorded in the medial septum and diagonal band of Broca (MSDB) in freely moving rats. The majority of single MSDB cells (60%) were significantly suppressed during SPWs. Most cells inhibited during SPW (80%) fired rhythmically and phase-locked to the negative peak of the CA1 pyramidal layer theta waves. Because both SPW and the negative peak of local theta waves correspond to the maximum discharge probability of CA1 pyramidal cells and interneuron classes, the findings indicate that the activity of medial septal neurons can be negatively (during SPW) or positively (during theta waves) correlated with the activity of hippocampal interneurons. We hypothesize that the functional coupling between medial septal neurons and hippocampal interneurons varies in a state-dependent manner.

Sustained activation of hippocampal pyramidal cells by 'space clamping' in a running wheel.

In contrast to sensory cortical areas of the brain, the relevant physiological inputs to the hippocampus, leading to selective activation of pyramidal cells, are largely unknown. Pyramidal cells are thought to be phasically activated by spatial cues and a variety of sensory and motor stimuli. Here, we used a behavioural 'space clamp' method, which involved the confinement of the actively running animal in a defined position in space (running wheel) and kept sensory inputs constant. Twelve percent of the recorded CA1 pyramidal cells were selectively active while the rat was running in the wheel. Cell firing was specific to the direction of running and disappeared after rotating the recording apparatus. The discharge frequency of pyramidal cells and interneurons was sustained as long as the rat ran continuously in the wheel. Furthermore, the discharge frequency of pyramidal cells and interneurons increased with increasing running velocity, even though the frequency of hippocampal theta waves remained constant. The discharge frequency of some 'wheel-related' pyramidal cells could increase more than 10-fold between 10 and 100 cm/s, whereas the firing rate of 'non-wheel' cells remained constantly low. We hypothesize that: (i) a necessary condition for place-specific discharge of hippocampal pyramidal cells is the presence of theta oscillation; and (ii) relevant stimuli can tonically and selectively activate hippocampal pyramidal cells as long as theta activity is present.

Oscillatory coupling of hippocampal pyramidal cells and interneurons in the behaving Rat.

We examined whether excitation and inhibition are balanced in hippocampal cortical networks. Extracellular field and single-unit activity were recorded by multiple tetrodes and multisite silicon probes to reveal the timing of the activity of hippocampal CA1 pyramidal cells and classes of interneurons during theta waves and sharp wave burst (SPW)-associated field ripples. The somatic and dendritic inhibition of pyramidal cells was deduced from the activity of interneurons in the pyramidal layer [int(p)] and in the alveus and st. oriens [int(a/o)], respectively. Int(p) and int(a/o) discharged an average of 60 and 20 degrees before the population discharge of pyramidal cells during the theta cycle, respectively. SPW ripples were associated with a 2.5-fold net increase of excitation. The discharge frequency of int(a/o) increased, decreased ("anti-SPW" cells), or did not change ("SPW-independent" cells) during SPW, suggesting that not all interneurons are innervated by pyramidal cells. Int(p) either fired together with (unimodal cells) or both before and after (bimodal cells) the pyramidal cell burst. During fast-ripple oscillation, the activity of interneurons in both the int(p) and int(a/o) groups lagged the maximum discharge probability of pyramidal neurons by 1-2 msec. Network state changes, as reflected by field activity, covaried with changes in the spike train dynamics of single cells and their interactions. Summed activity of parallel-recorded interneurons, but not of pyramidal cells, reliably predicted theta cycles, whereas the reverse was true for the ripple cycles of SPWs. We suggest that network-driven excitability changes provide temporal windows of opportunity for single pyramidal cells to suppress, enable, or facilitate selective synaptic inputs.

Reliability and state dependence of pyramidal cell-interneuron synapses in the hippocampus: an ensemble approach in the behaving rat.

Spike transmission probability between pyramidal cells and interneurons in the CA1 pyramidal layer was investigated in the behaving rat by the simultaneous recording of neuronal ensembles. Population synchrony was strongest during sharp wave (SPW) bursts. However, the increase was three times larger for pyramidal cells than for interneurons. The contribution of single pyramidal cells to the discharge of interneurons was often large (up to 0.6 probability), as assessed by the presence of significant (<3 ms) peaks in the cross-correlogram. Complex-spike bursts were more effective than single spikes. Single cell contribution was higher between SPW bursts than during SPWs or theta activity. Hence, single pyramidal cells can reliably discharge interneurons, and the probability of spike transmission is behavior dependent.

Epileptic afterdischarge in the hippocampal-entorhinal system: current source density and unit studies.

The contribution of the various hippocampal regions to the maintenance of epileptic activity, induced by stimulation of the perforant path or commissural system, was examined in the awake rat. Combination of multiple-site recordings with silicon probes, current source density analysis and unit recordings allowed for a high spatial resolution of the field events. Following perforant path stimulation, seizures began in the dentate gyrus, followed by events in the CA3-CA1 regions. After commissural stimulation, rhythmic bursts in the CA3-CA1 circuitry preceded the activation of the dentate gyrus. Correlation of events in the different subregions indicated that the sustained rhythmic afterdischarge (2-6 Hz) could not be explained by a cycle-by-cycle excitation of principal cell populations in the hippocampal-entorhinal loop. The primary afterdischarge always terminated in the CA1 region, followed by the dentate gyrus, CA3 region and the entorhinal cortex. The duration and pattern of the hippocampal afterdischarge was essentially unaffected by removal of the entorhinal cortex. The emergence of large population spike bursts coincided with a decreased discharge of interneurons in both CA1 and hilar regions. The majority of hilar interneurons displayed a strong amplitude decrement prior to the onset of population spike phase of the afterdischarge. These findings suggest that (i) afterdischarges can independently arise in the CA3-CA1 and entorhinal dentate gyrus circuitries, (ii) reverberation of excitation in the hippocampal-entorhinal loop is not critical for the maintenance of afterdischarges and (iii) decreased activity of the interneuronal network may release population bursting of principal cells.