Mark Stewart

Do septal neurons pace the hippocampal theta rhythm

The hippocampal theta rhythm (rhythmical slow activity, RSA) is one of the most thoroughly studied EEG phenomena. Much of this experimental interest has been stimulated by suggestions that the mnemonic functions of the hippocampus may depend upon theta-related neuronal activity. Inputs from the medial septal nuclei to the hippocampus were shown to be essential for the theta rhythm in the 1950s, but the role of these basal forebrain projections has not been clearly defined. Four models of the septo-hippocampal connections involved in theta rhythm production are reviewed as the precise roles of these projections are discussed. In our final, consolidated model both cholinergic and GABAergic septal projection cells fire in rhythmic bursts that entrain hippocampal interneurons. The resulting rhythmic inhibition of hippocampal projection cells, together with their excitatory interconnections, generates at least one component of the theta rhythm.

Current source density analysis of the hippocampal theta rhythm: associated sustained potentials and candidate synaptic generators

Single-electrode depth profiles of the hippocampal EEG were made in urethane-anesthetized rats and rats trained in an alternating running/drinking task. Current source density (CSD) was computed from the voltage as a function of depth. A problem inherent to AC-coupled profiles was eliminated by incorporating sustained potential components of the EEG. AC profiles force phasic current sinks to alternate with current sources at each lamina, changing the magnitude and even the sign of the computed membrane current. It was possible to include DC potentials in the profiles from anesthetized rats by using glass micropipettes for recording. A method of subtracting profiles of the non-theta EEG from theta profiles was developed as an approach to including sustained potentials in recordings from freely-moving animals implanted with platinum electrodes. DC profiles are superior to AC profiles for analysis of EEG activity because DC-CSD values can be considered correct in sign and more closely represent the actual membrane current magnitudes. Since hippocampal inputs are laminated, CSD analysis leads to straightforward predictions of the afferents involved. Theta-related activity in afferents from entorhinal neurons, hippocampal interneurons and ipsi- and contralateral hippocampal pyramids all appear to contribute to sources and sinks in CA1 and the dentate area. The largest theta-related generator was a sink at the fissure, having both phasic and tonic components. This sink may reflect activity in afferents from the lateral entorhinal cortex. The phase of the dentate mid-molecular sink suggests that medial entorhinal afferents drive the theta-related granule and pyramidal cell firing. The sustained components may be simply due to different average rates of firing during theta rhythm than during non-theta EEG in afferents whose firing rates are also phasically modulated.

A comparison of corticospinal activation by magnetic coil and electrical stimulation of monkey motor cortex

The effects of different orientations of a Cadwell round magnetic coil (MC) were compared with each other and with surface electrical stimulation of motor cortex in monkeys anesthetized with pentobarbital or urethane. Recordings were made from within the lateral corticospinal tract, either from axonal populations or with a microelectrode from individual axons. A lateral-sagittally orientated MC directly excited corticospinal neurons at lower stimulus intensity than was required for indirect, i.e., transsynaptic excitation via inputs to corticospinal neurons. By contrast, in 2 out of 3 macaques tested, a vertex-tangential orientation could excite corticospinal neurons indirectly at lower intensities than were required for direct excitation; at higher intensities, direct excitation also occurred. The site of direct corticospinal excitation by a lateral-sagittally orientated MC was inferred by comparing the response variability and latency to MC and surface electrical stimuli. Cathodal stimuli elicited more variable corticospinal population responses and later individual axonal responses than were obtained with anodal stimuli. The variability in response is attributed to interaction between nearby, on-going synaptic bombardment and the stimulus, implying that surface cathodal stimuli directly activate corticospinal neurons at the spike trigger zone (presumably the initial segment). By contrast, the consistency and reduced latency of the corticospinal responses to surface anodal stimuli are attributed to the direct excitation of corticospinal fibers within the white matter. When the stimulus intensity is clearly above threshold, surface anodal and cathodal stimuli can activate corticospinal neurons both directly and indirectly. Direct corticospinal excitation by the MC can resemble the effects of either surface anodal or surface cathodal stimuli. We conclude that the MC can activate corticospinal neurons at the spike trigger zone or their fibers deeper in white matter. The findings in the monkey are used to interpret the effects of different MC orientations in the human.

Different firing patterns generated in dendrites and somata of CA1 pyramidal neurones in guinea-pig hippocampus.

1. Intracellular recordings, taken from CA1 pyramidal cells in guinea‐pig hippocampal slices, were used to examine the origins of repetitive and burst firing in these cells. Single action potentials were elicited by depolarizing current injection at somatic recording sites. In contrast, current injection during intradendritic recordings initiated burst firing in the dendrites. Burst firing could be elicited in the soma by direct depolarization of distal apical dendrites (> 150 microns from the cell body layer) with large extracellular polarizing electrodes. 2. Intracellular recordings were taken simultaneously from the apical dendrites and pyramidal cell somata with the intention of impaling the same neurone with both electrodes. Paired dendrite‐soma recordings confirmed that rhythmic single action potentials were generated at the cell soma, whereas bursts of action potentials were initiated in the distal apical dendrites (> 150 microns from the cell body layer). Fast spikes in the dendrite often triggered fast spikes in the soma, but not all fast spikes in the dendritic burst were ‘relayed’ to the soma. 3. In paired recordings, when a dendritic action potential failed to elicit a full somatic action potential, a ‘d‐spike’ was commonly recorded in the soma. Somatic d‐spikes were uniform all‐or‐none responses that could be shown, in some cases, to trigger the full somatic action potentials. 4. Attenuated spikes could be recorded in the dendrites, triggered by action potentials initiated at the cell soma. Dendritic responses to somatic stimulation sometimes varied in amplitude, but always showed a direct correspondence with somatic action potentials. 5. Dendritic recordings taken closer to the pyramidal cell bodies (< 150 microns from the cell body layer) showed a ‘transitional’ region where single action potentials rather than burst discharges could be evoked. After‐potentials of these single spikes differed from those associated with somatic spikes in that proximal dendritic spikes had depolarizing after‐potentials. The observed shift from after‐hyperpolarization to depolarizing after‐potentials in intradendritic recordings taken progressively further from the cell body corresponds to the change from repetitive to burst firing. 6. The results indicate that activity of the CA1 pyramidal cell soma, presumably a reflection of its output, can be either burst or repetitive firing. Somatic ‘bursts,’ unlike the burst discharges seen in the apical dendrites or the burst discharges reported in CA3 cells, are not initiated locally. Rather, they appear to be simply a rapid spike‐for‐spike response by the soma to the fast spikes that form part of the apical dendritic burst.(ABSTRACT TRUNCATED AT 400 WORDS)

HIPPOCAMPAL THETA ACTIVITY IN MONKEYS

The hippocampal theta rhythm has been extensively studied in many subprimate mammals. Considering the technical difficulties involved in recording from freely moving animals during voluntary motion and REM sleep, it was thought that urethane anesthesia might be appropriate for initial studies of the primate hippocampal EEG. Three of three macaques and one of two squirrel monkeys showed clear rhythmic hippocampal EEG activity. One very old squirrel monkey (a 16-year-old female) showed no theta activity in the hippocampal EEG. Similarities of the monkey theta activity with theta rhythm of urethane-anesthetized rats included: (1) a high coherence between recordings from electrodes separated by several millimeters within the hippocampal formation; (2) sensitivity of the theta activity to muscarinic drugs; and (3) its correlation with spontaneous movements during light anesthesia. Important differences were: (1) the frequency of the monkey theta activity was 7-9 Hz compared to the 4-5 Hz found in rats; (2) theta activity was not detected in the distal apical dendritic regions of CA1 or dentate in the monkey; (3) considerable amounts of low-frequency EEG co-existed with the monkey theta activity; and (4) the durations of bouts of theta activity in monkeys were much shorter than in rats. We conclude that primates generate hippocampal theta activity homologous, but not identical, to that of rats.

Intrinsic connectivity of the rat subiculum : I. Dendritic morphology and patterns of axonal arborization by pyramidal neurons

The dendritic and axonal morphology of rat subicular neurons was studied in single cells labeled with Neurobiotin. Electrophysiological classification of cells as intrinsic burst firing or regular spiking neurons was correlated with morphologic patterns and cell locations. Every cell had dendritic branches that reached the outer molecular layer, with most cells having branches that reached the hippocampal fissure. All but two pyramidal cells had axon collaterals that entered the deep white matter (alveus). Branching patterns of apical dendrites varied as a function of the cells soma location along the fissure–alveus axis of the cell layer. The first major dendritic branch point for most cells occurred at the superficial edge of the cell layer giving deep cells long primary apical dendrites and superficial cells short or absent primary apical dendrites. In contrast, basal dendritic arbors were similar across cells regardless of cell position. Apical and basal dendrites of all cells had numerous spines. Superficial and deep cells also differed in axonal collateralization. Deep cells (mostly intrinsically bursting [IB] class) had one or more ascending axon collaterals that typically remained within the region circumscribed by their apical dendrites. Superficial cells (mostly regular spiking [RS] class) tended to have axon collaterals that reached longer distances in the cell layer. Numerous varicosities and axonal extensions were present on axon collaterals in the cell layer and in the apical dendritic region, suggesting intrinsic connectivity. Axonal varicosities and extensions were found on axons that entered presubiculum, entorhinal cortex or CA1, supporting the notion that these were projection cells. Local collaterals were distinctly thinner than collaterals that would leave the subiculum, suggesting little or no myelin on local collaterals and some myelin on efferent fibers. We conclude that both IB and RS classes of subicular principal cells make synaptic contacts in and apical to the cell layer. Based on the patterns of axonal arborization, we suggest that subiculum has at least a crude columnar and laminar architecture, with ascending collaterals of deep cells forming columns and broader axonal arbors of superficial cells serving to distribute activity across multiple columns. J. Comp. Neurol. 435:490–505, 2001.

Firing relations of medial entorhinal neurons to the hippocampal theta rhythm in urethane anesthetized and walking rats

SummaryThe firing of neurons from layers II and III of medial entorhinal cortex (MEC) was examined in relation to the hippocampal theta rhythm in urethane anesthetized and walking rats. 1) MEC neurons showed a significant phase relation to the hippocampal theta rhythm in both walking and urethane anesthetized rats, suggesting that this region contributes to the generation of both atropine-resistant and atropine-sensitive theta rhythm components. 2) The proportion of phase-locked cells was three times greater in walking rats (22/23 cells) as compared to anesthetized rats (8/23 cells), indicating that MEC cells made a greater contribution during walking theta rhythm. This difference was also manifest in the greater mean vector length for the group of phase-locked MEC cells during walking: 0.39 ± 0.13 versus 0.21 ± 0.08. Firing rate differences between walking and urethane conditions were not significant. 3) In walking rats, MEC cells fired on the positive peak of the dentate theta rhythm (group mean phase = 5°; 0° = positive peak at the hippocampal fissure). This is close to the reported phases for dentate granule and hippocampal pyramidal cells. The distribution of MEC cell phases in urethane anesthetized rats was broader (group mean phase = 90°), consistent with the phase data reported for hippocampal projection cells.These findings suggest that medial entorhinal neurons are the principal determinant of theta-related firing of hippocampal neurons and that their robust rhythmicity in walking as compared to urethane anesthesia accounts for EEG differences across the two conditions.

Intrinsic connectivity of the rat subiculum: II. Properties of synchronous spontaneous activity and a demonstration of multiple generator regions

Brain structures that can generate epileptiform activity possess excitatory interconnections among principal cells and a subset of these neurons that can be spontaneously active (“pacemaker” cells). We describe electrophysiological evidence for excitatory interactions among rat subicular neurons. Subiculum was isolated from presubiculum, CA1, and entorhinal cortex in ventral horizontal slices. Nominally zero magnesium perfusate, picrotoxin (100 μM), or NMDA (20 μM) was used to induce spontaneous firing in subicular neurons. Synchronous population activity and the spread of population events from one end of subiculum to the other in isolated subicular subslices indicate that subicular pyramidal neurons are coupled together by excitatory synapses. Both electrophysiological classes of subicular pyramidal cells (bursting and regular spiking) exhibited synchronous activity, indicating that both cell classes are targets of local excitatory inputs. Burst firing neurons were active in the absence of synchronous activity in field recordings, indicating that these cells may serve as pacemaker neurons for the generation of epileptiform activity in subiculum. Epileptiform events could originate at either proximal or distal segments of the subiculum from ventral horizontal slices. In some slices, events originated in both proximal and distal locations and propagated to the other location. Finally, propagation was supported over axonal paths through the cell layer and in the apical dendritic zone. We conclude that subicular burst firing and regular spiking neurons are coupled by means of glutamatergic synapses. These connections may serve to distribute activity driven by topographically organized inputs and to synchronize subicular cell activity. J. Comp. Neurol. 435:506–518, 2001.

PRESUBICULAR AND PARASUBICULAR CORTICAL NEURONS OF THE RAT: FUNCTIONAL SEPARATION OF DEEP AND SUPERFICIAL NEURONS IN VITRO

1 The presubiculum and parasubiculum are retrohippocampal structures bordered by the subiculum and medial entorhinal cortex. Deep layer (IV‐VI) neurons from this region exhibit stable synaptically triggered burst behaviour which distinguishes them from superficial layer (I‐III) cells. This functional separation was examined with intracellular and field potential recordings from horizontal slices of rat brain. Neurobiotin labelling and rapid Golgi techniques were used to obtain anatomical evidence of axonal trajectories. 2 Extracellular stimulation of the subiculum, deep medial entorhinal cortex or superficial pre‐or parasubiculum caused, in deep layer cells only, a short latency burst discharge which could be followed by one or more after‐discharges. Bursts appeared after repetitive stimulation and were stable for the life of the slice. Each event was supported by giant excitatory postsynaptic potentials (EPSPs). Events were similar whether they were evoked in horizontal slices or slices cut perpendicular to the horizontal plane. 3 Bath application of the NMDA receptor antagonist 3‐[2‐carboxypiperazin‐4‐yl]‐propyl‐1‐phosphonic acid (GPP; 5 μm) elevated the threshold for evoking the giant EPSP. Higher concentrations (10‐15 μm) reduced the amplitude and duration of the giant EPSP. Bath application of the AMPA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5 μm) eliminated the evoked EPSP. 4 In intact slices, superficial layer neurons of pre‐ and parasubiculum could exhibit EPSPs coincident with bursts recorded in the deep layers. However, in isolated subsections of horizontal slices or in ‘vertical slices’, both of which contained only pre‐ and/or parasubiculum, evoked or picrotoxin‐induced bursts occurred only in deep layer cells. Superficial layer cells in these subsections showed no response to deep layer events. 5 Antidromic population spikes confirmed projections from superficial cell layers of pre‐ and parasubiculum down to their deep cell layers. Reciprocal antidromic responses were absent. 6 Axons of superficial layer stellate and pyramidal cells had horizontal collaterals and at least one ascending and one descending collateral. Branches of the descending collaterals were given off in layer V and some axons were found to reach the angular bundle. Axons of deep layer stellate and pyramidal cells also had horizontal collaterals and descending collaterals which could be traced to the angular bundle. One ascending axon collateral was found among the thirty‐one deep layer cells examined morphologically. 7 We conclude that the deep layer cells of the presubiculum and parasubiculum are richly interconnected with excitatory synapses. These interconnections can generate giant excitatory synaptic potentials that support the bursting behaviour exhibited by these neurons. Any of the excitatory inputs to deep layer cells can trigger the population bursts and specific inputs from entorhinal cortex produce the after‐discharges. Further, connections between superficial and deep layer cells appear to be almost exclusively in the direction of superficial to deep. The absence of significant ascending input can account for the functional separation of superficial and deep layer neurons of presubiculum and parasubiculum.

Propagation of synchronous epileptiform events from subiculum backward into area CA1 of rat brain slices.

The hippocampal trisynaptic pathway is comprised of superficial entorhinal afferents (part of the perforant path) to dentate granule cells, dentate mossy fiber inputs to CA3 pyramidal neurons, and CA3 cell projections to CA1 pyramidal neurons. This CA1 output is among others to the subiculum, and both CA1 and subiculum project to the entorhinal cortex to close the loop. Smaller circuits involving fewer hippocampal and parahippocampal regions have also been described. We present morphological and electrophysiological evidence from rat brain slices for a projection from subiculum back into area CA1. Axons of neurobiotin-labeled subicular pyramidal neurons were visualized in the apical dendritic region of CA1. Spontaneous activity in isolated subiculum--CA1 slices was produced by bathing slices in reduced magnesium media. Events in CA1 always followed events in proximal subiculum. Disruption of this subiculum--CA1 circuit with a radially oriented knife cut in the apical dendritic region between subiculum and CA1 eliminated afterdischarges in subicular and CA1 events, but did not de-synchronize the two regions. Full transections between CA1 and subiculum were necessary to functionally isolate the two regions. Only subiculum remained spontaneously active. We conclude that a subiculum--CA1 circuit supports afterdischarges in both regions and synchronizes their activity. This circuit may serve to maintain a level of depolarization in subicular and CA1 pyramidal neurons well beyond the duration of excitatory synaptic potentials resulting from activation of the trisynaptic circuitry.