Limbic Gamma Rhythms. II. Synaptic and Intrinsic Mechanisms Underlying Spike Doublets in Oscillating Subicular Neurons

Ian M. Stanford, Roger D. Traub, and John G. R. Jefferys

Neuroscience Unit, Department of Physiology, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Stanford, Ian M., Roger D. Traub, and John G. R. Jefferys. Limbic gamma rhythms. II. Synaptic and intrinsic mechanisms underlying spike doublets in oscillating subicular neurons. J. Neurophysiol. 80: 162-171, 1998. Gamma oscillations were evoked in the subiculum in rat transverse hippocampal slices by tetanic stimulation (200 ms/100 Hz) of either CA1 or subiculum. Gamma oscillations in the subiculum differed from those in CA1 in containing population spike doublets as well as singlets. The present study addresses the origin of this more complex form of gamma oscillation in the subiculum. Intracellular recordings from subicular neurons revealed that 63% of them fired double action potentials on cycles of the gamma oscillation that generated population spike doublets after tetanic stimulation of either CA1 or subiculum. The remaining cells produced excitatory postsynaptic potentials (EPSPs), and occasional single spikes, on each cycle. Neurons that fired occasional single action potentials during gamma rhythms were "regular spiking" cells. They did not produce burst discharges during depolarizing steps, had minimal membrane potential sags on hyperpolarizing steps, and responded to single afferent volleys with a single action potential on an EPSP followed by a large inhibitory postsynaptic potential complex. Fast spiking cells were observed too infrequently to be studied in detail. Neurons that fired doublets during gamma rhythms were "intrinsic burst" (IB) cells. They generated bursts of action potentials on step membrane depolarizations, had significant membrane potential sags on step hyperpolarizations with an anodal break potential on return to rest, and fired multiple action potentials in response to high-intensity single afferent volleys. IB neurons did not fire action potential doublets during 1-s membrane depolarizations. Double action potentials, however, were evoked in these cells by depolarizing pulses at 40 Hz from hyperpolarized membrane potentials (-100 mV). Computer simulations suggest that the hyperpolarization between the depolarizations was essential for action potential doublets. The results in this and the previous paper suggest the following: either CA1 or subiculum alone can generate gamma oscillations gated by local networks of interneurons, oscillations in CA1 project through pyramidal cell axons to subiculum with a time lag expected from axon conduction delays, and oscillating sequences of EPSPs and intrinsic and/or synaptic hyperpolarizing potentials in IB subicular neurons generate gamma frequency spike doublets, which depend on both the intrinsic properties of these neurons and their temporally patterned synaptic input. This phenomenon could amplify gamma output from CA1 and modify its coupling to gamma oscillations in the wider limbic system.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The subiculum is a major output region of the hippocampal formation receiving projections from the CA1, CA2, and CA3 regions (Andersen et al. 1973; Finch and Babb 1980, 1981; Hjorth-Simonsen 1973; Swanson et al. 1978). It in turn sends efferents to the septal nuclei, mammillary bodies, thalamus, hypothalamus, and entorhinal cortex, from which it receives reciprocal inputs. The subiculum therefore is placed in a crucial position in the transmission and integration of information between the hippocampus and neocortical and subcortical areas. Indeed, the hippocampal-subiculum-entorhinal cortex long loop has been implicated in learning and the formation of memory (Squire 1986; Squire et al. 1993; Zola-Morgan et al. 1989). Synchronous neuronal discharges in hippocampal CA3/CA1 regions and subiculum have been shown to occur during physiological sharp wave activity (Chrobak and Buzsáki 1994). Furthermore, unit recordings from freely moving rats show that subicular neurons signal head direction and suggest that this information is integrated with the positional information coded by hippocampal units in the solution of spatial problems (Muller et al. 1996; Taube et al. 1990).

There appear to be two principal cell types in the subiculum (Mason 1993; Stewart and Wong 1993; Taube 1993) corresponding to intrinsic bursting and regular spiking neurons observed in the neocortex (Connors and Gutnick 1990). Interneurons, located mainly in the stratum radiatum, also have been reported that have highly branched axons ramifying throughout the pyramidal cell layers (Soriano et al. 1993).

Fast gamma rhythms (30-100 Hz) are prominent in the hippocampus and neocortex and have been implicated in information processing and higher cognitive function (Gray 1994; Singer and Gray 1995). Their putative role comes from the synchronization of groups of neurons in sites that can be many mm apart on the cortical surface (Gray et al. 1989). Such synchronized rhythms are ideally suited to provide a mechanism for the functional "binding" of sensory features along the lines proposed by von der Malsburg (von der Malsburg and Schneider 1986). In the hippocampus, in vitro gamma rhythms depend on the activity of networks of interneurons (Jefferys et al. 1996; Traub et al. 1996a; Whittington et al. 1995). The synaptic connections between the interneuronal network and pyramidal cells cause interneurons to fire doublets of action potentials when enough pyramidal cells are firing. These interneuronal doublets play a key role in synchronizing pyramidal cell firing at gamma frequencies over long distances. Such synchronization occurs with much shorter phase lags than expected from the estimated conduction delays (Traub et al. 1996b).

The existing evidence is that gamma oscillations in the neocortex may be more complex than in the hippocampus. We have shown that the neocortex can generate gamma rhythms when excitatory postsynaptic potentials (EPSPs) are blocked (Whittington et al. 1995). On the other hand, there is indirect evidence that intrinsic neuronal properties may play a greater role in generating oscillations in the cortex than in the hippocampus. This is shown by the existence of chattering cells (which are spiny and presumed to be excitatory), studied intracellularly by Gray and McCormick (1996), and by other classes of neuron that tend to oscillate at 40 Hz in response to tonic depolarizing currents. Some of these intrinsically oscillating neurons are inhibitory (Llinás et al. 1991).

In the previous paper, we showed that gamma rhythms can project across regional boundaries from hippocampal CA1 to the subiculum (but not from one part of CA1 to other distant parts of CA1 nor from subiculum to CA1), that gamma rhythms can be generated in subiculum by interneuron network mechanisms at least superficially similar to those in CA1, and that the subicular rhythm often contains population spike doublets. Here we show that the population spike doublets are due to action potential doublets in intrinsic burster neurons, which result from the interaction between their intrinsic membrane properties and the rhythmic drive from the sequence of synaptic potentials generated by the network. Intrinsic burster neurons preferentially participate in network-induced gamma rhythm in the subiculum, and we postulate that these neurons are able to amplify the oscillating signal and are able to transmit the hippocampal 40-Hz activity to other areas of the brain.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Intracellular and extracellular recordings were made in transverse hippocampal slices obtained from Sprague Dawley rats weighing 250-300 g. The animals were anesthetized with a mixture of medatamine hydrochloride (Domitor; 1 mg/kg, SmithKline Beecham) and ketamine hydrochloride (Vetalar; 100 mg/kg, Parke Davis Veterinary) and killed by cervical dislocation. The brain was removed quickly and 400-µm-thick hippocampal slices cut on a Vibroslice (Campden Instruments, Loughborough, UK). The slices were maintained in an interface chamber and constantly perfused at a rate of 2-3 ml/min with artificial cerebrospinal fluid containing (in mM) 135 NaCl, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 10 glucose, buffered to pH 7.4 with Na2HCO3 (16 mM) at 33-35°C. Recording electrodes were made from borosilicate glass pipettes. Intracellular electrodes were 40-60 MOmega in resistance containing 2 M potassium acetate. Signals were amplified by a high-input impedance amplifier (Axoclamp 2A), which allowed current injection and constant monitoring of bridge balance and capacitance compensation. Extracellular electrodes were filled with 3 M NaCl and were 6-10 MOmega in resistance.

Recordings were made in the CA1 pyramidal cell layer and in the subiculum. Gamma oscillations were evoked by tetanic stimulation (200 ms/100 Hz) using a bipolar stimulating electrodes placed in the CA1 layer and subiculum in close proximity to the recording sites. Recordings were amplified, low-pass filtered at 10 kHz, and digitized using a CED 1401-plus laboratory computer system using SIGAVG and SPIKE2 (Cambridge Electronic Design, Cambridge, UK). All numerical data are expressed as means ± SE unless otherwise stated.

Simulations of subicular intrinsic bursting neuron

We began with a branching dendritic model of a CA3 pyramidal neuron (Traub et al. 1994) that contained five compartments for the axon and initial segment, a soma, four branching basilar dendrites of seven compartments each, and a branching apical dendrite of 35 compartments. Electrotonic parameters included Rm = 50,000 Omega -cm2, Cm = 0.75 µF/cm2 (hence tau m = 37.5 ms), Ri = 200 Omega -cm, Rinput = 37.4 MOmega . Ionic membrane conductances included fast gNa (but not persistent gNa), high-threshold gCa, and three types of gK: delayed rectifier, fast voltage- and Ca- dependent gK(C), and the slow Ca-dependent gK(AHP). This model was rendered less prone to generate intrinsic bursts (but still capable of it) by reduction of gCa (twofold), increase of gK(AHP) (2.5-fold), and increase of gK(C) (twofold). The firing of this modified neuron, in response to injected depolarizing somatic currents, resembled that of intrinsically bursting subicular neurons but with two exceptions: the model cell produced more fast spikes on the initial burst than do real neurons and posthyperpolarization rebound action potentials were absent in the model (cf. Mattia et al. 1993). The model neuron was stimulated either with tonic currents or hyperpolarizing/depolarizing current pulses delivered to the soma or with excitatory postsynaptic currents (EPSCs) delivered at 40 Hz to the basal dendrites. Each EPSC consisted of a conductance 40 × t × exp (-t/2) nS, t in ms, to each of four compartments in the basal dendrites. The reversal potential of the EPSC was 60 mV positive to resting potential.

Network model

A distributed network model was constructed using an approach similar to that described in Whittington et al. (1997a), using component pyramidal cell and interneuron models as in Traub et al. (1994) and Traub and Miles (1995). Differences from the previous model were in intrinsic properties of the pyramidal cells, which were modified as described above, to resemble subicular intrinsic bursting cells; network topology; method used to generate axon conduction delays between cells; and unitary synaptic conductances.

Network topology

The network model contains 120 pyramidal cells and 120 interneurons, the latter presumed to be basket cells. We first constructed two overlying arrays, each 30 × 20, of pyramidal cells and interneurons, respectively. The lattice spacing in the array is taken to be 150 µm, so that the "proto"-array is ~4.5 × 3.0 mm. Cells are "wired up," so that each neuron receives 50 excitatory inputs, coming from a 5 × 10 band of pyramidal cells overlying the neuron; and it receives 49 inhibitory inputs, coming from a 7 × 7 square of interneurons overlying the cell. The input patterns were the same for pyramidal cells and interneurons. Finally, a "slice" was cut, consisting of the lower 6 × 20 part of the array.

Axon conduction delays in the model were calculated from the Euclidean distance between pre- and postsynaptic cells, assuming a conduction velocity of 0.2 m/s, for axons of pyramidal cells and of interneurons.

Unitary synaptic conductances

As each neuron now has more presynaptic neurons than in previous models, we used somewhat smaller unitary synaptic conductances. With units of nanoSiemens for conductance and milliseconds for t = time, the unitary conductances were as follows: e right-arrow i, 4 × t × exp(-t); i right-arrow e, 1.3 × exp(-t/10); i right-arrow i, 0.5 × exp(-t/10); e right-arrow e (to an apical dendritic compartment), 1.8 × t × exp(-t/2); e right-arrow e (to a basilar dendritic compartment), 0.9 × t × exp(-t/2). Only alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and GABAA-receptor-mediated conductances were simulated. The reversal potentials of AMPA and GABAA synaptic conductances were 60 mV positive and 15 mV negative to resting potential, respectively. In some simulations, GABAA conductances were blocked, to represent the effect of bicuculline.

Stimuli

The model neurons were stimulated in two different modes, alone or in combination. To simulate the effects of local subicular tetanus, depolarizing conductances were applied to both pyramidal neurons and interneurons (Whittington et al. 1997a). We used tonic conductances (resting potential 60 mV positive to rest): for pyramidal cells, this conductance was 50-70 nS total, spread over four apical dendritic compartments; for interneurons, it was 2-2.25 nS, applied to the soma. To simulate the effects of CA1 gamma oscillations on the subiculum, 40-Hz periodic EPSCs were applied to the distal basilar dendrites and to the middle apical dendrites. The total conductance to the basilar dendrites had time course: 48 × t × exp(-t/2) nS and to the apical dendrites: 120 × t × exp(-t/2) nS. The onset of the EPSCs was not simultaneous for all of the pyramidal cells but propagated along the long axis of the network at 0.5 m/s.

Simulations of networks were run using a 12-node IBM SP2 parallel computer. Simulation of 1 s of neuronal activity took 1,935 s. Simulations of single neurons were run on an individual node of the computer.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Gamma oscillations in subiculum

Tetanic stimulation (200 ms/100 Hz) of the stratum pyramidale in CA1 or subiculum evoked coherent rhythmic population responses (after a silent period of 18.6-183.0 ms) in the gamma frequency band (52.3 ± 2.6 Hz). The frequency peaked during the initial 200 ms of activity and then gradually slowed. The subicular population gamma rhythm differed from that of CA1 in that double population spikes occurred on many cycles in the subiculum (Fig. 1). The peaks of the individual population spikes within the doublets were separated by 4.4 ± 0.2 ms.


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FIG. 1. Subicular population spike doublets evoked by tetanic stimulation of the CA1 pyramidal cell layer. A: simultaneous extracellular recordings from the CA1 pyramidal cell layer (top trace) and the subiculum (bottom trace) in response to 100 Hz/200 ms stimulation of the CA1 pyramidal cell layer. Gamma activity at 62 Hz is evoked in CA1, which propagates to the subiculum where population spike doublets were observed. Subicular doublets lag the CA1 response by 5.0 ms as determined by cross-correlation analysis. Segments of the 2 traces have been expanded in B to show the single population spikes in CA1 and the slightly lagging doublets in the subiculum. Traces in A start at the end of the stimulus train and show the latent period before the onset of the gamma oscillation (traces in the remaining figures in this paper all start after the end of the stimulus train).

Subicular neuron electrophysiological characterization

Stable intracellular recordings were obtained from 50 subicular neurons. Subicular cells were characterized by their response to a step depolarization and orthodromic stimulation in the alveus in CA1. A 250-ms depolarizing current above threshold for action potentials elicited one of two responses depending on the type of neuron.

The first type of neuron had a resting membrane potential of -65.9 ± 0.6 mV and an input resistance of 29.6 ± 2.2 MOmega . These neurons evoked a burst of two to four action potentials followed by single spikes throughout the remainder of the depolarization step (Figs. 2Ai and 6A). Step hyperpolarizations of 250-ms duration elicited a slow time-dependent inward current sag in membrane potential (by a mean of 3.7 ± 0.3 mV for -0.7-nA pulses). At the end of the hyperpolarizing step, these cells responded with a depolarization that often could reach threshold for burst firing. Orthodromic stimulation of these neurons evoked an EPSP/IPSP complex and action potential firing at a threshold potential of -50.9 ± 0.9 mV. On suprathreshold stimulation a second action potential often could be evoked (Fig. 2Aii). These properties are identical to those previously described (Mason 1993; Stewart and Wong 1993; Taube 1993) and cells exhibiting these properties were considered intrinsic bursting (IB) neurons similar to those described in the neocortex (Connors and Gutnick 1990).


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FIG. 2. Electrophysiological characteristics of intrinsic bursting neurons and regular spiking neurons. Ai: voltage response of an intrinsic bursting neuron in response to 200-ms current steps from a resting membrane potential of -63 mV. Hyperpolarizing current steps elicited a pronounced sag in the membrane potential. On removal of the hyperpolarizing step, there was often an overshoot of the voltage ("anodal break," see arrow) that could evoke a burst of action potentials. Depolarizing steps to threshold for action potential firing caused bursts of action potentials. Aii: in response to a high-intensity single shock in the CA1 region, intrinsic bursting neurons often could fire multiple action potentials. Bi: voltage traces of a regular spiking neuron in response to 200-ms current steps from a resting membrane potential of -62 mV. A smaller sag in the voltage is seen during hyperpolarizing steps, and there is no evidence of anodal break and action potential firing afterward. Depolarizing steps to threshold for action potential firing evoked single action potentials. Bii: single shock orthodromic stimulation only evoked a single action potential riding on an excitatory postsynaptic potential (EPSP) followed by pronounced biphasic inhibitory postsynaptic potentials (IPSPs).

The second type of subicular cell encountered resembled regular spiking (RS) neurons (Taube 1993). These neurons had resting membrane potentials of -65.0 ± 0.9 mV, input resistances of 29.4 ± 2.6 MOmega , and an action potential thresholds of -51.8 ± 1.0 mV. These neurons fired single action potentials throughout a depolarizing pulse. They had smaller membrane potential sags during hyperpolarization steps than IB cells [a mean of 1.5 ± 0.4 mV for -0.7-nA pulses; t-test P = 0.0004; comparable with the difference between RS and IB cells reported previously (Taube 1993)]. There was no evidence of a rebound (anodal break) depolarization on return to rest. Orthodromic stimulation evoked only a single action potential riding on an EPSP, usually followed by a pronounced two phase IPSP complex (Fig. 2Bii). These cells had the characteristics of RS cells.

IB cells preferentially participate in gamma activity in the subiculum

The intracellular response of IB and RS cells to tetanic stimulation of either CA1 or subiculum was very different. IB cells fired in phase with the population spikes recorded extracellularly. Strong tetanic stimulation of the CA1 pyramidal cell layer caused IB cells to fire action potential doublets in the gamma frequency range. These doublets corresponded to the double population spike activity recorded extracellularly (Fig. 3A). High-intensity tetanic stimulation of the subiculum was not as effective as stimulation of CA1 for eliciting IB cell doublet spiking. However, intracellular records did reveal doublet spiking on 33% (3/9) of occasions. On the occasions where intracellular double spikes were not observed there was still clear evidence of extracellular double population spikes (Fig. 3Aii). In marked contrast, RS cells responded much more weakly to tetanic stimulation than did IB cells. High-intensity tetanic stimulation of the CA1 pyramidal cell layer produced EPSPs and single spikes in RS cells at gamma frequency even though simultaneous extracellular recording showed population responses with double spikes (Fig. 3Bi). Tetanic stimulation of the subiculum resulted in a lower level of excitation of RS cells. Even at high stimulus intensity, many RS cells only produced EPSPs at gamma frequency with the occasional single action potential (Fig. 3Bii).


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FIG. 3. Spike doublets are due to the activity of intrinsic bursting neurons. Simultaneous extracellular population recording and intracellular recordings from an intrinsic bursting neuron (A) and a regular spiking neuron (B). Activity of each neuron shown is in response to high-intensity tetanic stimulation (100 Hz/200 ms) delivered either to the CA1 pyramidal cell layer (i) or the subiculum (ii). On CA1 stimulation, the extracellular response is robust with both examples showing population doublets. This is due to the activity of intrinsic bursting neurons, which show doublet action potential firing on almost every cycle, whereas the regular spiking cell activity is limited to EPSPs and single action potentials. High-intensity tetanic stimulation of the subiculum can evoke population doublets although to a lesser degree. In this case intrinsic bursting neurons show sporadic doublet firing, and depolarizing afterpotentials following single action potentials, while regular spiking cells only reveal EPSPs with occasional action potentials.

The activity of IB cells during gamma rhythms depended on stimulus strength. Weak tetanic stimulation of CA1 produced repetitive EPSPs at gamma frequencies with only occasional action potentials (Fig. 4Ai). Increasing the stimulus intensity produced single action potentials on each cycle sometimes followed by an "aborted" spike (Fig. 4Aii). Further increases in stimulation strength resulted in double action potentials riding on large EPSPs on each cycle (Fig. 4Aiii). Similar results occurred with stimulation of the subiculum, although the IB cell activity was not quite as pronounced as with stimulation of CA1 (Fig. 4B). The extracellular field potential responded to the increasing stimulus strength, applied to either CA1 or subiculum, with a similar progression of repetitive field EPSPs, single population spikes, and population spike doublets (data not shown).


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FIG. 4. Intrinsic bursting neurons fire doublets in response to tetanic stimulation of CA1 and subiculum: effect of stimulus intensity. Ai: tetanic stimulation of the CA1 layer at low stimulus intensity evokes EPSPs at the gamma frequency along with the occasional action potential. ii: increasing the stimulus strength elicits larger EPSPs with spikes and occasional aborted second spikes. iii: further increase of the stimulation resulted in action potential spike doublets at a frequency of 37 Hz. Bi: low intensity tetanic stimulation of the subiculum evoked rhythmic EPSPs (ii), increasing the stimulation resulted in single action potential firing. iii: further increases of the stimulation intensity resulted in action potential firing on every EPSP with doublets at the end of the train of activity.

Subicular gamma rhythms evoked by subicular stimulation were blocked by 2 µM bicuculline and replaced by a transient period of epileptic responses (Fig. 5A), resembling the effect of bicuculline on subicular gamma evoked by CA1 stimulation (see Figs 6 and 7 in companion paper, Colling et al. 1998). The epileptic discharges were lost after 10 min in bicuculline. These epileptic discharges differed from gamma oscillations in the characteristic presence of paroxysmal depolarizations with multiple action potentials on each cycle (Fig. 5B). Gamma rhythms evoked by stimulation of the subiculum were blocked by perfusion with 20 µM 6nitro - 7 - sulphamoylbenzo[f]quinoxaline - 2, 3 - dione   (NBQX) and 100 µM D-2-amino-5-phosphonopentanoic acid (D-AP5) (see the companion paper, Colling et al. 1998, where we showed that gamma rhythms evoked in the subiculum by stimulation of CA1 were blocked by NBQX). Injection of depolarizing current into neurons impaled in the presence of these excitatory blockers revealed small (1-2 mV) rhythmic hyperpolarizing potentials in response to subicular stimulation that were not associated with a measurable field potential (Fig. 5C). Autocorrelation of this signal revealed a 31-Hz rhythm. These potentials were bicuculline sensitive and therefore were considered to be GABAA-receptor-mediated IPSPs evoked by activation of the local interneuron network, similar to those we reported previously for CA1 (Whittington et al. 1995).


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FIG. 5. Bicuculline disrupts and then blocks both CA1 and subicular induced 40 Hz activity. A: extracellular subicular recordings after tetanic stimulation of the subiculum within 150 µm of recording electrode. Perfusion with 2 µM bicuculline blocked the gamma rhythm and induced bursting activity similar to epileptic discharges (7 min) before it blocked the response entirely (12 min). This effect was reversed on wash out of the bicuculline. B: dual intracellular and extracellular recordings from an intrinsic bursting neuron in response to 200-ms/100-Hz stimulation of the CA1 pyramidal layer in the presence of 2 µM bicuculline, during the transient epileptic phase as shown at 7 min in A. This effect was reversible on wash. Ci: rhythmic inhibitory postsynaptic potentials (IPSPs) evoked by tetanic stimulation were recorded in the presence of 20 µM 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 100 µM D-2-amino-5-phosphonopentanoic acid (D-AP5). Cell was depolarized to -40 mV to reveal IPSPs more clearly; 5 mM QX314 was included in the micropipette to prevent action potential firing. ii: autocorrelation revealed a period of 32.5 ms, corresponding to a frequency of 31 Hz. iii: these IPSPs were blocked by 5 µM bicuculline.


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FIG. 6. Current injection patterns necessary for repetitive action potential doublets in intrinsic bursting neurons. A: 1-nA depolarization step, 1 s in duration, from a resting membrane potential of -65 mV, evoked a burst of action potentials followed by single spikes at a frequency of 14 Hz. Increasing the current to 2.0 nA evoked an initial larger burst followed by single spikes at a frequency of 33 Hz. There was no evidence of intrinsic bursting neurons firing doublets in response to a long depolarizing current step. B: current pulses, 20 ms in duration, delivered at 40 Hz (top) did evoke spike doublets in intrinsic bursting neurons. Train of 2-nA depolarizing pulses from a resting potential of -65 mV triggered an action potential on each cycle, followed by a broad 2nd spike (and a 3rd on the 1st cycle; 2nd trace). Second spike on each pulse became sharper when the baseline membrane potential was hyperpolarized to -80 mV (with a corresponding increase in the pulse height). Current pulses from a potential of -100 mV induced sharp spike doublets similar to those observed in gamma rhythm activity.


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FIG. 7. Model subicular neuron does not generate doublets in response to tonic depolarization of the soma but can do so in response to 40-Hz on-off current pulses or to 40-Hz EPSPs. Top: tonic 0.75-nA current injection into the soma elicits a burst, followed by single action potentials of steadily diminishing frequency. Middle: on/off current pulses at 40 Hz (+2.0 nA for 12.5 ms, -1.0 nA for 12.5 ms) lead to a few spike triplets, then a 40-Hz train of doublets, and eventually a singlet. Bottom: 40-Hz EPSPs [conductance in nS 10 × 2 × exp(-t/2) to each of 4 compartments in the basal dendrites] elicit an initial burst, then bursts with 4 right-arrow 3 right-arrow 2 spikes, and then a series of spike doublets at 40 Hz. Note the gradually increasing underlying afterhyperpolarization.

Intrinsic properties of IB cells alone do not explain double population spikes and action potentials

To determine whether intrinsic properties alone can explain the spike doublets seen in IB cells during gamma rhythms, long depolarizing pulses (1 s in duration) were injected. After the initial burst, IB cells showed frequency adaptation and then produced only single action potentials. The firing frequency appeared to depend on the magnitude of the step depolarization (Fig. 6A). Under these conditions, IB cells could not be made to fire repetitive spike doublets.

Could IB cells produce spike doublets if presented with depolarization steps at 40 Hz? This experiment is shown in Fig. 6B. Step depolarizations, 20 ms in duration, were applied every 25 ms to evoke action potentials. At resting membrane potential (~ -65 mV), an initial action potential was followed by a long spike, presumably mediated by calcium channels (Stewart and Wong 1993) and/or persistent sodium channels (Mattia et al. 1993). This second spike was evoked independently of the duration of the depolarization step. This pattern recurred on subsequent pulses as long as the depolarization was strong enough. When the depolarizing pulses were superimposed on a steady hyperpolarization, the second spike gradually sharpened (reduced time constant of decay). Step depolarizations at 40 Hz from -100 mV can evoke multiple sharp spike doublets in IB cells similar to those seen during the gamma rhythms.

The computer model of an intrinsically bursting neuron generated behaviors similar to those observed experimentally in subicular IB cells (Fig. 7). Specifically, injection of a steady depolarizing current into the soma, provided the current was large enough to elicit an initial burst, produced a series of single spikes at gradually decreasing frequency; the peak frequency and total number of these spikes depended on the magnitude of the injected current (not shown). Spike adaptation was caused by intrinsic K+ currents, particularly the afterhyperpolarization (AHP) current. In contrast, doublet firing could be induced in either of two ways: 1) by somatic injection of depolarizing/hyperpolarizing current pulses (Fig. 7, middle). Depending on how K+ conductances were scaled, injection of on/zero-current pulses (i.e., 12.5 ms on, 12.5 ms off) also could elicit doublets, but there would first occur a series of periodic bursts (not shown). 2) Doublets also could be induced by 40-Hz EPSPs into the basal dendrites (bottom). A similar pattern could be evoked with periodic EPSPs delivered to both basal and apical dendrites (data not shown).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Gamma rhythms generated in the subiculum differ from those in the CA1 region in the doublet firing of the subicular principal cells. In other respects, the rhythms evoked by local tetanic stimulation are similar. In both regions, tetanic stimulation resulted in gamma-frequency population spikes (Colling et al. 1998; Whittington et al. 1997a). Here we show that the subiculum also resembles CA1 in the persistence of rhythmic IPSPs after the block of EPSPs and rhythmic population spikes with NBQX and AP5. This shows that inhibition mediated by GABAA receptors plays a crucial role in organizing the synchronous rhythm in the subiculum as it does in CA1 (Whittington et al. 1995).

The double spikes originate from the IB neurons, which are the most common neurons in the subiculum. These neurons fire action potential doublets reliably in phase with the double population spikes seen in the gamma response. However, the intrinsic properties of the IB cells do not, by themselves, determine the pattern of activity in the same way as, for instance, the CA3 pyramidal cells shape secondary bursts during epileptic discharges (Traub et al. 1993). Rather the activity of subicular IB cells has to be conditioned by patterned inputs, either pulsed current injection or rhythmic synaptic activity, if it is to replicate the rhythmic doublet firing. The implication is that one way for the IB cells to fire doublets is by the removal of deactivation of an inward current (whether Na+ or Ca2+) or by removal of the activation of an outward K+ current; we show in the computer model that another way is by e right-arrow e connections. During evoked gamma activity, this patterned hyperpolarization can be provided by several mechanisms, including dendritic IPSPs or intrinsic potassium currents.

RS cells rarely fired during gamma rhythms. The low rate of firing during subicular gamma activity, evoked from either CA1 or the subiculum itself, is not obviously related to the resting potential, input resistance, or threshold of RS cells. It is more likely related to weaker calcium spiking mechanisms (which differentiate them from IB cells), which promote action potentials after each inhibitory phase. The intermittent response to CA1 gamma activation also could reflect a weaker excitatory input from CA1 or greater feed-forward inhibition.

The differences between the participation of IB and RS cells in subicular gamma rhythms may be related to their different patterns of connections with other structures. There are preliminary data that deep subicular neurons preferentially project to the anterior thalamus (N. Tamamaki, personal communication). As disproportionately many IB cells are found in the deeper layers of the subiculum (Greene and Totterdell 1997), it is possible that IB cells preferentially contact anterior thalamic neurons; if so, the anterior thalamic neurons would receive a strong rhythmic afferent input during subicular gamma oscillations.

Doublet firing of interneurons in the CA1 region plays a key role in maintaining tight synchronization in the face of relatively long conduction delays (Traub et al. 1996b). The role of doublet firing in the excitatory IB cells in the subiculum is likely to differ. The most obvious role would be as an amplifier, strengthening the output from CA1 and relaying it on to the entorhinal cortex and elsewhere. This activity may be similar in form and purpose to that of the chattering cell, which appears to be characteristic of gamma rhythms in the neocortex (Gray and McCormick 1996). In the subiculum, however, the pyramidal doublet activity is the product of intrinsic and synaptic mechanisms, whereas chattering cells can fire high-frequency bursts when tonically depolarized. To understand the effects of the subicular doublets will require further experiments on brain regions "downstream" from the subiculum.

Taking the previous and current papers together, we conclude that both the subiculum and the CA1 region have similar mechanisms to generate gamma rhythms locally and that networks of inhibitory neurons play a key role in both. In addition, gamma rhythms in the CA1 region can project to subiculum through excitation mediated by AMPA receptors, where they generate gamma with a phase lag explicable by conduction delays. The tight synchrony seen between pairs of sites in CA1 does not occur here because of the asymmetry of the connections between CA1 and subiculum. When oscillations are induced in the subiculum by oscillations in CA1, then the oscillating sequence of EPSPs and IPSPs produce in subicular IB cells action potential doublets at gamma frequency, which depend both on the intrinsic properties of these cells and the temporally patterned synaptic input they receive.


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FIG. 8. Two alternative means for generating gamma rhythms (with principal cell spike doublets) in the same network. A: network contains E cells (principal cells) and I cells (inhibitory neurons), synaptically interconnected. Tonic depolarizing inputs to E and I cells together lead to synchronized oscillations (Jefferys et al. 1996; Traub et al. 1996a, 1997; Whittington et al. 1995, 1997a), with recurrent E/E connections inducing the pyramidal cell spike doublets (Whittington et al. 1997b). This would correspond to local subicular stimulation. B: periodic synchronized EPSPs to the principal neurons, providing these latter are designed like subicular IB cells, also can evoke gamma oscillations with principal cell doublets. Network connections are still present but do not play a major role in patterning the network output. This would correspond to the subiculum responding to CA1 gamma oscillations.

The above scheme does not, by itself, account for one crucial experimental observation: the occurrence of spike doublets in subicular IB cells after local tetanic subicular stimulation. If such local stimulation leads to a tonic depolarization of pyramidal neurons (Whittington et al. 1997a) and if tonic depolarization of IB cells fails to generate doublets (Fig. 6), why do doublets occur during the locally generated population oscillation? One possibility is that a patterned synaptic excitatory input can be produced in the subiculum via recurrent excitatory collaterals between IB cells; the existence of such collaterals appears likely, given that epileptiform bursts occur in the disinhibited subiculum after local stimulation (Fig. 5A). Furthermore, pyramidal cell spike doublets can occur in CA1 after intense tetanization that leads to the enhancement of recurrent EPSPs (Whittington et al. 1997b). We propose, therefore, the following scheme to account for the diverse experiments (Fig. 8).


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FIG. 9. Network simulations demonstrating 2 alternative means of generating synchronized gamma oscillations with principal cell spike doublets. Network was designed as in METHODS and was intended to represent a population of subicular IB principal cells and inhibitory neurons. Three patterns of stimulation were used: tonic excitatory conductances to the principal cells and inhibitory neurons, corresponding to subicular stimulation (A); periodic EPSPs to the basal and apical dendrites of the principal neurons, corresponding to CA1 stimulation (B); and tonic excitatory conductances and periodic EPSPs together, corresponding to stimulation of both subiculum and CA1 (C). Synchronized gamma oscillations occur in each case with at least some doublets. Doublets are most prominent after CA1 stimulation as is true experimentally. For A-C, we show the somatic potential of a principal neuron (top), and the average somatic potential of 9 nearby principal neurons (below).

In this scheme, the most essential synaptic interactions are GABAA-receptor-mediated between interneurons and from interneurons to pyramidal cells, and AMPA-receptor-mediated from pyramidal cells to interneurons. Tonic depolarization of mutually inhibitory interneurons, in the absence of phasic excitatory synaptic inputs, can generate gamma oscillations (Traub et al. 1996a; Whittington et al. 1995), whereas in the full system of pyramidal cells and interneurons, tonic depolarization of both cell types together leads to gamma oscillations in which the two cell types fire in phase (Traub et al. 1997; Whittington et al. 1997a). Finally, tonic depolarization of both cell types together, with recurrent excitation to pyramidal cells, can lead to gamma oscillations in which the pyramidal cells fire doublets (Whittington et al. 1997b) (Fig. 8A). This is one possible way that local stimulation of the subiculum might lead to gamma oscillations with doublet population spikes.

On the other hand, periodic EPSPs in the pyramidal cells alone, arriving from afferents impinging from CA1, can generate doublets in CA1 because the EPSPs are synchronized (as CA1 cells oscillate synchronously after tetanic stimulation to them), and because subicular IB cells can respond to periodic excitation with periodic doublets (Figs. 5, 6, and 8B).

That this scheme is logically self-consistent was shown by network simulations (see METHODS) in which a network of IB cells and interneurons was stimulated in three different ways (Fig. 9): tonic depolarization of principal cells and interneurons together, corresponding to local subicular stimulation (Fig. 9A); 40-Hz EPSPs to basal and apical dendrites of the pyramidal cells (Fig. 9B), corresponding to a synchronized afferent input from CA1; and both stimuli simultaneously. In each case, the network responds with doublets both at the single-cell and at the population level. As seen experimentally (Fig. 3), the doublets are less robust after local stimulation than after CA1 stimulation.

There are two major experimental questions posed by our results: how to determine the functional role of doublets and spike multiplets in gamma oscillations and how to determine, in the case of in vivo gamma oscillations, whether the oscillations are being generated endogenously in local networks or are instead being projected from elsewhere.

    ACKNOWLEDGEMENTS

  We thank Drs. Menno Witter and Nobuaki Tamamaki for helpful discussions.

  This work was funded by the Wellcome Trust and the Human Frontier. R. D. Traub is a Wellcome Trust Principal Research Fellow.

    FOOTNOTES

   Present address of I. M. Stanford: Dept. of Pharmacology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

  Address reprint requests to: J.G.R. Jefferys.

  Received 17 October 1997; accepted in final form 20 February 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society