High-frequency organization and synchrony of activity in the purkinje cell layer of the cerebellum

The cerebellum controls complex, coordinated, and rapid movements, a function requiring precise timing abilities. However, the network mechanisms that underlie the temporal organization of activity in the cerebellum are largely unexplored, because in vivo recordings have usually targeted single units. Here, we use tetrode and multisite recordings to demonstrate that Purkinje cell activity is synchronized by a high-frequency (approximately 200 Hz) population oscillation. We combine pharmacological experiments and modeling to show how the recurrent inhibitory connections between Purkinje cells are sufficient to generate these oscillations. A key feature of these oscillations is a fixed population frequency that is independent of the firing rates of the individual cells. Convergence in the deep cerebellar nuclei of Purkinje cell activity, synchronized by these oscillations, likely organizes temporally the cerebellar output.     

Period doubling of calcium spike firing in a model of a Purkinje cell dendrite

Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained current injection, the cell firing pattern can move from tonic firing of Ca²⁺ spikes to doublet firing and even to quadruplet firing or more complex firing. These firing patterns are not modified substantially if Na⁺ currents are blocked. We show that the experimental results can be viewed as a slow transition of the neuronal dynamics through a period-doubling bifurcation. To further support this conclusion and to understand the underlying mechanism that leads to doublet firing, we develop and study a simple, one-compartment model of Purkinje cell dendrite. The neuron can also exhibit quadruplet and chaotic firing patterns that are similar to the firing patterns that some of the Purkinje cells exhibit experimentally. The effects of parameters such as temperature, applied current, and potassium reversal potential in the model resemble their effects in experiments. The model dynamics involve three time scales. Ca²⁺- dependent K⁺ currents, with intermediate time scales, are responsible for the appearance of doublet firing, whereas a very slow hyperpolarizing current transfers the neuron from tonic to doublet firing. We use the fast-slow analysis to separate the effects of the three time scales. Fast-slow analysis of the neuronal dynamics, with the activation variable of the very slow, hyperpolarizing current considered as a parameter, reveals that the transitions occurs via a cascade of period-doubling bifurcations of the fast and intermediate subsystem as this slow variable increases. We carry out another analysis, with the Ca²⁺ concentration considered as a parameter, to investigate the conditions for the generation of doublet firing in systems with one effective variable with intermediate time scale, in which the rest state of the fast subsystem is terminated by a saddle-node bifurcation. We find that the scenario of period doubling in these systems can occur only if (1) the time scale of the intermediate variable (here, the decay rate of the calcium concentration) is slow enough in comparison with the interspike interval of the tonic firing at the transition but is not too slow and (2) there is a bistability of the fast subsystem of the spike-generating variables.     

The Sodium-Potassium Pump Controls the Intrinsic Firing of the Cerebellar Purkinje Neuron

In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na⁺/K⁺ pump activity sets the Purkinje cell''s operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na⁺/K⁺ pump. The model can replicate these recordings. We propose that Na⁺/K⁺ pump activity controls the intrinsic firing mode of cerbellar Purkinje cells.     

On the Firing Rate Dependency of the Phase Response Curve of Rat Purkinje Neurons In Vitro

Synchronous spiking during cerebellar tasks has been observed across Purkinje cells: however, little is known about the intrinsic cellular mechanisms responsible for its initiation, cessation and stability. The Phase Response Curve (PRC), a simple input-output characterization of single cells, can provide insights into individual and collective properties of neurons and networks, by quantifying the impact of an infinitesimal depolarizing current pulse on the time of occurrence of subsequent action potentials, while a neuron is firing tonically. Recently, the PRC theory applied to cerebellar Purkinje cells revealed that these behave as phase-independent integrators at low firing rates, and switch to a phase-dependent mode at high rates. Given the implications for computation and information processing in the cerebellum and the possible role of synchrony in the communication with its post-synaptic targets, we further explored the firing rate dependency of the PRC in Purkinje cells. We isolated key factors for the experimental estimation of the PRC and developed a closed-loop approach to reliably compute the PRC across diverse firing rates in the same cell. Our results show unambiguously that the PRC of individual Purkinje cells is firing rate dependent and that it smoothly transitions from phase independent integrator to a phase dependent mode. Using computational models we show that neither channel noise nor a realistic cell morphology are responsible for the rate dependent shift in the phase response curve.     

The Output Signal of Purkinje Cells of the Cerebellum and Circadian Rhythmicity

Measurement of clock gene expression has recently provided evidence that the cerebellum, like the master clock in the SCN, contains a circadian oscillator. The cerebellar oscillator is involved in anticipation of mealtime and possibly resides in Purkinje cells. However, the rhythmic gene expression is likely transduced into a circadian cerebellar output signal to exert an effective control of neuronal brain circuits that are responsible for feeding behavior. Using electrophysiological recordings from acute and organotypic cerebellar slices, we tested the hypothesis whether Purkinje cells transmit a circadian modulated signal to their targets in the brain. Extracellular recordings from brain slices revealed the typical discharge pattern previously described in vivo in single cell recordings showing basically a tonic or a trimodal-like firing pattern. However, in acute sagittal cerebellar slices the average spike rate of randomly selected Purkinje cells did not exhibit significant circadian variations, irrespective of their specific firing pattern. Also, frequency and amplitude of spontaneous inhibitory postsynaptic currents and the amplitude of GABA- and glutamate-evoked currents did not vary with circadian time. Long-term recordings using multielectrode arrays (MEA) allowed to monitor neuronal activity at multiple sites in organotypic cerebellar slices for several days to weeks. With this recording technique we observed oscillations of the firing rate of cerebellar neurons, presumably of Purkinje cells, with a period of about 24 hours which were stable for periods up to three days. The daily renewal of culture medium could induce circadian oscillations of the firing rate of Purkinje cells, a feature that is compatible with the behavior of slave oscillators. However, from the present results it appears that the circadian expression of cerebellar clock genes exerts only a weak influence on the electrical output of cerebellar neurons.     

Interspike background activity in extracelullarly recorded Purkinje neurons: spectral analysis

The aim of this study was to investigate the spectral characteristics of Purkinje cell interspike background activity caused by the occurrence of particular action potentials or by electrically induced enhancement of cerebellar inhibitory and excitatory input drive. Spontaneously active Purkinje neurons were extracellularly recorded in anesthetized rats before and after cessation of stimulation from the inferior olive (10) or locus coeruleus (LC). After A/D conversion (30 kHz), direct spectral analysis of extracted interspike background activity was done. Our results have shown that, in contrast to simple spikes, the occurrence of complex spikes induces changes in the spectra of interspike background activity. The different spectral changes of interspike background activity induced by LC and 10 stimulation also indicated the importance of this extracellularly recorded phenomenon.     

Blockade of central beta-adrenergic receptors by tazolol (1-isopropylamino-3-(2-thiazoloxy)-2-propanol).

Tazolol, a β₁-adrenergic agonist in heart, had no intrinsic β-adrenergic agonist activity with respect to cyclic AMP-generating systems in rat cerebral cortical slices or with respect to firing of rat cerebellar Purkinje cells. Instead, tazolol proved to be a relatively potent and specific β-adrenergic antagonist. The IC₅₀ for (±) tazolol in antagonizing (-) isoproterenol-elicited accumulation of cyclic AMP in rat cortical slices was 7 × 10^?7M. The IC₅₀ in antagonizing [³H] dihydroalprenolol-binding in rat cortical homogenates was 2.9 × 10^?7 M. Tazolol was about 10 fold more potent in both cases than the β-antagonist, (±) sotalol. Tazolol antagonized the inhibitory, β-adrenergically mediated effects of iontophoretically applied norepinephrine on firing of cerebellar Purkinje cells. The inhibitory effects of γ-aminobutyric acid on firing of Purkinje cells were not altered by tazolol. Tazolol appeared to lack significant local anesthetic activity as evidenced by its lack of effect on spike height in spontaneous firing Purkinje cells.     

Activity-dependent induction of facilitation,depression, and post-tetanic potentiation at an insect central synapse

Summary In Manduca sexta larvae, sensory neurons innervating planta hairs on the tips of the prolegs make monosynaptic excitatory connections with motoneurons innervating proleg retractor muscles. Tactile stimulation of the hairs evokes reflex retraction of the proleg. In this study we examined activity-dependent changes in the amplitude of the excitatory postsynaptic potentials (EPSPs) evoked in a proleg motoneuron by stimulation of individual planta hair sensory neurons. Deflection of a planta hair caused a phasic-tonic response in the sensory neuron, with a mean peak instantaneous firing frequency of >300 Hz, and a tonic firing rate of 10–20 Hz. Direct electrical stimulation was used to activate individual sensory neurons to fire at a range of frequencies including those observed during natural stimulation of the hair. At relatively low firing rates (e.g., 1 Hz), EPSP amplitude was stable indefinitely. At higher instantaneous firing frequencies (>10 Hz), EPSPs were initially facilitated, but continuous stimulation led rapidly to synaptic depression. High-frequency activation of a sensory neuron could also produce post-tetanic potentiation, in which EPSP amplitude remained elevated for several min following a stimulus train. Facilitation, depression, and post-tetanic potentiation all appeared to be presynaptic phenomena. These activity-dependent changes in sensory transmission may contribute to the behavioral plasticity of the proleg withdrawal reflex observed in intact insects.Abbreviations ACh acetylcholine - AChE acetylcholine esterase - CNS central nervous system - EPSP excitatory postsynaptic potential - I _h injected hyperpolarizing current - LTP long-term potentiation - PPR principal planta retractor motoneuron - PTP post-tetanic potentiation - R _in input resistance - V _h hyperpolarized potential - V _m membrane potential - VN ventral nerve - VNA anterior branch of the ventral nerve - V _r resting potential.     

The direct excitatory effect of IL-1β on cerebellar Purkinje cell

Interleukin (IL)-1β is one of the important proinflammatory cytokines in neural as well as immune systems, and plays a pivotal role in the neuroinflammation. We previously demonstrated that cerebellar IL-1β is involved in kainate-induced ataxia, i.e., IL-1β was activated in the cerebellum with systemic administration of kainate, and its type I receptor (IL-1R) was expressed at a soma of cerebellar Purkinje cells. In this study, we examined the effect of IL-1β on cerebellar Purkinje cell function by recording extracellular neuronal activities in anesthetized mice. Systemic administration of kainate increased the firing rates of cerebellar Purkinje cells in normal mice but showed little effect in IL-1R-knockout (IL-1R-KO) mice. Moreover, microiontophoretic administration of IL-1β to cerebellar Purkinje cells increased the firing rates promptly in response to IL-1β. The present results demonstrate that IL-1 system exerts a direct modulatory effect on cerebellar Purkinje cells.     

Causes and consequences of oscillations in the cerebellar cortex

Cerebellar high-frequency oscillations have been observed for many decades, but their underlying mechanisms have remained enigmatic. In this issue of Neuron, two papers indicate that specific intrinsic mechanisms in the cerebellar cortex contribute to the generation of these oscillations. Middleton et al. show that GABA(A) receptor activation and nonchemical transmission are required for nicotine-dependent oscillations at 30-80 Hz and 80-160 Hz, respectively, while de Solages et al. provide evidence that recurrent inhibition by Purkinje cells is essential for oscillations around 200 Hz.     

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development^1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity^3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons^5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function⁷, interaction with other purified cerebellar cells^8,9 or cell death⁷.Download video file.^{(101M, flv)}     

Immunological studies on the Purkinje cells from rat and mouse cerebella. I. Evidence for antibodies characteristic of the Purkinje cells.

Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with ¹²⁵I and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the ¹²⁵I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the ¹²⁵I-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched ¹²⁵I-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively.     

Effects of the selective noradrenergic neurotoxin DSP4 on cerebellar Purkinje neuron electrophysiology

The effect of the adrenergic neurotoxin DSP4 on cerebellar electrophysiology was studied in the rat. DSP4, administered parenterally, depleted cerebellar norepinephrine by 76%. The depressant response of cerebellar Purkinje neurons to phencyclidine, a drug which acts on adrenergic presynaptic terminals to release NE, was markedly reduced after DSP4 pretreatment. In contrast with 60HDA, which increased firing rates of the Purkinje cells, DSP4 did not change the rate or pattern of Purkinje cell discharge. Taken together these results suggest that DSP4 may be a valuable tool for studying central adrenergic pathways, but that this drug has properties which differ from 60HDA.     

Alterations in the Intrinsic Burst Activity of Purkinje Neurons in Offspring Maternally Exposed to the CB1 Cannabinoid Agonist WIN 55212-2

Burst firing plays an important role in normal neuronal function and dysfunction. In Purkinje neurons, where the firing rate and discharge pattern encode the timing signals necessary for motor function, any alteration in firing properties, including burst activity, may affect the motor output. Therefore, we examined whether maternal exposure to the cannabinoid receptor agonist WIN 55212-2 (WIN) may affect the burst firing properties of cerebellar Purkinje cells in offspring. Whole-cell somatic patch-clamp recordings were made from cerebellar slices of adult male rats that were exposed to WIN prenatally. WIN exposure during pregnancy induced long-term alterations in the burst firing behavior of Purkinje neurons in rat offspring as evidenced by a significant increase in the mean number of spikes per burst (p < 0.05) and the prolongation of burst firing activity (p < 0.01). The postburst afterhyperpolarization potential (p < 0.001), the mean intraburst interspike intervals (p < 0.001) and the mean intraburst firing frequency (p < 0.001) were also significantly increased in the WIN-treated group. Prenatal exposure to WIN enhanced the firing irregularity as reflected by a significant decrease in the coefficient of variation of the intraburst interspike interval (p < 0.05). Furthermore, whole-cell voltage-clamp recordings revealed that prenatal WIN exposure significantly enhanced Ca²⁺ channel current amplitude in offspring Purkinje neurons compared to control cells. Overall, the data presented here strongly suggest that maternal exposure to cannabinoids can induce long-term changes in complex spike burst activity, which in turn may lead to alterations in neuronal output.     

p75 Regulates Purkinje Cell Firing by Modulating SK Channel Activity through Rac1

p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75^−/− mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca²⁺-activated potassium (SK) channel activity in Purkinje cells from p75^−/− mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75^−/− cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75^−/− cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels.     

Formation of a functional adrenergic input to intraocular cerebellar grafts: Ingrowth of inhibitory sympathetic fibers

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10⁻⁶ or 10⁻⁵ M α-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic β-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the β-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.     

Serotonin fiber innervation of cerebellar cell suspensions intraparenchymally grafted to the cerebellum ofpcd mutant mice

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of Purkinje cell degeneration (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss inpcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11–13 normal mouse embryos and were intraparenchymally grafted into the cerebellum ofpcd mutants either directly or after pre-treatment with 5, 7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca²⁺-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells. On the other hand, in grafts pre-treated with 5,7-DHT, no 5-HT cell bodies were seen in the grafted cerebellum; 5-HT fibre innervation of the grafts occurred, but it appeared to be slightly less robust compared to situations of co-grafted 5-HT cell bodies. These findings suggest that host 5-HT fibers are able to provide afferent innervation to donor cerebellar tissue; the presence of co-grafted 5-HT cells may augment such an innervation.Special issue dedicated to Dr. Morris H. Aprison.     

Modeling of torsion dystonia through electric stimulation of the vermis cerebellum cortex

It was shown in acute experiments on cats that electrical stimulation (ES) (100-300 Hz, 5.0-10.0 V) of cat's cerebellar vermal cortex (lobules V and VI) was followed by head deviation in the direction opposite to that side on which the animal was laying, posture and movement disturbances and also by simultaneously occurred contraction of musculus-antagonists of extremities. The tonic and posture disturbances were observed during 40-60 s after ES cessation. During this time in the zone of ES in cerebellar cortex the high-amplitude synchronized activity was registered which was due to generator of pathologically enhanced excitation (GPEE) formation. Intraperitoneal diazepam (0.5-1.0 mg/kg, 30 min before the observation) pretreatment suppressed GPEE formation that correlated with suppression of syndrome manifestations. The conclusion was made that cerebellar hyperactive cortex, which was due to GPEE induction, might have played the role of pathological hyperactive determinant structure of the described syndrome.     

Involvement of Ca2 + and flowing endoplasm in recovery of cytoplasmic streaming after K+-induced cessation

Summary When K⁺ of high concentration (50 mM) was applied toNitella cells, the cytoplasmic streaming stopped instantly as in the case of electrical stimulation. Recovery of the streaming after chemical stimulation was much slower than after electrical stimulation. When the endoplasm content was modified by centrifugation, streaming recovery was accelerated in the centrifugal cell fragments rich in endoplasm and deccelerated in those poor in it. The recovery was also accelerated either by permeabilizing the plasmalemma in the presence of EGTA in the external solution or by removing the tonoplast by vacuolar perfusion with the EGTA-containing medium. We concluded that the streaming was recovered due to decrease of the cytoplasmic Ca²⁺ concentration, which seems to be accelerated by sequestering of Ca²⁺ by endoplasmic components. The slow recovery of the streaming after KCl-stimulated cessation is assumed to be caused by continuous influx of Ca^{2 +} during the prolonged membrane depolarization.Abbreviations ATP adenosine 5-triphosphoric acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

New roles for the gamma rhythm: population tuning and preprocessing for the Beta rhythm

Gamma (30–80 Hz) and beta (12–30 Hz) oscillations such as those displayed by in vitro hippocampal (CA1) slice preparations and by in vivo neocortical EEGs often occur successively, with a spontaneous transition between them. In the gamma rhythm, pyramidal cells fire together with the interneurons, while in the beta rhythm, pyramidal cells fire on a subset of cycles of the interneurons. It is shown that gamma and beta rhythms have different properties with respect to creation of cell assemblies. In the presence of heterogeneous inputs to the pyramidal cells, the gamma rhythm creates an assembly of firing pyramidal cells from cells whose drive exceeds a threshold. During the gamma to beta transition, a slow outward potassium current is activated, and as a result the cell assembly vanishes. The slow currents make each of the pyramidal cells fire with a beta rhythm, but the field potential of the network still displays a gamma rhythm. Hebbian changes of connections among the pyramidal cells give rise to a beta rhythm, and the cell assemblies are recovered with a temporal separation between cells firing in different cycles. We present experimental evidence showing that such a separation can occur in hippocampal slices.