Mode of activation of pyramidal neurons by mossy fiber stimulation in thin hippocampal slices in vitro

During slow wave sleep and consummatory behaviors, electroencephalographic recordings from the rodent hippocampus reveal large amplitude potentials called sharp waves. The sharp waves originate from the CA3 circuitry and their generation is correlated with coherent discharges of CA3 pyramidal neurons and dependent on activities mediated by AMPA glutamate receptors. To model sharp waves in a relatively large hippocampal circuitry in vitro, we developed thick (1 mm) mouse hippocampal slices by separating the dentate gyrus from the CA2/CA1 areas while keeping the functional dentate gyrus-CA3-CA1 connections. We found that large amplitude (0.3–3 mV) sharp wave-like field potentials occurred spontaneously in the thick slices without extra ionic or pharmacological manipulation and they resemble closely electroencephalographic sharp waves with respect to waveform, regional initiation, pharmacological manipulations, and intracellular correlates. Through measuring tissue O2, K+, and synaptic and single cell activities, we verified that the sharp wave-like potentials are not a consequence of anoxia, nonspecific elevation of extracellular K+ and dissection-related tissue damage. Our data suggest that a subtle but crucial increase in the CA3 glutamatergic activity effectively recruits a population of neurons thus responsible for the generation of the sharp wave-like spontaneous field potentials in isolated hippocampal circuitry.

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Chaotic mossy fiber stimulation efficiently increases the frequency of epileptiform bursts in rat CA3 hippocampal slices

International Congress Series ◽

10.1016/j.ics.2006.02.004 ◽

2006 ◽

Vol 1291 ◽

pp. 93-96

Author(s):

Satoru Ishizuka ◽

Hatsuo Hayashi

Keyword(s):

Mossy Fiber ◽

Hippocampal Slices ◽

Fiber Stimulation

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Hyperexcitability in Combined Entorhinal/Hippocampal Slices of Adult Rat After Exposure to Brain-Derived Neurotrophic Factor

Journal of Neurophysiology ◽

10.1152/jn.1997.78.2.1082 ◽

1997 ◽

Vol 78 (2) ◽

pp. 1082-1095 ◽

Cited By ~ 111

Author(s):

Helen E. Scharfman

Keyword(s):

Entorhinal Cortex ◽

Neurotrophic Factor ◽

Mossy Fiber ◽

Hippocampal Slices ◽

Brain Derived Neurotrophic Factor ◽

Field Potentials ◽

Adult Rat ◽

Bath Application ◽

Fiber Stimulation ◽

Area Ca3

Scharfman, Helen E. Hyperexcitability in combined entorhinal/hippocampal slices of adult rat after exposure to brain-derived neurotrophic factor. J. Neurophysiol. 78: 1082–1095, 1997. Effects of brain-derived neurotrophic factor (BDNF) in area CA3, the dentate gyrus, and medial entorhinal cortex were examined electrophysiologically by bath application of BDNF in slices containing the hippocampus and entorhinal cortex. Bath application of 25–100 ng/ml BDNF for 30–90 min increased responses to single afferent stimuli in selective pathways in the majority of slices. In area CA3, responses to mossy fiber stimulation increased in 73% of slices and entorhinal cortex responses to white matter stimulation increased in 64% of slices. After exposure to BDNF, these areas also demonstrated evidence of hyperexcitability, because responses to repetitive stimulation (1-Hz paired pulses for several s) produced multiple population spikes in response to mossy fiber stimulation in CA3 or multiple field potentials in response to white matter stimulation in the entorhinal cortex. Repetitive field potentials persisted after repetitive stimulation ended and usually were followed by spreading depression. Enhancement of responses to single stimuli and hyperexcitability were never evoked in untreated slices or after bath application of boiled BDNF or cytochrome C. The tyrosine kinase antagonist K252a (2 μM) blocked the effects of BDNF. In area CA3, both the potentiation of responses to single stimuli and hyperexcitability showed afferent specificity, because responses to mossy fiber stimulation were affected but responses to fimbria or Schaffer collateral stimulation were not. In addition, regional specificity was demonstrated in that the dentate gyrus was much less affected. The effects of BDNF in area CA3 were similar to those produced by bath application of low doses of kainic acid, which is thought to modulate glutamate release from mossy fiber terminals by a presynaptic action. These results suggest that BDNF has acute effects on excitability in different areas of the hippocampal-entorhinal circuit. These effects appear to be greatest in areas that are highly immunoreactive for BDNF, such as the mossy fibers and the entorhinal cortex. Although the present experiments do not elucidate mechanism(s) definitively, the afferent specificity, similarity to the effects of kainic acid, and block by K252a are consistent with previous hypotheses that BDNF affects acute excitability by a presynaptic action on trkB receptors that modulate excitatory amino acid transmission. However, we cannot rule out actions on inhibitory synapses or postsynaptic processes.

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Calcium Dynamics in Thorny Excrescences of CA3 Pyramidal Neurons

Journal of Neurophysiology ◽

10.1152/jn.1997.78.1.10 ◽

1997 ◽

Vol 78 (1) ◽

pp. 10-18 ◽

Cited By ~ 14

Author(s):

David B. Jaffe ◽

Thomas H. Brown

Keyword(s):

Laser Scanning ◽

Pyramidal Neurons ◽

Mossy Fiber ◽

Hippocampal Slices ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

High Temporal Resolution ◽

Calcium Dynamics ◽

Intracellular Ca ◽

Ca3 Pyramidal Neurons

Jaffe, David B. and Thomas H. Brown. Calcium dynamics in thorny excrescences of CA3 pyramidal neurons. J. Neurophysiol. 78: 10–18, 1997. Confocal laser scanning microscopy was used to visualize Ca2+ transients in a particular type of dendritic spine, known as a thorny excrescence, in hippocampal CA3 pyramidal neurons. These large excrescences or thorns, which serve as the postsynaptic target for the mossy-fiber synaptic inputs, were identified on the basis of their location, frequency, and size. Whole cell recordings were made from superficial CA3 pyramidal neurons in thick hippocampal slices with the use of infrared video microscopy; cells with proximal apical dendrites close to the surface of the slice were selected. Changes in intracellular Ca2+ levels were monitored by imaging changes in fluorescence of the dyes Calcium Green-1 and Fluo-3. Dual-emission fluorescence imaging was also employed with the use of a combination of Fluo-3 and the Ca2+insensitive dye seminaphthorhodafluor-1. This method was used todecrease the potential influence of background fluorescence on the calculated changes in intracellular Ca2+ concentration ([Ca2+]i). Somatic depolarization produced increases in [Ca2+]i in both the thorn and the immediately adjacent dendrite. Changes in [Ca2+]i were time locked with the onset of depolarization and the decay began immediately after the termination of depolarization. The peak increase in the Ca2+ signal was significantly greater in the thorns than in the adjacent dendritic shafts. With the use of high-temporal-resolution methods (line scans), differences were also seen in the time course of Ca2+ signals in these two regions. The decay time constants of the Ca2+ signal were faster in thorns than in the adjacent dendritic shafts. These observations suggest that voltage-gated Ca2+ channels are localized directly on the dendritic spines receiving mossy-fiber input. Furthermore, Ca2+ homeostasis within thorny excrescences is distinct from Ca2+ regulation in the dendritic shaft, at least over brief time periods, a finding that could have important implications for synaptic plasticity and signaling.

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Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices

Journal of Neurophysiology ◽

10.1152/jn.1991.65.4.771 ◽

1991 ◽

Vol 65 (4) ◽

pp. 771-785 ◽

Cited By ~ 201

Author(s):

P. Perreault ◽

M. Avoli

Keyword(s):

Mossy Fiber ◽

Epileptiform Activity ◽

Hippocampal Slices ◽

Extracellular Recording ◽

Neuronal Membrane ◽

Frequency Of Occurrence ◽

Receptor Antagonists ◽

Postsynaptic Potentials ◽

Ca3 Neurons

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

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Ethanol Alters the Frequency, Amplitude, and Decay Kinetics of Sr2+-Supported, Asynchronous NMDAR mEPSCs in Rat Hippocampal Slices

Journal of Neurophysiology ◽

10.1152/jn.00997.2003 ◽

2004 ◽

Vol 91 (6) ◽

pp. 2568-2577 ◽

Cited By ~ 36

Author(s):

Adam W. Hendricson ◽

John R. Sibbald ◽

Richard A. Morrisett

Keyword(s):

Pyramidal Neurons ◽

Hippocampal Slices ◽

Mean Amplitude ◽

Long Term Potentiation ◽

Exponential Fitting ◽

Decay Kinetics ◽

Current Decay ◽

Event Frequency

To discriminate between pre- and postsynaptic effects of ethanol on N-methyl-d-aspartate receptor (NMDAR) signaling in hippocampus, we adapted the technique of Sr2+ substitution to the hippocampal blind slice patch-clamp preparation. Hippocampal slices were isolated from 12- to 20-day-old rats that were killed in accordance with University of Texas Institutional Animal Care and Use Committee guidelines. NMDAR miniature excitatory postsynaptic currents (mEPSCs) were evoked from CA1 pyramidal neurons in the presence of Sr2+ (4 mM), causing the synchronous EPSC observed in the presence of Ca2+ to be supplanted by asynchronous mEPSCs. Amplitudes typically ranged from 5 to 40 pA and responded to the NMDAR antagonist (DL)-APV (50 μM), with a statistically significant reduction in mean amplitude. Ethanol (25, 50, and 75 mM) exerted dose-dependent effects on mEPSC amplitude and frequency. Peak amplitude inhibition was observed at 75 mM ethanol. Notably, ethanol significantly decreased event frequency at 50 and 75 mM ethanol. Ethanol (75 mM) also significantly increased the paired-pulse ratio of NMDAR EPSCs. Cumulative comparisons of decay time constants derived from single-exponential fitting of mEPSCs revealed significantly accelerated current decay kinetics in the presence of 75 mM ethanol. Taken together, these reductions in miniature event frequency and amplitude, concurrent with an increased rate of decay, suggest that the acute effects of ethanol on NMDAR signaling at hippocampal synapses are multifocal in nature. This finding of pre- and postsynaptic effects of ethanol on NMDAR signal strength in a brain region central to cognition is wholly consistent with previous reports of ethanol inhibition of NMDAR–long-term potentiation in vitro and with the profound cognitive deficits associated with binge-level intoxication in vivo.

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Enhanced Neuronal Damage After Ischemic Insults in Mice Lacking Kir6.2-Containing ATP-Sensitive K+ Channels

Journal of Neurophysiology ◽

10.1152/jn.00970.2005 ◽

2006 ◽

Vol 95 (4) ◽

pp. 2590-2601 ◽

Cited By ~ 62

Author(s):

Hong-Shuo Sun ◽

Zhong-Ping Feng ◽

Takashi Miki ◽

Susumu Seino ◽

Robert J. French

Keyword(s):

Hippocampal Neurons ◽

Pyramidal Neurons ◽

Neuronal Damage ◽

Focal Cerebral Ischemia ◽

Hippocampal Slices ◽

Immunocytochemical Staining ◽

Sulfonylurea Receptor ◽

Ca1 Pyramidal Neurons

Adenosine triphosphate (ATP)–sensitive potassium (KATP) channels, incorporating Kir6.x and sulfonylurea receptor subunits, are weak inward rectifiers that are thought to play a role in neuronal protection from ischemic insults. However, the involvement of Kir6.2-containing KATP channel in hippocampus and neocortex has not been tested directly. To delineate the physiological roles of Kir6.2 channels in the CNS, we used knockout (KO) mice that do not express Kir6.2. Immunocytochemical staining demonstrated that Kir6.2 protein was expressed robustly in hippocampal neurons of the wild-type (WT) mice and absent in the KO. To examine neuronal sensitivity to metabolic stress in vitro, and to ischemia in vivo, we 1) exposed hippocampal slices to transient oxygen and glucose deprivation (OGD) and 2) produced focal cerebral ischemia by middle cerebral artery occlusion (MCAO). Both slice and whole animal studies showed that neurons from the KO mice were severely damaged after anoxia or ischemia, whereas few injured neurons were observed in the WT, suggesting that Kir6.2 channels are necessary to protect neurons from ischemic insults. Membrane potential recordings from the WT CA1 pyramidal neurons showed a biphasic response to OGD; a brief hyperpolarization was followed by a small depolarization during OGD, with complete recovery within 30 min after returning to normoxic conditions. By contrast, CA1 pyramidal neurons from the KO mice were irreversibly depolarized by OGD exposure, without any preceding hyperpolarization. These data suggest that expression of Kir6.2 channels prevents prolonged depolarization of neurons resulting from acute hypoxic or ischemic insults, and thus protects these central neurons from the injury.

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Delayed Signal Propagation via CA2 in Rat Hippocampal Slices Revealed by Optical Recording

Journal of Neurophysiology ◽

10.1152/jn.1997.78.3.1662 ◽

1997 ◽

Vol 78 (3) ◽

pp. 1662-1668 ◽

Cited By ~ 50

Author(s):

Yuko Sekino ◽

Kunihiko Obata ◽

Manabu Tanifuji ◽

Makoto Mizuno ◽

Jin Murayama

Keyword(s):

Mossy Fiber ◽

Hippocampal Slices ◽

Mossy Fibers ◽

Signal Propagation ◽

Middle Part ◽

Optical Recording ◽

Stratum Radiatum ◽

Ca1 Neurons ◽

Optical Signals ◽

Fiber Stimulation

Sekino, Yuko, Kunihiko Obata, Manabu Tanifuji, Makoto Mizuno, and Jin Murayama. Delayed signal propagation via CA2 in rat hippocampal slices revealed by optical recording. J. Neurophysiol. 78: 1662–1668, 1997. Signal propagation from mossy fibers to CA1 neurons was investigated in rat hippocampal slices by a combination of electrical and optical recordings. The slices were prepared by oblique sectioning of the middle part of the hippocampus to preserve fiber connections. The mossy fibers were stimulated to induce population spikes (PSs) and excitatory postsynaptic potentials in the middle part of the CA1 region. Latencies of maximal PSs in CA1 varied widely among slices; they ranged from 7 to 13.5 ms, with two maxima at 9 and 11.5 ms. The fastest PSs probably are evoked by the Schaffer collaterals that connect the CA3 and CA1 regions in the well-known trisynaptic circuit. However, the slower PSs suggest the existence of additional delayed inputs. To determine the source of the delayed input, slices were stained with a voltage-sensitive dye, RH482, and the optical signals relevant to membrane potential changes were detected by a high-resolution optical imaging system. Optical recording of responses to mossy fiber stimulation indicated two distinct types of signal propagation from CA3 to CA1. In preparations evincing the fast type of propagation, signals spread to CA1 within 7.2 ms after the mossy fiber stimulation. During such propagation, activity flowed directly from CA3 to the stratum radiatum of CA1. Other preparations illustrated slow signal propagation, in which optical signals were generated in CA2 before spreading to CA1. During such slow signal transmission, activity persisted in CA2 and its surrounding area for 3 ms before propagating to the strata radiatum and oriens in CA1. In such cases, CA1 activity was detected within 10.8 ms of mossy fiber stimulation. In some slices, a mixture of the fast and slow propagation patterns was observed, indicating that these two transmission modes can coexist. Our data reveal that CA2 neurons can transmit delayed excitatory signals to CA1 neurons. We therefore conclude that consideration of electrical signal propagation through the hippocampus should include flow through the CA2 region in addition to the traditional dentate gyrus–CA3–CA1 trisynaptic circuit.

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