Faculty Bibliography

1. The hypothesis that dentate hilar 'mossy' cells are excitatory was tested by simultaneous intracellular recording in rat hippocampal slices. Mossy cells were recorded simultaneously with their potential targets, granule cells and interneurons. The gamma-amino-butyric acid-A (GABAA) receptor antagonist bicuculline was used in most experiments to block the normally strong inhibitory inputs to granule cells that could mask excitatory effects of mossy cells. Some cells were recorded with electrodes containing the marker Neurobiotin so that their identity could be confirmed morphologically. 2. A mossy cell action potential was immediately followed by a brief depolarization in a granule cell in 20 of 1,316 pairs (1.5%) that were recorded in the presence of bicuculline. The mean amplitude of depolarizations was 1.99 +/- 0.24 (SE) mV when the postsynaptic membrane potential was -55 to -65 mV. Depolarizations could trigger an action potential if the granule cell was depolarized from its resting potential so that its membrane potential was -50 to -60 mV. These data suggest that mossy cells excite granule cells monosynaptically. 3. Monosynaptic excitation of an interneuron by a mossy cell was recorded in 4 of 47 (8.5%) simultaneously recorded mossy cells and interneurons, also in the presence of bicuculline. The mean interneuron depolarization was 1.64 +/- 0.29 mV when the interneuron membrane potential was approximately -60 mV. When an interneuron was at its resting potential (-52 to -63 mV), action potentials were often triggered by the depolarizations. 4. Without bicuculline present, mossy cells had no apparent monosynaptic effects on granule cells, as has been previously reported. However, effects that appeared to be polysynaptic were observed in 5 of 92 pairs (5.4%). Specifically, a small, brief hyperpolarization occurred in granule cells 2.5-7.3 ms after the peak of a mossy cell action potential. Given the results indicating that mossy cells excite interneurons, and the long latency to onset of the hyperpolarization, one possible explanation for the hyperpolarization is that mossy cells excited interneurons that inhibited granule cells. 5. The results suggest that mossy cells are excitatory neurons. In addition, mossy cells appear to innervate both granule cells and interneurons that are located within several hundred micrometers of the mossy cell soma. The only detectable effect on granule cells in this area under normal conditions appears to be disynaptic and inhibitory. However, when GABAA-receptor-mediated inhibition is blocked, monosynaptic excitation of granule cells by mossy cells can be detected

In the rat dentate gyrus, pyramidal-shaped cells located on the border of the granule cell layer and the hilus are one of the most common types of gamma-aminobutyric acid (GABA)-immunoreactive neurons. This study describes their electrophysiological characteristics. Membrane properties, patterns of discharge, and synaptic responses were recorded intracellularly from these cells in hippocampal slices. Each cell was identified as pyramidal-shaped by injecting the marker Neurobiotin intracellularly (n = 17). In several respects the membrane properties of the sampled cells were similar to 'fast-spiking' cells (putative inhibitory interneurons) that have been described in other areas of the hippocampus. For example, input resistance was high (mean 91.3 megohms), the membrane time constant was short (mean 7.7 ms), and there was a large afterhyperpolarization following a single action potential (mean 10.5 mV at resting potential). However, the action potentials of most pyramidal-shaped cells were not as brief (mean 1.2 ms total duration) as those of most previously described fast-spiking cells. Many pyramidal-shaped neurons had strong spike frequency adaptation relative to other fast-spiking cells. Although these latter two characteristics were apparent in the majority of the sampled cells, there were exceptional pyramidal-shaped neurons with fast action potentials and weak adaptation, demonstrating the electrophysiological variability of pyramidal-shaped cells. Responses to outer molecular layer stimulation were composed primarily of excitatory postsynaptic potentials (EPSPs) rather than inhibitory postsynaptic potentials (IPSPs), and were usually small (EPSPs evoked at threshold were often less than 2 mV), and brief (less than 30 ms). There was variability, because in a few cells EPSPs evoked at threshold were much larger. However, regardless of EPSP amplitude, suprathreshold stimulation (up to 4 times the threshold stimulus strength) rarely evoked more than one action potential in any cell. The results suggest that stimulation of perforant path axons produces limited excitatory synaptic responses in pyramidal-shaped neurons. This may be one of the reasons why they are relatively resistant to prolonged perforant path stimulation. The pyramidal-shaped neurons located at the base of the granule cell layer have been associated historically with a basket plexus around granule cell somata, and have been called pyramidal 'basket' cells. However, basket-like endings were rare and axon collaterals outside the granule cell layer as the outer molecular layer and the central hilus, and antidromic action potentials could be recorded in some cells in response to weak stimulation of these areas. Taken together with the electrophysiological variability, the results indicate that these cells are physiologically heterogeneous

1. Simultaneous intracellular recordings of area CA3 pyramidal cells and dentate hilar 'mossy' cells were made in rat hippocampal slices to test the hypothesis that area CA3 pyramidal cells excite mossy cells monosynaptically. Mossy cells and pyramidal cells were differentiated by location and electrophysiological characteristics. When cells were impaled near the border of area CA3 and the hilus, their identity was confirmed morphologically after injection of the marker Neurobiotin. 2. Evidence for monosynaptic excitation of a mossy cell by a pyramidal cell was obtained in 7 of 481 (1.4%) paired recordings. In these cases, a pyramidal cell action potential was followed immediately by a 0.40 to 6.75 (mean, 2.26) mV depolarization in the simultaneously recorded mossy cell (mossy cell membrane potentials, -60 to -70 mV). Given that pyramidal cells used an excitatory amino acid as a neurotransmitter (Cotman and Nadler 1987; Ottersen and Storm-Mathisen 1987) and recordings were made in the presence of the GABAA receptor antagonist bicuculline (25 microM), it is likely that the depolarizations were unitary excitatory postsynaptic potentials (EPSPs). 3. Unitary EPSPs of mossy cells were prone to apparent 'failure.' The probability of failure was extremely high (up to 0.72; mean = 0.48) if the effects of all presynaptic action potentials were examined, including action potentials triggered inadvertently during other spontaneous EPSPs of the mossy cell. Probability of failure was relatively low (as low as 0; mean = 0.24) if action potentials that occurred during spontaneous activity of the mossy cell were excluded. These data suggest that unitary EPSPs produced by pyramidal cells are strongly affected by concurrent synaptic inputs to the mossy cell. 4. Unitary EPSPs were not clearly affected by manipulation of the mossy cell's membrane potential. This is consistent with the recent report that area CA3 pyramidal cells innervate distal dendrites of mossy cells (Kunkel et al. 1993). Such a distal location also may contribute to the high incidence of apparent failures. 5. Characteristics of unitary EPSPs generated by pyramidal cells were compared with the properties of the unitary EPSPs produced by granule cells. In two slices, pyramidal cell and granule cell inputs to the same mossy cell were compared. In other slices, inputs to different mossy cells were compared. In all experiments, unitary EPSPs produced by granule cells were larger in amplitude but similar in time course to unitary EPSPs produced by pyramidal cells. Probability of failure was lower and paired-pulse facilitation more common among EPSPs triggered by granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)

When hippocampal slices are exposed to GABAA antagonists, area CA3 pyramidal cells and dentate hilar 'mossy' cells discharge in synchronized epileptiform bursts (Muller and Misgeld, 1991; Scharfman, 1994b). Dentate interneurons are excited simultaneously, but the degree of discharge varies (Scharfman, 1994b). This study primarily examined the activity of dentate granule cells simultaneous to the epileptiform bursts of pyramidal cells and mossy cells. EPSPs followed by large GABAB receptor-mediated IPSPs were generated in granule cells during all epileptiform bursts of pyramidal cells and mossy cells, regardless of whether they were evoked or spontaneous. By simultaneous recording it was determined that granule cell EPSPs began several milliseconds after the start of pyramidal cell bursts (n = 48 simultaneous recordings) and immediately after the first action potential of a mossy cell burst (n = 77). Interneurons were similar to granule cells in the timing of their depolarizations relative to the onset of pyramidal cell (n = 24; Scharfman, 1994b) and mossy cell (n = 9) bursts. All excitatory activity was blocked by bath application of the glutamatergic AMPA/kainate receptor antagonist CNQX (5 microM, n = 5), but not the NMDA receptor antagonist D-APV (25-50 microM, n = 9). Granule cell EPSPs were decreased after focal application of CNQX to the molecular layer at a site close to the impaled granule cell (n = 5), whereas D-APV had no effect (n = 3). EPSPs also decreased after focal application of CNQX to the hilus, in two of four slices tested. The extracellularly recorded EPSP of granule cells was maximal in the inner molecular layer (n = 33), the site of the mossy cell axonal plexus. Severing the junction of the dentate gyrus and area CA3 blocked all spontaneous and evoked activity of dentate neurons without affecting burst discharges in area CA3a and CA3b (n = 6). None of the excitatory activity of any cell type was affected by cholinergic antagonists (atropine and mecamylamine, 25 microM each, n = 5; pirenzipine and dihydro-beta-erythroidine, 25 microM each, n = 5). The results suggest that there is a glutamatergic, AMPA/kainate receptor-mediated, excitatory pathway from area CA3 to the dentate gyrus in disinhibited slices. The pharmacological results, analyses of latency, as well as the known axonal projections of the sampled cells, suggest that the excitatory pathway begins within area CA3 and leads to granule cells via mossy cells. The data also suggest that dentate interneurons are excited by mossy cells, and possibly by pyramidal cells as well.(ABSTRACT TRUNCATED AT 400 WORDS)

A recent study has described synchronous burst discharges of dentate hilar neurons and area CA3 pyramidal cells in the presence of the convulsants 4-aminopyridine and picrotoxin in guinea-pig hippocampal slices [Muller W. and Misgeld U. (1991) J. Neurophysiol. 65, 141-147]. To examine the synchronous activity of dentate cells and area CA3 pyramidal cells further, epileptiform burst discharges were examined in morphologically and/or electrophysiologically identified non-granule cells in the hilus and granule cell layer of the rat dentate gyrus and compared to simultaneously-recorded pyramidal cells of area CA3a, b, and c. Specifically, the types of dentate cells and the types of discharge were examined, as well as the timing of burst discharge of dentate cells relative to different cells of area CA3. In the presence of the GABAA receptor antagonist bicuculline (30 microM), all dentate cell types discharged in rhythmic, spontaneous bursts that were synchronized with area CA3 pyramidal cell epileptiform bursts. The sampled cells included hilar 'mossy' cells, hilar fast-spiking cells (putative interneurons) as well as interneurons located in the granule cell layer, such as the pyramidal 'basket' cells. Simultaneous recording from dentate non-granule cells and area CA3 pyramidal cells during exposure to bicuculline demonstrated that stimulus-evoked and spontaneous epileptiform bursts occurred almost exactly at the same time; there were only a few milliseconds between the onsets of pyramidal cell bursts and dentate cell bursts, with the pyramidal cell preceding the dentate cell in almost every case. There were no systematic differences among dentate cell types in the extent they lagged behind pyramidal cells, and there were no detectable differences among area CA3 pyramidal cells. In slices that were cut between area CA3 and the dentate gyrus, epileptiform bursts occurred in area CA3 but not in the dentate. These findings suggest that, in the absence of GABAA receptor-mediated inhibition, excitatory pathways from area CA3 to the dentate gyrus are strong and widespread. These pathways, and possibly other mechanisms, can lead to tightly synchronized action potential discharge of pyramidal cells and dentate non-granule cells. The results also suggest that disinhibition alone is insufficient to cause synchronous bursts in the dentate gyrus, in contrast to area CA3

Stimulation of the fimbria in rat hippocampal slices evoked an extracellular negativity in the granule cell layer and a small depolarization in granule cells at their resting potentials. The intracellular potentials appeared to be GABAA receptor-mediated IPSPs because they reversed at -69.1 +/- 1.0 mV (mean +/- S.E.M., n = 14) and were blocked by the GABAA receptor antagonist bicuculline (10-50 microM, n = 14). However, during the first few minutes of perfusion with bicuculline, IPSPs transiently and paradoxically increased in amplitude. As IPSPs increased, the reversal potential and latency to onset remained the same. These effects were reversible, and during the wash period IPSPs first increased and then stabilized at a smaller amplitude, similar to IPSPs evoked in control conditions. As the GABAA receptor-mediated IPSP decreased, it was followed by a second hyperpolarization. This late hyperpolarization appeared to be a GABAB receptor-mediated IPSP, because it reversed near the equilibrium potential for potassium (mean -81.8 +/- 2.3 mV, n = 12, [K+]o = 5 mM) and was blocked by the GABAB receptor antagonist 2-hydroxy saclofen (250-500 microM, n = 5). The results suggest that GABAA and GABAB receptor-mediated IPSPs evoked in granule cells by fimbria stimulation are normally inhibited by activation of GABAA receptors. The inhibition by GABAA receptors is strong enough that, in control conditions, the GABAA IPSPs are barely detectable and the GABAB IPSPs are undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Excitation of the spiny subtype of hilar neurons in the fascia dentata was characterized by intracellular recording from hilar cells in hippocampal slices. Stimulation of the outer molecular layer was used to activate the perforant path. Evoked responses were examined, as well as the large spontaneous excitatory potentials that are a distinctive characteristic of spiny hilar cells. 2. Excitatory potentials that occurred spontaneously, as well as those that occurred in response to outer molecular layer stimulation, were similar among the cells that were sampled, regardless of morphological variations such as the presence or absence of thorny excrescences. Spontaneous and evoked excitatory postsynaptic potentials (EPSPs) were complex depolarizations that often had several discrete peaks. Spontaneous EPSPs increased in amplitude slightly with hyperpolarization, and evoked EPSPs clearly increased with hyperpolarization. 3. Applications of selective antagonists of excitatory amino acid receptors were used to determine which excitatory amino acid receptor mediates EPSPs of these cells. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was used to block the receptor subtype selective for the agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (the 'AMPA/kainate' receptor). 2-amino-5-phosphonovaleric acid (APV) was used to block receptors specific for the agonist N-methyl-D-aspartate (NMDA; the 'NMDA' receptor). Perfusion with CNQX (5-25 microM) completely blocked all spontaneous and evoked excitation, even when activity was examined at relatively depolarized membrane potentials and a low concentration of extracellular magnesium (0.5 mM) was used. Under these conditions, APV (25-50 microM) had no detectable effect on spontaneous activity but did increase the stimulus strength required to elicit responses to outer molecular layer stimulation. 4. When extracellular magnesium was lowered to 0 mM (nominally), there was strong evidence for a contribution of NMDA receptors to spontaneous and evoked EPSPs. Thus, when cells were perfused with 0 mM extracellular magnesium and 5 microM CNQX, spontaneous depolarizations were present and EPSPs could be triggered by stimulation of the outer molecular layer. Both the spontaneous and evoked EPSPs were blocked by 25 microM APV. 5. Because gamma-aminobutyric acid (GABA)A receptors can cause depolarizations in hippocampal neurons, the GABAA receptor antagonist bicuculline was used to determine whether some of the EPSPs were mediated by GABAergic neurons that are normally activated by spontaneous release of excitatory amino acids. Bicuculline (5-25 microM) had no effect on spontaneous depolarizations, and led to an enhancement of evoked depolarizations. Therefore it does not appear that GABAA receptor-mediated depolarizations contribute to hilar cell depolarizations.(ABSTRACT TRUNCATED AT 400 WORDS)

It is has been shown that the major afferent input to the dentate gyrus, the perforant path, excites dentate hilar neurons. However, little is known about the other inputs to hilar cells. Therefore, we examined the responses of hilar neurons to stimulation of the fimbria. We positioned our stimulating electrodes so that granule cells were not excited antidromically by fimbria stimulation, although action potentials were easily triggered in area CA3b and CA3c pyramidal cells by such stimulation. In these experiments, fimbria stimulation evoked responses from every hilar cell tested, including examples of both of the major cell types, the spiny hilar 'mossy' cells (n = 15) and the relatively aspiny, 'fast-spiking' cells (putative interneurons, n = 5). Hilar cell responses consisted primarily of EPSPs that could trigger action potentials, but small IPSPs were also evoked in some cases, particularly in the fast-spiking cells. Excitation was blocked by an antagonist of the AMPA/kainate receptor subtype of excitatory amino acid receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM, n = 5), whereas the cholinergic antagonist atropine (10 microM) had no effect (n = 4). When sequential intracellular recordings were made from hilar cells and area CA3 pyramidal cells in the same slice, hilar cell EPSPs began after action potentials of CA3b pyramidal cells, and stimulus strengths required to evoke hilar cell EPSPs were above threshold for area CA3b pyramidal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron-dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)-labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)-/Neurobiotin (NB)-labeled neurons were recognized by avidin-HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron-dense reaction product via immunocytochemical staining with a rabbit anti-LY antibody, followed by goat anti-rabbit IgG-HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level

Area CA3c is an area of morphologically diverse neurons. In addition to the presence of interneurons and pyramidal cells that are similar to those found in other subfields of area CA3, many neurons of area CA3c are different. They do not resemble interneurons, since they bear numerous spines, yet they also differ substantially from pyramidal cells in their morphology. To determine if the variants of area CA3c spiny cells are distinct physiologically as well as morphologically, intracellular recordings were made to record the electrophysiological properties of area CA3c cells in rat hippocampal slices, and each cell was identified morphologically following intracellular dye injection. The results show that the spiny cells, regardless of their often extensive morphological variation, have relatively uniform, pyramidal-like electrophysiological properties. The aspiny cells are quite different from the spiny cells morphologically (i.e., in their paucity or complete lack of spines), and are also extremely different electrophysiologically, exhibiting features of 'fast-spiking' cells. Thus, spiny cells in area CA3c correspond to cells with pyramidal-like electrophysiology, and the aspiny cells in area CA3c correspond to cells with interneuronal physiological properties. This correlation between structure and function appears to be a rule that pertains to each of the subfields of the hippocampus