Synaptic target selectivity and input of GABAergic basket and bistratified interneurons in the CA1 area of the rat hippocampus

To assess the position of interneurons in the hippocampal network, fast spiking cells were recorded intracellularly in vitro and filled with biocytin. Sixteen non-principal cells were selected on the basis of 1) cell bodies located in the pyramidal layer and in the middle of the slice, 2) extensive labeling of their axons, and 3) a branching pattern of the axon indicating that they were not axo-axonic cells. Examination of their efferent synapses (n = 400) demonstrated that the cells made synapses on cell bodies, dendritic shafts, spines, and axon initial segments (AIS). Statistical analysis of the distribution of different postsynaptic elements, together with published data (n = 288) for 12 similar cells, showed that the interneurons were heterogeneous with regard to the frequency of synapses given to different parts of pyramidal cells. When the cells were grouped according to whether they had less or more than 40% somatic synaptic targets, each population appeared homogeneous. The population (n = 19) innervating a high proportion of somata (53 ± 10%, SD) corresponds to basket cells. They also form synapses with proximal dendrites (44 ± 12%) and rarely with AISs and spines. One well-filled basket cell had 8,859 boutons within the slice, covering an area of 0.331 mm² of pyramidal layer tangentially and containing 7,150 pyramidal cells, 933 (13%) of which were calculated to be innervated, assuming that each pyramidal cell received nine to ten synapses. It was extrapolated that the intact axon probably had about 10,800 boutons innervating 1,140 pyramids. The proportion of innervated pyramidal cells decreased from 28% in the middle to 4% at the edge of the axonal field. The other group of neurons, the bistratified cells (n = 9), showed a preference for dendritic shafts (79 ± 8%) and spines (17 ± 8%) as synaptic targets, rarely terminating on somata (4 ± 8%). Their axonal field was significantly larger (1,250 ± 180 μm) in the medio-lateral direction than that of basket cells (760 ± 130 μm). The axon terminals of bistratified cells were smaller than those of basket cells. Furthermore, in contrast to bistratified cells, basket cells had a significant proportion of dendrites in stratum lacunosum-moleculare suggesting a direct entorhinal input. The results define two distinct types of GABAergic neuron innervating pyramidal cells in a spatially segregated manner and predict different functions for the two inputs. The perisomatic termination of basket cells is suited for the synchronization of a subset of pyramidal cells that they select from the population within their axonal field, whereas the termination of bistratified cells in conjunction with Schaffer collateral/commissural terminals may govern the timing of CA3 input and/or voltage-dependent conductances in the dendrites. © 1996 Wiley-Liss, Inc.     

Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus

Hippocampal pyramidal cells express several alpha-subunits, which determine the affinity of GABAA (gamma-aminobutyric acid) receptors for benzodiazepine site ligands. This study asked whether inhibitory postsynaptic potentials (IPSPs) elicited by specific interneuronal subclasses were differentially sensitive to the alpha1-preferring agonist Zolpidem, i.e. whether different receptors mediate different inhibitory connections. Paired intracellular recordings in which the presynaptic cell was an interneuron and the postsynaptic cell a CA1 pyramid were performed in slices of adult rat hippocampus. Resultant IPSPs were challenged with Zolpidem, cells filled with biocytin and identified morphologically. IPSPs elicited by fast spiking (FS) basket cells (n = 9) were enhanced more than IPSPs elicited by regular spiking (RS) basket cells (n = 10). At FS basket cell synapses the efficacy of Zolpidem was equivalent to that of Diazepam, while RS basket cell IPSPs are enhanced 50% less by Zolpidem than by Diazepam. Thus, while alpha1 subunits may dominate at synapses supplied by FS basket cells, RS basket cell synapses also involve alpha2/3 subunits. Two bistratified cell IPSPs tested with Zolpidem did not increase in amplitude, despite powerful enhancements of bistratified cell IPSPs by Diazepam, consistent with previous indications that these synapses utilize alpha5-containing receptors. Enhancements of basket cell IPSPs by Zolpidem and Diazepam were bi- or triphasic with steep amplitude increases separated by plateaux, occurring 10-15, 25-30 and 45-55 min after adding the drug to the bath. The entire enhancement was, however, blocked by the antagonist Flumazenil (n = 7). Flumazenil, either alone (n = 3), or after Zolpidem, reduced IPSP amplitude to approximately 90% of control, suggesting that alpha4-containing receptors were not involved.     

Dendritic amplification of inhibitory postsynaptic potentials in a model Purkinje cell

In neurons with large dendritic arbors, the postsynaptic potentials interact in a complex manner with active and passive membrane properties, causing not easily predictable transformations during the propagation from synapse to soma. Previous theoretical and experimental studies in both cerebellar Purkinje cells and neocortical pyramidal neurons have shown that voltage-dependent ion channels change the amplitude and time-course of postsynaptic potentials. We investigated the mechanisms involved in the propagation of inhibitory postsynaptic potentials (IPSPs) along active dendrites in a model of the Purkinje cell. The amplitude and time-course of IPSPs recorded at the soma were dependent on the synaptic distance from the soma, as predicted by passive cable theory. We show that the effect of distance on the amplitude and width of the IPSP was significantly reduced by the dendritic ion channels, whereas the rise time was not affected. Somatic IPSPs evoked by the activation of the most distal synapses were up to six times amplified owing to the presence of voltage-gated channels and the IPSP width became independent of the covered distance. A transient deactivation of the Ca(2+) channels and the Ca(2+)-dependent K(+) channels, triggered by the hyperpolarization following activation of the inhibitory synapse, was found to be responsible for these dynamics. Nevertheless, the position of activated synapses had a marked effect on the Purkinje cell firing pattern, making stellate cells and basket cells most suitable for controlling the firing rate and spike timing, respectively, of their target Purkinje cells.     

Depression of early and late monosynaptic inhibitory postsynaptic potentials in hippocampal CA1 neurons following prolonged benzodiazepine administration: Role of a reduction in Cl driving force

GABAergic synaptic responses were studied by direct, monosynaptic activation of GABAergic interneurons in the CA1 region of in vitro hippocampal slices from rats made tolerant to the benzodiazepine, flurazepam. Monosynaptic IPSPs were elicited in CA1 pyramidal neurons, following 1 week oral flurazepam administration, by electrical stimulation at the stratum oriens/stratum pyramidale or stratum radiatum/ stratum-lacanosum border ≤ 0.5 mm from the recording electrode plane. Excitatory input to pyramidal cells and interneurons was eliminated by prior superfusion of the glutamate receptor antagonists, APV (50 μM) and DNQX (10 μM). GABA_A receptor-mediated early IPSPs were further isolated by perfusion of the GABA_B antagonist, CGP 35348 (25 μM) or by diffusion of Cs⁺ from the recording electrode. GABA_B receptor-mediated late IPSPs were pharmacologically isolated by perfusion of the GABA_A antagonist, picrotoxin (50 μM). There was a significant decrease in the amplitude of pharmacologically isolated early and late IPSPs in FZP-treated neurons without a change in passive membrane properties. A shift of the early IPSP, but not the late IPSP, reversal potential in FZP-treated neurons suggested that a change in the driving force for anions, presumably Cl, in CA1 neurons was one important factor related to the decreased early IPSP amplitude after prolonged activation of GABA_A receptors by flurazepam. A decreased early IPSP amplitude accompanied by a decreased late IPSP amplitude suggested that presynaptic GABA release onto FZP-treated pyramidal cells may also be reduced. We conclude from these data that an impairment of GABAergic transmission in CA1 pyramidal neurons associated with the development of tolerance during chronic benzodiazepine treatment may be related to the regulation of both pre- and postsynaptic mechanisms at the GABA synapse. Synapse 25:125–136, 1997. © 1997 Wiley-Liss, Inc.     

Hyperpolarizing synaptic potentials evoked in CA1 pyramidal cells by glutamate stimulation of interneurons from the oriens/alveus border of rat hippocampal slices. I. Electrophysiological response properties

To examine the inhibitory postsynaptic potentials (IPSPs) elicited in pyramidal cells by interneurons situated at the stratum oriens/alveus border (O/A), glutamate was applied by micropressure to this area during intracellular recordings from CA1 pyramidal cells. Glutamate stimulation evoked IPSPs (glut-IPSPs) of small amplitude (4 mV), delayed peak latency (100–110 ms), and long duration (300–400 ms). Recurrent activation of interneurons via glutamate stimulation of pyramidal cells by local application in stratum pyramidale (PYR) evoked recurrent IPSPs (PYR glut-IPSPs) with similar amplitude and time course as O/A glut-IPSPs. The mean equilibrium potential of O/A glut-IPSPs (?77 mV) was significantly different from that of the PYR glut-IPSPs (?71 mV), however, neither equilibrium potential was significantly different from that of the electrically evoked early IPSP in the same cells. Glutamate-evoked IPSPs elicited from O/A displayed some response reversal (27% reversal) like those evoked from PYR (41% reversal). The early IPSP evoked by electrical stimulation displayed significantly more response reversal (67% reversal) than glut-IPSPs. Both types of glut-IPSPs (O/A and PYR) were associated with moderate increases in membrane conductance (5.9 and 6.6 nS, respectively), which were significantly less than the conductance change associated with the early IPSP (45.8 nS). In interneurons within PYR, glutamate stimulation in PYR readily elicited a flurry of excitatory postsynaptic potentials, whereas glutamate stimulation in O/A elicited IPSPs. The electrophysiological properties of IPSPs elicited in pyramidal cells by glutamate stimulation of interneurons in O/A were similar to those of recurrent IPSPs evoked from PYR. Given that both of these types of glutamate-evoked IPSPs were mostly mediated via GABA_A receptor channels (Samulack DD, Lacaille J-C, 1993, Hippocampus 3:345–358), the small differences observed between equilibrium potentials, response reversals, and conductance changes could be due to a more electrotonically distant location from the soma of the synapses involved in O/A glut-IPSPs as compared to those of recurrent IPSPs elicited from PYR.     

Modulation of bistratified cell IPSPs and basket cell IPSPs by pentobarbitone sodium, diazepam and Zn2+: dual recordings in slices of adult rat hippocampus

Simultaneous intracellular recordings from presynaptic Stratum pyramidale interneurons and postsynaptic pyramidal cells in adult rat hippocampal slices were performed to investigate the strength of the modulation of single-axon inhibitory postsynaptic potentials (IPSPs) by the GABAA receptor modulators pentobarbitone, diazepam and zinc. The processing of biocytin-filled interneurons for light microscopy revealed that these single-axon IPSPs were generated by basket cells (n = 33), bistratified cells (n = 18) and axo-axonic cells (n = 2). The IPSPs generated by these three groups of interneurons had amplitudes and widths at half amplitude with similar ranges, but when bistratified cell IPSPs were compared with basket cell IPSPs with similar half widths their rise times were slower. Pentobarbitone sodium (250 microM) powerfully enhanced 13 tested IPSPs generated by all three cell types. Amplitudes were enhanced by 82 +/- 56%, 10-90% rise times by 150 +/- 101% and the widths at half amplitude by 71 +/- 29%. Diazepam (1-2 microM) also increased all IPSPs tested, although the changes were more moderate in basket cell IPSPs (amplitudes increased by 19 +/- 11%, n = 8) than in bistratified cell IPSPs (amplitudes increased by 66 +/- 48%, n = 5). Basket cell IPSP 10-90% rise times and widths at half amplitude were not significantly increased. Bistratified cell IPSP 10-90% rise times were increased by 44 +/- 24% and the widths at half amplitude by 32 +/- 35%. The one tested IPSP generated by an axo-axonic cell was also diazepam-sensitive. Zinc, 250 microM, decreased four out of 10 IPSPs generated by basket cells and four out of five IPSPs generated by bistratified cells. The one tested axo-axonic cell IPSP was zinc-insensitive. These data suggest that IPSPs generated in CA1 pyramidal cells by basket and bistratified cells display different pharmacologies and may be mediated by different receptors or receptor combinations.     

Precision and Variability in Postsynaptic Target Selection of Inhibitory Cells in the Hippocampal CA3 Region

Non-pyramidal cells were filled intracellularly with biocytin in the CA3 region of the guinea-pig hippocampus in vitro, within or close to stratum pyramidale. On the basis of camera lucida reconstructions and electron microscopy, six different cell types with distinct laminar distribution of axon terminals could be distinguished. The axon of three axo-axonic cells, three typical basket cells, and atypical basket cells of two types arborized in the perisomatic and proximal dendritlc region of CA3 pyramidal cells. Two cells with axons innervating the distal dendritlc segments of pyramidal cells were also found; one terminated in stratum radiatum and the other in stratum lacunosum-moleculare. Electron microscopy demonstrated that symmetrical synapses were formed by the labelled boutons on axon initial segments, somata, and proximal or distal dendrites of mostly pyramidal neurons. Axo-axonic cells showed absolute target selectivity for axon initial segments, whereas for the other cells the distribution of contacted elements was determined by the laminar distribution of axon terminals. In two cases, where additional cells were labelled with biocytin, multiple (up to nine) light microscopically identified contacts (presumed synaptic contacts) were established by the interneurons on several pyramidal cells and on an axo-axonic cell. Our results show that a restricted set of inhibitory cells, with somata within or close to CA3 stratum pyramldale, possess variable patterns of axonal arborization. Various types of postsynaptic elements are contacted, but precision in selecting certain targets and ignoring others is maintained within a particular cell type and layer. In contrast to the diversity of axonal arbors the structure of the dendritic trees shows no consistent differences, suggesting that the cells may be activated by a similar set of afferents. It seems probable that the innervation of precise regions of postsynaptic pyramidal cells by different types of interneurons–often in conjunction with particular excitatory afferents (Han et at., Eur. J. Neurosci., 5, 395–410, 1993)–underlies functional differences in inhibitory synaptic actions.     

IPSPs elicited in CA1 pyramidal cells by putative basket cells in slices of adult rat hippocampus

CA1 basket cells are identifiable by an axonal arbour largely confined to, and spanning, the entire depth of stratum pyramidale where they innervate pyramidal somata and proximal dendrites. Basket cells display a range of electrophysiological properties and the inhibitory postsynaptic potentials (IPSPs) they elicit in pyramidal cells vary widely in duration. To determine whether these parameters are correlated, we used paired intracellular recordings, with biocytin filling, in pyramidal cells of adult hippocampal slices, and studied gamma-aminobutyric acid (GABAA) IPSPs (n = 43) elicited by putative basket cells (n = 35) with axons largely confined to stratum pyramidale in simultaneously recorded pyramidal cells. Fast-spiking interneurons elicited relatively brief IPSPs, while IPSPs elicited by burst-firing cells were amongst the slowest. Regular spiking interneurons elicited fast and slow GABAA IPSPs, but any one interneuron elicited IPSPs with remarkably similar durations in two to four pyramidal targets. However, with different types of target for a single putative basket cell, IPSPs elicited in postsynaptic interneurons were briefer than in pyramidal cells. Vertical oriens cells with somata in stratum oriens and a narrow, sparse axonal arbour in stratum pyramidale in transverse hippocampal slices, elicited IPSPs whose rise times and half widths clustered around intermediate values. Durations of IPSPs in pyramidal cells thus correlate, to a degree, with the physiological properties of presynaptic basket cells. The seven-fold range of durations observed (10-70 ms half widths) may underlie contributions made by different basket cells to hippocampal rhythms of different frequencies.     

The Neuroactive Steroid 5α-Tetrahydrodeoxycorticosterone Increases GABAergic Postsynaptic Inhibition in Rat Neocortical Neurons in vitro

The neuroactive steroid 5α-pregnane-3α,21-diol-20-one (5α-tetrahydrodeoxycorticosterone; 5α-THDOC) has been shown to potentiate GABA-induced chloride currents in cell cultures and subcellular preparations. In this study, we recorded from pyramidal neurons in an in vitro slice preparation of the adult rat frontal neocortex using intracellular microelectrodes. 5α-THDOC (10 μM) increased and prolonged the inhibitory postsynaptic potential (IPSP). The mean maximal synaptic conductance of the early, GABA_A receptor-mediated, IPSP was enhanced to more than 700%, the one at the maximum of the late, partially GABA_A receptor-mediated, IPSP to approximately 400%. The progesterone/glucocorticoid receptor antagonist RU 38486 did not prevent the IPSP increase. At a concentration of 1 μM 5α-THDOC increased only the early IPSP to about 125%. Responses to the iontophoretically applied specific GABA_A receptor agonist muscimol but not to the specific GABA_B receptor agonist L-baclofen were enhanced by 5α-THDOC (10 μM). In the giga-seal whole-cell configuration when the GABA_B receptor-mediated IPSP component was absent due to intracellular perfusion, 5α-THDOC (10 μM) increased IPSPs to a similar extent as in the conventional microelectrode recordings. Excitatory postsynaptic potentials, resting membrane potential, input resistance and action potential amplitude were not affected by 5α-THDOC (10 μM). These data demonstrate that in neocortical tissue of the rat 5α-THDOC enhances GABAergic inhibition by interacting with postsynaptic GABA_A receptors while synaptic excitation and parameters of electric excitability remain unchanged.     

Reduced GABAergic transmission and number of hippocampal perisomatic inhibitory synapses in juvenile mice deficient in the neural cell adhesion molecule L1

Cell adhesion molecules have been implicated in neural development and hippocampal synaptic plasticity. Here, we investigated the role of the neural cell adhesion molecule L1 in regulation of basal synaptic transmission and plasticity in the CA1 area of the hippocampus of juvenile mice. We show that theta-burst stimulation (TBS) and pairing of low-frequency presynaptic stimulation with depolarization of postsynaptic CA1 pyramidal cells induced similar levels of LTP in L1-deficient and wild-type mice. The basal excitatory synaptic transmission and density of asymmetric excitatory synapses in the stratum radiatum were also normal in L1-deficient mice. Since L1 is expressed not only by principal cells but also by inhibitory interneurons, we recorded inhibitory postsynaptic currents (IPSCs) evoked in CA1 pyramidal cells by minimal stimulation of perisomatic interneurons. L1-deficient mice showed a reduction in the mean amplitude of putative unitary IPSCs, higher values of the coefficient of amplitude variation, higher number of failures in transmitter release, and a reduction in frequency but not amplitude of miniature IPSCs. The use-dependent modulation of inhibitory transmission by paired-pulse or short tetanic stimulation was, however, normal in L1-deficient mice. The physiological abnormalities correlated with a strong reduction in the density of inhibitory active zones, indicating that L1 is involved in establishing inhibitory perisomatic synapses in the hippocampus.     

Differential effects of a benzodiazepine on synaptic transmissions in rat hippocampal neurons in vitro

The effects of midazolam, one of the most popular benzodiazepines, on synaptic transmissions were compared with intracellular recordings between CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) in rat hippocampal slices. First, we studied the effects of midazolam on orthodromically evoked spikes, membrane properties and synaptic potentials. Secondly, the effects of a GABA_A receptor agonist, muscimol, were examined on membrane properties to determine whether or not the densities of GABA_A receptors are different between CA1-PCs and DG-GCs. Midazolam (75 μM) markedly depressed orthodromically evoked spikes in CA1-PCs, compared with those in DG-GCs. A GABA_A receptor antagonist, bicuculline (10 μM), almost completely antagonized the depressant effects of midazolam on spike generation in CA1-PCs, whereas it had little effect on midazolam in dentate gyrus granule cells. Midazolam produced either depolarizing or hyperpolarizing effects on resting membrane potentials (V_m) with an input resistance decrease in CA1-PCs, whereas it produced depolarized V_m in DG-GCs. Midazolam significantly increased the amplitude of monosynaptic inhibitory postsynaptic potentials in CA1-PCs, whereas midazolam slightly decreased these in DG-GCs. Midazolam significantly decreased the amplitude of excitatory postsynaptic potentials both in CA1-PCs and DG-GCs. Muscimol (100 μM) produced either depolarizing or hyperpolarizing effects on V_m with an input resistance decrease in CA1-PCs, and it depolarized V_m with an input resistance decrease in DG-GCs. These results demonstrate that midazolam has differential effects on excitatory and inhibitory synaptic transmissions in hippocampal neurons. The mechanism of this difference could be partly due to the different types of GABA_A receptors between CA1-PCs and DG-GCs.     

GABA receptor-mediated post-synaptic potentials in the retrohippocampal cortices: regional, laminar and cellular comparisons

Inhibitory post-synaptic potentials (IPSPs) were studied in neurons of presubiculum, parasubiculum and medial entorhinal cortex in horizontal slices from rat brains. Isolated IPSPs were evoked by extracellular electrical stimuli in the presence of glutamate receptor antagonists. Cellular morphology was identified using Neurobiotin labeling. IPSPs were compared: (a) across morphological cell types, (b) across laminae within regions, and (c) across regions. IPSPs were visible in stellate and pyramidal cells from layers II, III, and V of all retrohippocampal areas during bath application of glutamate antagonists. Qualitative and quantitative differences in IPSPs were only found when comparing responses by superficial layer II, III cells to responses by deep layer V cells. Responses by stellate and pyramidal cells within the same or adjacent layers did not differ, nor did responses differ from region to region. All cell types exhibited an early hyperpolarizing response. The majority (85%) of superficial layer cells in all regions, regardless of cell shape, exhibited a second hyperpolarizing component. Fewer (50%) deep layer cells exhibited the late peak with similar long latencies. IPSPs were typically larger in superficial layer cells. IPSPs were comprised of GABA_A and GABA_B (γ-aminobutyric acid) receptor-mediated components. With repetitive stimulation, the peak amplitude of the GABA_A receptor-mediated component decreased with successive stimuli, but stabilized during the first five or fewer stimuli to a level that did not vary with stimulation frequency. The GABA_B receptor-mediated component also stabilized, but the final amplitude appeared to decrease as the stimulation frequency increased. With high-frequency repetitive stimulation, both components of the IPSP showed summation. We conclude that the most meaningful distinction for IPSPs among retrohippocampal neurons is a laminar distinction, between superficial and deep layer neurons, and not one across cell shape or retrohippocampal subregion. These laminar differences can contribute to synchronous activity by deep layer neurons and restrict the activity of superficial layer neurons.     

Synaptic inhibition in the isolated respiratory network of neonatal rats

Gramicidin-perforated patch-clamp recording revealed phasic Cl^–-mediated hyperpolarizations in respiratory neurons of the brainstem–spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of γ-aminobutyric acid (GABA), i.e. GABA_A, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO₂/HCO₃^–-free solutions, but shifted from about ?65 mV to values more positive than ?20 mV upon dialysis of the cells with 144 instead of 4 mm Cl^–. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABA_B receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K⁺ channel-mediated hyperpolarization (reversal potential ?88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABA_A and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABA_B receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.     

Subdivisions in the Multiple GABAergic Innervation of Granule Cells in the Dentate Gyrus of the Rat Hippocampus

The sources of GABAergic innervation to granule cells were studied to establish how the basic cortical circuit is implemented in the dentate gyrus. Five types of neuron having extensive local axons were recorded electrophysiologically in vitro and filled intracellularly with biocytin (Han et al., 1993). They were processed for electron microscopy in order to reveal their synaptic organization and postsynaptic targets, and to test whether their terminals contained GABA. (1) The hilar cell, with axon terminals in the commissural and association pathway termination field (HICAP cell), formed Gray's type 2 (symmetrical) synapses with large proximal dendritic shafts (n= 18), two-thirds of which could be shown to emit spines, and with small dendritic branches (n= 6). Other boutons of the HICAP neuron were found to make either Gray's type 1 (asymmetrical) synapses (n= 4) or type 2 synapses (n= 6) with dendritic spines. Using a highly sensitive silver-intensified immunogold method for the postembedding visualization of GABA immunoreactivity, both the terminals and the dendrites of the HICAP cell were found to be immunopositive, whereas its postsynaptic targets were GABA-immunonegative. The dendritic shafts of the HICAP cell received synapses from both GABA-negative and GABA-positive boutons; the dendritic spines which densely covered the main apical dendrite in the medial one-third of the molecular layer received synapses from GABA-negative boutons. (2) The hilar cell, with axon terminals distributed in conjunction with the perforant path termination field (HIPP cell), established type 2 synapses with distal dendritic shafts (n= 17), most of which could be shown to emit spines, small-calibre dendritic profiles (n= 2) and dendritic spines (n= 6), all showing characteristics of granule cell dendrites. The sparsely spiny dendrites of the HIPP cell were covered with many synaptic boutons on both their shafts and their spines. (3) The cell with soma in the molecular layer had an axon associated with the perforant path termination field (MOPP cell). This GABA-immunoreactive cell made type 2 synapses exclusively on dendritic shafts (n= 20), 60% of which could be shown to emit spines. The smooth dendrites of the MOPP cell were also restricted to the outer two-thirds of the molecular layer, where they received both GABA-negative and GABA-positive synaptic inputs. (4) The extensive axonal arborization of the dentate basket cell terminated mainly on somata (n= 26) and proximal dendrites (n= 9) in the granule cell layer, and some boutons made synapses on somatic spines (n= 6); all boutons established type 2 synapses. (5) The dentate axo-axonic cell established type 2 synapses (n= 14) exclusively on axon initial segments of granule cells in the granule cell layer, and on initial segments of presumed mossy cells in the hilus. The results demonstrate that granule cells receive inputs from the local circuit axons of at least five distinct types of dentate neuron terminating in mutually exclusive domains of the cell's surface in four out of five cases. Four of the cell types (HICAP cell, MOPP cell, basket cell, axo-axonic cell) contain GABA, and the HIPP cell may also be inhibitory. The specific local inhibitory neurons terminating in conjunction with particular excitatory amino acid inputs to the granule cells (types 1 – 3) are in a position to interact selectively with the specific inputs on the same dendritic segment. This arrangement provides a possibility for the independent regulation of the gain and long-term potentiation of separate excitatory inputs, through different sets of GABAergic local circuit neurons. The pairing of excitatory and inhibitory inputs may also provide a mechanism for the downward reseating of excitatory postsynaptic potentials, thereby extending their dynamic range.     

Selective suppression of intrinsic but not afferent fiber synaptic transmission by baclofen in the piriform (olfactory) cortex

The GABA_B agonist baclofen has been shown to suppress synaptic transmission in subregions of the hippocampus and in the piriform (olfactory) cortex. Here we report a laminar selectivity of suppression of synaptic potentials in the olfactory cortex. In brain slice preparations, baclofen suppresses extracellularly recorded field potentials at the intrinsic fiber synapses proximal to the superficial pyramidal cell bodies (layer Ib) while leaving the af ferent fiber synaptic potentials recorded at the distal dendrites (layer Ia) little affected. This dose-dependent selective suppression of intrinsic fiber synaptic transmission is also correlated with an increase of paired-pulse facilitation. These results suggest that afferent and intrinsic synaptic inputs may be differentially modulated by the activation of GABA_B receptors and that this selective suppression is at least partially mediated via a presynaptic mechanism.     

GABA-dependent generation of ectopic action potentials in the rat hippocampus

Intracellular recordings from CA3 pyramidal cells of rat hippocampus in a slice preparation revealed the occurrence of interictal epileptiform discharges and synchronous GABA-mediated potentials during application of 4-aminopyridine (4AP, 50 μm ). The synchronous GABA-mediated potential consisted of a sequence of early hyperpolarization, long-lasting depolarization (LLD), and late hyperpolarization. Action potentials of variable amplitude occurred at the peak of the early hyperpolarization and during the LLD rising phase (48 of 64 cells); they were not prevented by membrane hyperpolarization and displayed inflections that were reminiscent of the initial segment-somatodendritic (IS-SD) fractionation. Interictal discharges were blocked by excitatory amino acid receptor antagonists, while both GABA-mediated potentials and action potentials of variable amplitude continued to occur (n = 10). The latter events were still recorded in the presence of the GABA_B receptor antagonist CGP-35348 (0.5–1 mm , n = 4), but were abolished by the GABA_A receptor antagonist bicuculline methiodide (BMI, 10 μm , n = 5). Localized application of BMI (20 μm , n = 6) or tetrodotoxin (TTX, 5 μm , n = 3) to the CA1 stratum radiatum blocked the variable amplitude action potentials; these effects were not seen when BMI (n = 4) or TTX (n = 4) were applied to the CA3 stratum radiatum, although both procedures made LLDs disappear. Our findings indicate that action potentials of variable amplitude recorded from CA3 pyramidal cells in the 4AP model are generated at or near the terminal region of the Schaffer collaterals and that they represent TTX-sensitive ectopic events. These action potentials are generated at this site by a BMI-sensitive (and thus GABA_A-mediated) mechanism. We propose that the ectopic action potentials reflect an increased excitability of axon terminals that is presumably caused by [K⁺]_o elevations associated with the 4AP-induced synchronous GABA-mediated potential.     

On the sites of presynaptic inhibition by neuropeptide y in rat hippocampus in vitro

Neuropeptide Y (NPY) reduces excitatory synaptic transmission between stratum radiatum and CA1 pyramidal cells in rat hippocampal slice in vitro by a presynaptic action. To understand NPY's role in the control of excitability in hippocampus, its actions on excitatory and inhibitory synaptic transmission were examined, using intracellular, sharp microelectrode, and tight-seal, whole cell recordings from principal neurons in areas CA1, CA3, and dentate. Bath application of 1 μM NPY reversibly inhibited excitatory postsynaptic potentials (EPSPs) evoked in CA1 pyramidal cells from either stratum radiatum or stratum oriens by about 50%. Neuropeptide Y also inhibited EPSPs at mossy fiber-CA3, stratum oriens-CA3, and CA3-CA3 synapses by between 45% and 55%. As in CA1, the action of NPY was presynaptic. By contrast, NPY did not inhibit EPSPs evoked in dentate granule cells from either perforant path or commissural inputs. Neuropeptide Y did not alter postsynaptic membrane properties in any cell type. Although NPY attenuated the orthodromically evoked (stratum radiatum) inhibitory postsynaptic potentials in CA1 pyramidal cells by about the same amount as it inhibited the EPSPs, it did not affect the IPSPs evoked in the same cells by antidromic stimulation from alveus. Inhibitory postsynaptic potentials evoked in pharmacological isolation in CA1, CA3, or dentate were also not significantly affected by NPY. The evidence supports the hypothesis that NPY acts at feedforward excitatory synapses to presynaptically reduce the amplitude of excitation as it travels through hippocampal circuits. By contrast, synaptically mediated inhibition is not directly affected by NPY. Neuropeptide Y is the only known endogenous substance that selectively reduces feedforward excitatory transmission without causing changes in other properties of the hippocampal circuitry.     

Intrinsic subthreshold oscillations extend the influence of inhibitory synaptic inputs on cortical pyramidal neurons

Fast inhibitory synaptic inputs, which cause conductance changes that typically last for 10–100 ms, participate in the generation and maintenance of cortical rhythms. We show here that these fast events can have influences that outlast the duration of the synaptic potentials by interacting with subthreshold membrane potential oscillations. Inhibitory postsynaptic potentials (IPSPs) in cortical neurons in vitro shifted the oscillatory phase for several seconds. The phase shift caused by two IPSPs or two current pulses summed non‐linearly. Cholinergic neuromodulation increased the power of the oscillations and decreased the magnitude of the phase shifts. These results show that the intrinsic conductances of cortical pyramidal neurons can carry information about inhibitory inputs and can extend the integration window for synaptic input.     

Norepinephrine decreases synaptic inhibition in the rat hippocampus

The effects of norepinephrine (NE) on inhibitory synaptic potentials were studied on CA1 pyramidal neurons in the hippocampal slice in vitro. Norepinephrine caused the appearance of multiple population spikes in the CA1 region of the hippocampal slice, reminiscent of the actions of gamma-aminobutyric acid (GABA) antagonists. Intracellular recording revealed that NE causes a marked and reversible reduction in inhibitory postsynaptic potentials (IPSPs) recorded in CA1 pyramidal cells. This reduced IPSP results in a larger intracellular excitatory postsynaptic potential (EPSP), which can cause the cell to fire more than one action potential. This disinhibitory effect of NE appears to be mediated by an alpha-receptor, and occurs at a site presynaptic to the pyramidal cell, since NE does not change the reversal potential of the IPSP nor does it affect the amplitude or the reversal potential of iontophoretic GABA responses. In addition to reducing evoked IPSPs, NE causes an increase in the frequency of spontaneous IPSPs, suggesting that inhibition of interneuronal firing may not account for this disinhibitory action of NE.     

Long‐term depression of inhibitory synaptic transmission induced by spike‐timing dependent plasticity requires coactivation of endocannabinoid and muscarinic receptors

The precise timing of pre‐postsynaptic activity is vital for the induction of long‐term potentiation (LTP) or depression (LTD) at many central synapses. We show in synapses of rat CA1 pyramidal neurons in vitro that spike timing dependent plasticity (STDP) protocols that induce LTP at glutamatergic synapses can evoke LTD of inhibitory postsynaptic currents or STDP‐iLTD. The STDP‐iLTD requires a postsynaptic Ca²⁺ increase, a release of endocannabinoids (eCBs), the activation of type‐1 endocananabinoid receptors and presynaptic muscarinic receptors that mediate a decreased probability of GABA release. In contrast, the STDP‐iLTD is independent of the activation of nicotinic receptors, GABA_BRs and G protein‐coupled postsynaptic receptors at pyramidal neurons. We determine that the downregulation of presynaptic Cyclic adenosine monophosphate/protein Kinase A pathways is essential for the induction of STDP‐iLTD. These results suggest a novel mechanism by which the activation of cholinergic neurons and retrograde signaling by eCBs can modulate the efficacy of GABAergic synaptic transmission in ways that may contribute to information processing and storage in the hippocampus. © 2013 Wiley Periodicals, Inc.