Efficient control of principal neuron firing by basket cells is critical for information processing in cortical microcircuits, however, the relative contribution of their perisomatic and dendritic synapses to spike inhibition is still unknown. Using in vitro electrophysiological paired recordings we reveal that in the mouse basal amygdala cholecystokinin- and parvalbumin-containing basket cells provide equally potent control of principal neuron spiking. We performed pharmacological manipulations, light and electron microscopic investigations to show that, although basket cells innervate the entire somato-denditic membrane surface of principal neurons, the spike controlling effect is achieved primarily via the minority of synapses targeting the perisomatic region. As the innervation patterns of individual basket cells on their different postsynaptic partners show high variability, the impact of inhibitory control accomplished by single basket cells is also variable. Our results show that both basket cell types can powerfully regulate the activity in amygdala networks predominantly via their perisomatic synapses.

Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study, we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6-expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed-forward and feed-back inhibitory circuits in this brain region. © 2012 Wiley Periodicals, Inc.

Information processing in neural networks depends on the connectivity among excitatory and inhibitory neurons. The presence of parallel, distinctly controlled local circuits within a cortical network may ensure an effective and dynamic regulation of microcircuit function. By applying a combination of optogenetics, electrophysiological recordings, and high resolution microscopic techniques, we uncovered the organizing principles of synaptic communication between principal neurons and basket cells in the basal nucleus of the amygdala. In this cortical structure, known to be critical for emotional memory formation, we revealed the presence of 2 parallel basket cell networks expressing either parvalbumin or cholecystokinin. While the 2 basket cell types are mutually interconnected within their own category via synapses and gap junctions, they avoid innervating each other, but form synaptic contacts with axo-axonic cells. Importantly, both basket cell types have the similar potency to control principal neuron spiking, but they receive excitatory input from principal neurons with entirely diverse features. This distinct feedback synaptic excitation enables a markedly different recruitment of the 2 basket cell types upon the activation of local principal neurons. Our data suggest fundamentally different functions for the 2 parallel basket cell networks in circuit operations in the amygdala.

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.

The release of GABA from cholecystokinin-containing interneurons is modulated by type-1 cannabinoid receptors (CB1). Here we tested the hypothesis that the strength of CB1-mediated modulation of GABA release is related to the CB1 content of axon terminals. Basket cell boutons have on average 78% higher CB1 content than those of dendritic-layer-innervating (DLI) cells, a consequence of larger bouton surface and higher CB1 density. The CB1 antagonist AM251 caused a 54% increase in action potential-evoked [Ca(2+)] in boutons of basket cells, but not in DLI cells. However, the effect of AM251 did not correlate with CB1 immunoreactivity of individual boutons. Moreover, a CB1 agonist decreased [Ca(2+)] in a cell type- and CB1-content-independent manner. Replica immunogold labelling demonstrated the colocalization of CB1 with the Cav2.2 Ca(2+) channel subunit. Our data suggest that only a subpopulation of CB1s, within nanometre distances from their target Cav2.2 channels, are responsible for endocannabinoid-mediated modulation of GABA release.

Spike generation is most effectively controlled by inhibitory inputs that target the perisomatic region of neurons. Despite the critical importance of this functional domain, very little is known about the organization of the GABAergic inputs contacting the perisomatic region of principal cells (PCs) in the basolateral amygdala. Using immunocytochemistry combined with in vitro single-cell labeling we determined the number and sources of GABAergic inputs of PCs at light and electron microscopic levels in mice. We found that the soma and proximal dendrites of PCs were innervated primarily by two neurochemically distinct basket cell types expressing parvalbumin (PVBC) or cholecystokinin and CB1 cannabinoid receptors (CCK/CB1BC). The innervation of the initial segment of PC axons was found to be parceled out by PVBCs and axo-axonic cells (AAC), as the majority of GABAergic inputs onto the region nearest to the soma (between 0 and 10 μm) originated from PVBCs, while the largest portion of the axon initial segment was innervated by AACs. Detailed morphological investigations revealed that the three perisomatic region-targeting interneuron types significantly differed in dendritic and axonal arborization properties. We found that, although individual PVBCs targeted PCs via more terminals than CCK/CB1BCs, similar numbers (15-17) of the two BC types converge onto single PCs, whereas fewer (6-7) AACs innervate the axon initial segment of single PCs. Furthermore, we estimated that a PVBC and a CCK/CB1BC may target 800-900 and 700-800 PCs, respectively, while an AAC can innervate 600-650 PCs. Thus, BCs and AACs innervate ~10 and 20% of PC population, respectively, within their axonal cloud. Our results collectively suggest, that these interneuron types may be differently affiliated within the local amygdalar microcircuits in order to fulfill specific functions in network operation during various brain states.

Potassium channels comprise the most diverse family of ion channels and play critical roles in a large variety of physiological and pathological processes. In addition to their molecular diversity, variations in their distributions and densities on the axo-somato-dendritic surface of neurons are key parameters in determining their functional impact. Despite extensive electrophysiological and anatomical investigations, the exact location and densities of most K(+) channels in small subcellular compartments are still unknown. Here we aimed at providing a quantitative surface map of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K(+) channel subunits on hippocampal CA1 pyramidal cells (PCs). Freeze-fracture replica immunogold labelling was employed to determine the relative densities of these K(+) channel subunits in 18 axo-somato-dendritic compartments. Significant densities of the Kv1.1 subunit were detected on axon initial segments (AISs) and axon terminals, with an approximately eight-fold lower density in the latter compartment. The Kv2.1 subunit was found in somatic, proximal dendritic and AIS plasma membranes at approximately the same densities. This subunit has a non-uniform plasma membrane distribution; Kv2.1 clusters are frequently adjacent to, but never overlap with, GABAergic synapses. A quasi-linear increase in the Kir3.2 subunit density along the dendrites of PCs was detected, showing no significant difference between apical dendritic shafts, oblique dendrites or dendritic spines at the same distance from the soma. Our results demonstrate that each subunit has a unique cell-surface distribution pattern, and predict their differential involvement in synaptic integration and output generation at distinct subcellular compartments.

We investigated the effect of a synthetic cannabinoid, WIN 55,212-2 on excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collaterals in CA1 pyramidal cells. Bath application of WIN 55,212-2 reduced the amplitude of EPSCs in dose-dependent manner tested between 0.01 nM and 30 microM. In rats and mice, this cannabinoid ligand inhibited excitatory synapses in two steps at the nM and muM concentrations. When the function of CB(1) cannabinoid receptors (CB(1)R) was impaired, either by the application of a CB(1)R antagonist AM251, or by using CB(1)R knockout mice, WIN 55,212-2 in microM concentrations could still significantly reduced the amplitude of EPSCs. WIN 55,212-2 likely affected the efficacy of excitatory transmission only at presynaptic sites, since both at low and high doses the paired pulse ratio of EPSC amplitude was significantly increased. The inactive enantiomer, WIN 55,212-3, mimicked the effect of WIN 55,212-2 applied in high doses. In further experiments we found that the CB(1)R-independent effect of 10 microM WIN 55,212-2 at glutamatergic synapses was fully abolished, when slices were pre-treated with omega-conotoxin GVIA, but not with omega-agatoxin IVA. These data suggest that, in the hippocampus, WIN 55,212-2 reduces glutamate release from Schaffer collaterals solely via CB(1)Rs in the nM concentration range, whereas in microM concentrations, WIN 55,212-2 suppresses excitatory transmission, in addition to activation of CB(1)Rs, by directly blocking N-type voltage-gated Ca(2+) channels independent of CB(1)Rs.

In cortical structures, principal cell activity is tightly regulated by different GABAergic interneurons (INs). Among these INs are vasoactive intestinal polypeptide-expressing (VIP+) INs, which innervate preferentially other INs, providing a structural basis for temporal disinhibition of principal cells. However, relatively little is known about VIP+ INs in the amygdaloid basolateral complex (BLA). In this study, we report that VIP+ INs have a variable density in the distinct subdivisions of the mouse BLA. Based on different anatomical, neurochemical, and electrophysiological criteria, VIP+ INs could be identified as IN-selective INs (IS-INs) and basket cells expressing CB1 cannabinoid receptors. Whole-cell recordings of VIP+ IS-INs revealed three different spiking patterns, none of which was associated with the expression of calretinin. Genetic targeting combined with optogenetics and in vitro recordings enabled us to identify several types of BLA INs innervated by VIP+ INs, including other IS-INs, basket and neurogliaform cells. Moreover, light stimulation of VIP+ basket cell axon terminals, characterized by CB1 sensitivity, evoked IPSPs in ∼20% of principal neurons. Finally, we show that VIP+ INs receive a dense innervation from both GABAergic inputs (although only 10% from other VIP+ INs) and distinct glutamatergic inputs, identified by their expression of different vesicular glutamate transporters.In conclusion, our study provides a wide-range analysis of single-cell properties of VIP+ INs in the mouse BLA and of their intrinsic and extrinsic connectivity. Our results reinforce the evidence that VIP+ INs are structurally and functionally heterogeneous and that this heterogeneity could mediate different roles in amygdala-dependent functions.SIGNIFICANCE STATEMENT We provide the first comprehensive analysis of the distribution of vasoactive intestinal polypeptide-expressing (VIP+) interneurons (INs) across the entire mouse amygdaloid basolateral complex (BLA), as well as of their morphological and physiological properties. VIP+ INs in the neocortex preferentially target other INs to form a disinhibitory network that facilitates principal cell firing. Our study is the first to demonstrate the presence of such a disinhibitory circuitry in the BLA. We observed structural and functional heterogeneity of these INs and characterized their input/output connectivity. We also identified several types of BLA INs that, when inhibited, may provide a temporal window for principal cell firing and facilitate associative plasticity, e.g., in fear learning.

Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence--using whole-cell recording--that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light--and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.

CB(1) cannabinoid receptor (CB(1)R) activation by exogenous ligands can impair memory processes, which critically depend on synchronous neuronal activities that are temporarily structured by oscillations. In this study, we aimed to reveal the mechanisms underlying the cannabinoid-induced decrease in gamma oscillations. We first verified that cannabinoids (CP55,940 and WIN55,212-2) readily suppressed carbachol-induced gamma oscillations in the CA3 region of hippocampal slices via activation of CB(1)Rs. The cannabinoid-induced decrease in the peak power of oscillations was accompanied by reduced and less precise firing activity in CA3 pyramidal cells and fast spiking basket cells. By examining the cannabinoid sensitivity of synaptic inputs we found that the amplitude of evoked excitatory postsynaptic currents was significantly suppressed upon CB(1)R activation in both CA3 pyramidal cells and fast spiking basket cells. In contrast, evoked inhibitory postsynaptic currents in CA3 pyramidal cells were unaltered. Furthermore, we observed that a CB(1)R agonist-induced decrease in the oscillation power at the beginning of the drug application was accompanied primarily by the reduced discharge of fast spiking basket cells, while pyramidal cell firing was unaltered. This result implies that the dampening of cholinergically induced gamma oscillations in the hippocampus by cannabinoids can be explained by a reduced excitatory input predominantly onto fast spiking basket cells, which leads to a reduction in neuronal firing frequency and precision, and thus to smaller field potentials. In addition, we uncovered that the spontaneously occurring sharp wave-ripple activities in hippocampal slices could also be suppressed by CB(1)R activation suggesting that cannabinoids profoundly reduce the intrinsically generated oscillatory activities at distinct frequencies in CA3 networks by reducing synaptic neurotransmission.

Studies in brain slices have provided a wealth of data on the basic features of neurons and synapses. In the intact brain, these properties may be strongly influenced by ongoing network activity. Although physiologically realistic patterns of network activity have been successfully induced in brain slices maintained in interface-type recording chambers, they have been harder to obtain in submerged-type chambers, which offer significant experimental advantages, including fast exchange of pharmacological agents, visually guided patch-clamp recordings, and imaging techniques. Here, we investigated conditions for the emergence of network oscillations in submerged slices prepared from the hippocampus of rats and mice. We found that the local oxygen level is critical for generation and propagation of both spontaneously occurring sharp wave-ripple oscillations and cholinergically induced fast oscillations. We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice. These improvements to the recording conditions enable detailed studies of neurons under more realistic conditions of network activity, which are essential for a better understanding of neuronal network operation.

Perisomatic inhibition originates from three types of GABAergic interneurons in cortical structures, including parvalbumin-containing fast-spiking basket cells (FSBCs) and axo-axonic cells (AACs), as well as cholecystokinin-expressing regular-spiking basket cells (RSBCs). These interneurons may have significant impact in various cognitive processes, and are subjects of cholinergic modulation. However, it is largely unknown how cholinergic receptor activation modulates the function of perisomatic inhibitory cells. Therefore, we performed paired recordings from anatomically identified perisomatic interneurons and pyramidal cells in the CA3 region of the mouse hippocampus. We determined the basic properties of unitary inhibitory postsynaptic currents (uIPSCs) and found that they differed among cell types, e.g. GABA released from axon endings of AACs evoked uIPSCs with the largest amplitude and with the longest decay measured at room temperature. RSBCs could also release GABA asynchronously, the magnitude of the release increasing with the discharge frequency of the presynaptic interneuron. Cholinergic receptor activation by carbachol significantly decreased the uIPSC amplitude in all three types of cell pairs, but to different extents. M2-type muscarinic receptors were responsible for the reduction in uIPSC amplitudes in FSBC- and AAC-pyramidal cell pairs, while an antagonist of CB(1) cannabinoid receptors recovered the suppression in RSBC-pyramidal cell pairs. In addition, carbachol suppressed or even eliminated the short-term depression of uIPSCs in FSBC- and AAC-pyramidal cell pairs in a frequency-dependent manner. These findings suggest that not only are the basic synaptic properties of perisomatic inhibitory cells distinct, but acetylcholine can differentially control the impact of perisomatic inhibition from different sources.

The intrinsic properties of distinct types of neuron play important roles in cortical network dynamics. One crucial determinant of neuronal behaviour is the cell's response to rhythmic subthreshold input, characterised by the input impedance, which can be determined by measuring the amplitude and phase of the membrane potential response to sinusoidal currents as a function of input frequency. In this study, we determined the impedance profiles of anatomically identified neurons in the CA1 region of the rat hippocampus (pyramidal cells as well as interneurons located in the stratum oriens, including OLM cells, fast-spiking perisomatic region-targeting interneurons and cells with axonal arbour in strata oriens and radiatum). The basic features of the impedance profiles, as well as the passive membrane characteristics and the properties of the sag in the voltage response to negative current steps, were cell-type specific. With the exception of fast-spiking interneurons, all cell types showed subthreshold resonance, albeit with distinct features. The HCN channel blocker ZD7288 (10 microM) eliminated the resonance and changed the shape of the impedance curves, indicating the involvement of the hyperpolarization-activated cation current I(h). Whole-cell voltage-clamp recordings uncovered differences in the voltage-dependent activation and kinetics of I(h) between different cell types. Biophysical modelling demonstrated that the cell-type specificity of the impedance profiles can be largely explained by the properties of I(h) in combination with the passive membrane characteristics. We conclude that differences in I(h) and passive membrane properties result in a cell-type-specific response to inputs at given frequencies, and may explain, at least in part, the differential involvement of distinct types of neuron in various network oscillations.

Nanoscale distribution of molecules within small subcellular compartments of neurons critically influences their functional roles. Although, numerous ways of analyzing the spatial arrangement of proteins have been described, a thorough comparison of their effectiveness is missing. Here we present an open source software, GoldExt, with a plethora of measures for quantification of the nanoscale distribution of proteins in subcellular compartments (e.g. synapses) of nerve cells. First, we compared the ability of five different measures to distinguish artificial uniform and clustered patterns from random point patterns. Then, the performance of a set of clustering algorithms was evaluated on simulated datasets with predefined number of clusters. Finally, we applied the best performing methods to experimental data, and analyzed the nanoscale distribution of different pre- and postsynaptic proteins, revealing random, uniform and clustered sub-synaptic distribution patterns. Our results reveal that application of a single measure is sufficient to distinguish between different distributions.

Hippocampal place cells are activated sequentially as an animal explores its environment. These activity sequences are internally recreated ('replayed'), either in the same or reversed order, during bursts of activity (sharp wave-ripples [SWRs]) that occur in sleep and awake rest. SWR-associated replay is thought to be critical for the creation and maintenance of long-term memory. In order to identify the cellular and network mechanisms of SWRs and replay, we constructed and simulated a data-driven model of area CA3 of the hippocampus. Our results show that the chain-like structure of recurrent excitatory interactions established during learning not only determines the content of replay, but is essential for the generation of the SWRs as well. We find that bidirectional replay requires the interplay of the experimentally confirmed, temporally symmetric plasticity rule, and cellular adaptation. Our model provides a unifying framework for diverse phenomena involving hippocampal plasticity, representations, and dynamics, and suggests that the structured neural codes induced by learning may have greater influence over cortical network states than previously appreciated.

GABAergic neurons are key circuit elements in cortical networks. Despite growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types has been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22%) is significantly higher than in the LA (16%) in both male and female mice. No difference was observed between the right and left hemispheres in either sex. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5%-6%), basket cells expressing parvalbumin (17%-20%) or cholecystokinin (7%-9%), dendrite-targeting inhibitory cells expressing somatostatin (10%-16%), NPY-containing neurogliaform cells (14%-15%), VIP and/or calretinin-expressing interneuron-selective interneurons (29%-38%), and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (5.5%-8%). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathologic conditions, and to model functioning amygdalar networks in health and disease.SIGNIFICANCE STATEMENT GABAergic cells in cortical structures, as in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyperexcitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions, including anxiety, post-traumatic stress disorder, schizophrenia, and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathologic brain states.

Several studies have shown that capsaicin could effectively regulate excitatory synaptic transmission in the central nervous system, but the assumption that this effect is mediated by TRPV1 vanilloid receptors (TRPV1Rs) has not been tested directly. To provide direct evidence, we compared the effect of capsaicin on excitatory synapses in wild type mice and TRPV1R knockouts. Using whole-cell patch-clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded in granule cells of the dentate gyrus. First, we investigated the effect of capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare. Bath application of 10 microM capsaicin reduced the amplitude of evoked EPSCs both in wild type and TRPV1R knockout animals to a similar extent. Treatment of the slices with the TRPV1R antagonist capsazepine (10 microM) alone, or together with the agonist capsaicin, also caused a decrease in the EPSC amplitude both in wild type and TRPV1R knockout animals. Both drugs appeared to affect the efficacy of excitatory synapses at presynaptic sites, since a significant increase was observed in paired-pulse ratio of EPSC amplitude after drug treatment. Next we examined the effect of capsaicin on spontaneously occurring EPSCs. This prototypic vanilloid ligand increased the frequency of events without changing their amplitude in wild type mice. Similar enhancement in the frequency without altering the amplitude of spontaneous EPSCs was observed in TRPV1R knockout mice. These data strongly argue against the hypothesis that capsaicin modulates excitatory synaptic transmission by activating TRPV1Rs, at least in the hippocampal network.

Replay of neuronal activity during hippocampal sharp wave-ripples (SWRs) is essential in memory formation. To understand the mechanisms underlying the initiation of irregularly occurring SWRs and the generation of periodic ripples, we selectively manipulated different components of the CA3 network in mouse hippocampal slices. We recorded EPSCs and IPSCs to examine the buildup of neuronal activity preceding SWRs and analyzed the distribution of time intervals between subsequent SWR events. Our results suggest that SWRs are initiated through a combined refractory and stochastic mechanism. SWRs initiate when firing in a set of spontaneously active pyramidal cells triggers a gradual, exponential buildup of activity in the recurrent CA3 network. We showed that this tonic excitatory envelope drives reciprocally connected parvalbumin-positive basket cells, which start ripple-frequency spiking that is phase-locked through reciprocal inhibition. The synchronized GABA(A) receptor-mediated currents give rise to a major component of the ripple-frequency oscillation in the local field potential and organize the phase-locked spiking of pyramidal cells. Optogenetic stimulation of parvalbumin-positive cells evoked full SWRs and EPSC sequences in pyramidal cells. Even with excitation blocked, tonic driving of parvalbumin-positive cells evoked ripple oscillations. Conversely, optogenetic silencing of parvalbumin-positive cells interrupted the SWRs or inhibited their occurrence. Local drug applications and modeling experiments confirmed that the activity of parvalbumin-positive perisomatic inhibitory neurons is both necessary and sufficient for ripple-frequency current and rhythm generation. These interneurons are thus essential in organizing pyramidal cell activity not only during gamma oscillation, but, in a different configuration, during SWRs.

We analyzed the origin and relevance of the perisomatic excitatory inputs on the parvalbumin interneurons of the granule cell layer in mouse. Confocal analysis of the glutamatergic innervation showed that it represents ∼50% of the perisomatic synapses that parvalbumin cells receive. This excitatory input may originate from granule cell collaterals, the mossy cells, or even supramammillary nucleus. First, we assessed the input from the mossy cells on parvalbumin interneurons. Axon terminals of mossy cells were visualized by their calretinin content. Using multicolor confocal microscopy, we observed that less than 10% of perisomatic excitatory innervation of parvalbumin cells could originate from mossy cells. Correlative light and electron microscopy revealed that innervation from mossy cells, although present, was indeed infrequent, except for those parvalbumin cells whose somata were located in the inner molecular layer. Second, we investigated the potential input from supramammillary nucleus on parvalbumin cell somata using anterograde tracing or immunocytochemistry against vesicular glutamate transporter 2 (VGLUT2) and found only occasional contacts. Third, we intracellularly filled dentate granule cells in acute slice preparations using whole-cell recording and examined whether their axon collaterals target parvalbumin interneurons. We found that typical granule cells do not innervate the perisomatic region of these GABAergic cells. In sharp contrast, semilunar granule cells (SGCs), a scarce granule cell subtype often contacted the parvalbumin cell soma and proximal dendrites. Our data, therefore, show that perisomatic excitatory drive of parvalbumin interneurons in the granular layer of the dentate gyrus is abundant and originates primarily from SGCs.