Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.

Cell Surface Protein mRNAs Show Differential Transcription in Pyramidal and Fast-Spiking Cells as Revealed by Single-Cell Sequencing.

The prefrontal cortex (PFC) plays a key role in higher order cognitive functions and psychiatric disorders such as autism, schizophrenia, and depression. In the PFC, the two major classes of neurons are the glutamatergic pyramidal (Pyr) cells and the GABAergic interneurons such as fast-spiking (FS) cells. Despite extensive electrophysiological, morphological, and pharmacological studies of the PFC, the therapeutically utilized drug targets are restricted to dopaminergic, glutamatergic, and GABAergic receptors. To expand the pharmacological possibilities as well as to better understand the cellular and network effects of clinically used drugs, it is important to identify cell-type-selective, druggable cell surface proteins and to link developed drug candidates to Pyr or FS cell targets. To identify the mRNAs of such cell-specific/enriched proteins, we performed ultra-deep single-cell mRNA sequencing (19 685 transcripts in total) on electrophysiologically characterized intact PFC neurons harvested from acute brain slices of mice. Several selectively expressed transcripts were identified with some of the genes that have already been associated with cellular mechanisms of psychiatric diseases, which we can now assign to Pyr (e.g., Kcnn2, Gria3) or FS (e.g., Kcnk2, Kcnmb1) cells. The earlier classification of PFC neurons was also confirmed at mRNA level, and additional markers have been provided.

Local apoptotic-like mechanisms underlie complement-mediated synaptic pruning.

C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Behavior-dependent activity patterns of GABAergic long-range projecting neurons in the rat hippocampus.

Long-range glutamatergic and GABAergic projections participate in temporal coordination of neuronal activity in distributed cortical areas. In the hippocampus, GABAergic neurons project to the medial septum and retrohippocampal areas. Many GABAergic projection cells express somatostatin (SOM+) and, together with locally terminating SOM+ bistratified and O-LM cells, contribute to dendritic inhibition of pyramidal cells. We tested the hypothesis that diversity in SOM+ cells reflects temporal specialization during behavior using extracellular single cell recording and juxtacellular neurobiotin-labeling in freely moving rats. We have demonstrated that rare GABAergic projection neurons discharge rhythmically and are remarkably diverse. During sharp wave-ripples, most projection cells, including a novel SOM+ GABAergic back-projecting cell, increased their activity similar to bistratified cells, but unlike O-LM cells. During movement, most projection cells discharged along the descending slope of theta cycles, but some fired at the trough jointly with bistratified and O-LM cells. The specialization of hippocampal SOM+ projection neurons complements the action of local interneurons in differentially phasing inputs from the CA3 area to CA1 pyramidal cell dendrites during sleep and wakefulness. Our observations suggest that GABAergic projection cells mediate the behavior- and network state-dependent binding of neuronal assemblies amongst functionally-related brain regions by transmitting local rhythmic entrainment of neurons in CA1 to neuronal populations in other areas. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc.

Retino-cortical stimulus frequency-dependent gamma coupling: evidence and functional implications of oscillatory potentials.

Long-range gamma band EEG oscillations mediate information transmission between distant brain regions. Gamma band-based coupling may not be restricted to cortex-to-cortex communication but may include extracortical parts of the visual system. The retinogram and visual event-related evoked potentials exhibit time-locked, forward propagating oscillations that are candidates of gamma oscillatory coupling between the retina and the visual cortex. In this study, we tested if this gamma coupling is present as indicated by the coherence of gamma-range (70-200 Hz) oscillatory potentials (OPs) recorded simultaneously from the retina and the primary visual cortex in freely moving, adult rats. We found significant retino-cortical OP coherence in a wide range of stimulus duration (0.01-1000 msec), stimulus intensity (800-5000 mcd/mm2), interstimulus interval (10-400 msec), and stimulus frequency (0.25-25 Hz). However, at low stimulus frequencies, the OPs were time-locked, flickering light at 25 Hz entrained continuous OP coherence (steady-state response, SSR). Our results suggest that the retina and the visual cortex exhibit oscillatory coupling at high-gamma frequency with precise time locking and synchronization of information transfer from the retina to the visual cortex, similar to cortico-cortical gamma coupling. The temporal fusion of retino-cortical gamma coherence at stimulus rates of theater movies may explain the mechanism of the visual illusion of continuity. How visual perception depends on early transformations of ascending sensory information is incompletely understood. By simultaneous measurement of flash-evoked potentials in the retina and the visual cortex in awake, freely moving rats, we demonstrate for the first time that time-locked gamma oscillatory potentials exhibit stable retino-cortical synchrony across a wide range of stimulus parameters and that the temporal continuity of coherence changes with stimulus frequency according to the expected change in the visual illusion of continuity.

Divisions of Identified Parvalbumin-Expressing Basket Cells during Working Memory-Guided Decision Making.

Parvalbumin-expressing basket cells tightly control cortical networks and fire remarkably stereotyped during network oscillations and simple behaviors. How can these cells support multifaceted situations with different behavioral options and complex temporal sequences? We recorded from identified parvalbumin-expressing basket cells in prefrontal cortex of freely moving rats performing a multidimensional delayed cue-matching-to-place task, juxtacellularly filled recorded neurons for unequivocal histological identification, and determined their activity during temporally structured task episodes, associative working-memory, and stimulus-guided choice behavior. We show that parvalbumin-expressing basket cells do not fire homogenously, but individual cells were recruited or inhibited during different task episodes. Firing of individual basket cells was correlated with amount of presynaptic VIP (vasoactive intestinal polypeptide)-expressing GABAergic input. Together with subsets of pyramidal neurons, activity of basket cells differentiated for sequential actions and stimulus-guided choice behavior. Thus, interneurons of the same cell type can be recruited into different neuronal ensembles with distinct firing patterns to support multi-layered cognitive computations.

Divergent in vivo activity of non-serotonergic and serotonergic VGluT3-neurones in the median raphe region.

KEY POINTS: The median raphe is a key subcortical modulatory centre involved in several brain functions, such as regulation of the sleep-wake cycle, emotions and memory storage. A large proportion of median raphe neurones are glutamatergic and implement a radically different mode of communication compared to serotonergic cells, although their in vivo activity is unknown. We provide the first description of the in vivo, brain state-dependent firing properties of median raphe glutamatergic neurones identified by immunopositivity for the vesicular glutamate transporter type 3 (VGluT3) and serotonin (5-HT). Glutamatergic populations (VGluT3+/5-HT- and VGluT3+/5-HT+) were compared with the purely serotonergic (VGluT3-/5-HT+ and VGluT3-/5-HT-) neurones. VGluT3+/5-HT+ neurones fired similar to VGluT3-/5-HT+ cells, whereas they significantly diverged from the VGluT3+/5-HT- population. Activity of the latter subgroup resembled the spiking of VGluT3-/5-HT- cells, except for their diverging response to sensory stimulation. The VGluT3+ population of the median raphe may broadcast rapidly varying signals on top of a state-dependent, tonic modulation. ABSTRACT: Subcortical modulation is crucial for information processing in the cerebral cortex. Besides the canonical neuromodulators, glutamate has recently been identified as a key cotransmitter of numerous monoaminergic projections. In the median raphe, a pure glutamatergic neurone population projecting to limbic areas was also discovered with a possibly novel, yet undetermined function. In the present study, we report the first functional description of the vesicular glutamate transporter type 3 (VGluT3)-expressing median raphe neurones. Because there is no appropriate genetic marker for the separation of serotonergic (5-HT+) and non-serotonergic (5-HT-) VGluT3+ neurones, we utilized immunohistochemistry after recording and juxtacellular labelling in anaesthetized rats. VGluT3+/5-HT- neurones fired faster, more variably and were permanently activated during sensory stimulation, as opposed to the transient response of the slow firing VGluT3-/5-HT+ subgroup. VGluT3+/5-HT- cells were also more active during hippocampal theta. In addition, the VGluT3-/5-HT- population, comprising putative GABAergic cells, resembled the firing of VGluT3+/5-HT- neurones but without any significant reaction to the sensory stimulus. Interestingly, the VGluT3+/5-HT+ group, spiking slower than the VGluT3+/5-HT- population, exhibited a mixed response (i.e. the initial transient activation was followed by a sustained elevation of firing). Phase coupling to hippocampal and prefrontal slow oscillations was found in VGluT3+/5-HT- neurones, also differentiating them from the VGluT3+/5-HT+ subpopulation. Taken together, glutamatergic neurones in the median raphe may implement multiple, highly divergent forms of modulation in parallel: a slow, tonic mode interrupted by sensory-evoked rapid transients, as well as a fast one capable of conveying complex patterns influenced by sensory inputs.

Three axonal projection routes of individual pyramidal cells in the ventral CA1 hippocampus.

Pyramidal cells of the ventral hippocampal CA1 area have numerous and diverse distant projections to other brain regions including the temporal and parietal association areas, visual, auditory, olfactory, somatosensory, gustatory, and visceral areas, and inputs to the amygdalar and prefrontal-orbital-agranular insular region. In addition, their differential expression of proteins like calbindin provides further indications for cellular diversity. This raises the possibility that the pyramidal cells may form subpopulations participating in different brain circuitries. To address this hypothesis we applied the juxtacellular labeling technique to fill individual pyramidal cells in the ventral hippocampus with neurobiotin in urethane anesthetized rats. For each labeled pyramidal cell we determined soma location, dendritic arborizations and selective expression of calbindin and norbin. Reconstruction and mapping of long-range axonal projections were made with the Neurolucida system. We found three major routes of ventral CA1 pyramidal cell projections. The classical pathway run caudo-ventrally across and innervating the subiculum, further to the parahippocampal regions and then to the deep and superficial layers of entorhinal cortex. The other two pathways avoided subiculum by branching from the main axon close to the soma and either traveled antero- and caudo-ventrally to amygdaloid complex, amygdalopiriform-transition area and parahippocampal regions or run antero-dorsally through the fimbria-fornix to the septum, hypothalamus, ventral striatum and olfactory regions. We found that most pyramidal cells investigated used all three major routes to send projecting axons to other brain areas. Our results suggest that the information flow through the ventral hippocampus is distributed by wide axonal projections from the CA1 area.

Sleep and movement differentiates actions of two types of somatostatin-expressing GABAergic interneuron in rat hippocampus.

Neuropeptides acting on pre- and postsynaptic receptors are coreleased with GABA by interneurons including bistratified and O-LM cells, both expressing somatostatin but innervating segregated dendritic domains of pyramidal cells. Neuropeptide release requires high-frequency action potentials, but the firing patterns of most peptide/GABA-releasing interneurons during behavior are unknown. We show that behavioral and network states differentiate the activities of bistratified and O-LM cells in freely moving rats. Bistratified cells fire at higher rates during sleep than O-LM cells and, unlike O-LM cells, strongly increase spiking during sharp wave-associated ripples (SWRs). In contrast, O-LM interneurons decrease firing during sleep relative to awake states and are mostly inhibited during SWRs. During movement, both cell types fire cooperatively at the troughs of theta oscillations but with different frequencies. Somatostatin and GABA are differentially released to distinct dendritic zones of CA1 pyramidal cells during sleep and wakefulness to coordinate segregated glutamatergic inputs from entorhinal cortex and CA3.

DAG-sensitive and Ca(2+) permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation.

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.

Temporal dynamics of parvalbumin-expressing axo-axonic and basket cells in the rat medial prefrontal cortex in vivo.

Axo-axonic interneurons, innervating exclusively axon initial segments, and parvalbumin-expressing basket interneurons, targeting somata, dendrites, and spines of pyramidal cells, have been proposed to control neuronal activity in prefrontal circuits. We recorded the spike-timing of identified neurons in the prelimbic cortex of anesthetized rats, and show that axo-axonic cells increase their firing during tail pinch-induced brain state-activation. In addition, axo-axonic cells differ from other GABAergic parvalbumin-expressing cells in their spike timing during DOWN- to UP-state transitions of slow oscillations and in their coupling to gamma and spindle oscillations. The distinct firing dynamics and synaptic targets of axo-axonic and other parvalbumin-expressing cells provide differential contributions to the temporal organization of prefrontal networks.

Behavior-dependent specialization of identified hippocampal interneurons.

A large variety of GABAergic interneurons control information processing in the hippocampal circuits governing the formation of neuronal representations. Whether distinct hippocampal interneuron types contribute differentially to information processing during behavior is not known. We employed a new technique for recording and labeling interneurons and pyramidal cells in drug-free, freely moving rats. Recorded parvalbumin-expressing basket interneurons innervated somata and proximal pyramidal cell dendrites, whereas nitric oxide synthase- and neuropeptide Y-expressing ivy cells provided synaptic and extrasynaptic dendritic modulation. Basket and ivy cells showed distinct spike-timing dynamics, firing at different rates and times during theta and ripple oscillations. Basket, but not ivy, cells changed their firing rates during movement, sleep and quiet wakefulness, suggesting that basket cells coordinate cell assemblies in a behavioral state-contingent manner, whereas persistently firing ivy cells might control network excitability and homeostasis. Different interneuron types provide GABA to specific subcellular domains at defined times and rates, thereby differentially controlling network activity during behavior.

Glutamatergic input from specific sources influences the nucleus accumbens-ventral pallidum information flow.

The nucleus accumbens (NAc) is positioned to integrate signals originating from limbic and cortical areas and to modulate reward-related motor output of various goal-directed behaviours. The major target of the NAc GABAergic output neurons is the ventral pallidum (VP). VP is part of the reward circuit and controls the ascending mesolimbic dopamine system, as well as the motor output structures and the brainstem. The excitatory inputs governing this system converge in the NAc from the prefrontal cortex (PFC), ventral hippocampus (vHC), midline and intralaminar thalamus (TH) and basolateral nucleus of the amygdala (BLA). It is unclear which if any of these afferents innervate the medium spiny neurons of the NAc, that project to the VP. To identify the source of glutamatergic afferents that innervate neurons projecting to the VP, a dual-labelling method was used: Phaseolus vulgaris leucoagglutinin for anterograde and EGFP-encoded adenovirus for retrograde tract-tracing. Within the NAc, anterogradely labelled BLA terminals formed asymmetric synapses on dendritic spines that belonged to medium spiny neurons retrogradely labelled from the VP. TH terminals also formed synapses on dendritic spines of NAc neurons projecting to the VP. However, dendrites and dendritic spines retrogradely labelled from VP received no direct synaptic contacts from afferents originating from mPFC and vHC in the present material, despite the large number of fibres labelled by the anterograde tracer injections. These findings represent the first experimental evidence for a selective glutamatergic innervation of NAc neurons projecting to the VP. The glutamatergic inputs of different origin (i.e. mPFC, vHC, BLA, TH) to the NAc might thus convey different types of reward-related information during goal-directed behaviour, and thereby contribute to the complex regulation of nucleus accumbens functions.

Fast synaptic subcortical control of hippocampal circuits.

Cortical information processing is under state-dependent control of subcortical neuromodulatory systems. Although this modulatory effect is thought to be mediated mainly by slow nonsynaptic metabotropic receptors, other mechanisms, such as direct synaptic transmission, are possible. Yet, it is currently unknown if any such form of subcortical control exists. Here, we present direct evidence of a strong, spatiotemporally precise excitatory input from an ascending neuromodulatory center. Selective stimulation of serotonergic median raphe neurons produced a rapid activation of hippocampal interneurons. At the network level, this subcortical drive was manifested as a pattern of effective disynaptic GABAergic inhibition that spread throughout the circuit. This form of subcortical network regulation should be incorporated into current concepts of normal and pathological cortical function.

GABAergic neurons of the medial septum lead the hippocampal network during theta activity.

Information processing in the hippocampus critically relies on its reciprocal interaction with the medial septum (MS). Synchronization of the septo-hippocampal system was demonstrated during both major hippocampal activity states, the regular theta rhythm and the large amplitude irregular activity. Previous experimental and modeling data suggest that the MS provides rhythmic drive to the hippocampus, and hippocampo-septal feedback synchronizes septal pacemaker units. However, this view has recently been questioned based on the possibility of intrahippocampal theta genesis. Previously, we identified putative pacemaker neurons expressing parvalbumin (PV) and/or the pacemaker hyperpolarization-activated and cyclic nucleotide-gated nonselective cation channel (HCN) in the MS. In this study, by analyzing the temporal relationship of activity between the PV/HCN-containing medial septal neurons and hippocampal local field potential, we aimed to uncover whether the sequence of events during theta formation supports the classic view of septal drive or the challenging theory of hippocampal pacing of theta. Importantly, by implementing a circular statistical method, a temporal lead of these septal neurons over the hippocampus was observed on the course of theta synchronization. Moreover, the activity of putative hippocampal interneurons also preceded hippocampal local field theta, but by a shorter time period compared with PV/HCN-containing septal neurons. Using the concept of mutual information, the action potential series of PV/HCN-containing neurons shared higher amount of information with hippocampal field oscillation than PV/HCN-immunonegative cells. Thus, a pacemaker neuron population of the MS leads hippocampal activity, presumably via the synchronization of hippocampal interneurons.

The presence of pacemaker HCN channels identifies theta rhythmic GABAergic neurons in the medial septum.

The medial septum (MS) is an indispensable component of the subcortical network which synchronizes the hippocampus at theta frequency during specific stages of information processing. GABAergic neurons exhibiting highly regular firing coupled to the hippocampal theta rhythm are thought to form the core of the MS rhythm-generating network. In recent studies the hyperpolarization-activated, cyclic nucleotide-gated non-selective cation (HCN) channel was shown to participate in theta synchronization of the medial septum. Here, we tested the hypothesis that HCN channel expression correlates with theta modulated firing behaviour of MS neurons by a combined anatomical and electrophysiological approach. HCN-expressing neurons represented a subpopulation of GABAergic cells in the MS partly overlapping with parvalbumin (PV)-containing neurons. Rhythmic firing in the theta frequency range was characteristic of all HCN-expressing neurons. In contrast, only a minority of HCN-negative cells displayed theta related activity. All HCN cells had tight phase coupling to hippocampal theta waves. As a group, PV-expressing HCN neurons had a marked bimodal phase distribution, whereas PV-immunonegative HCN neurons did not show group-level phase preference despite significant individual phase coupling. Microiontophoretic blockade of HCN channels resulted in the reduction of discharge frequency, but theta rhythmic firing was perturbed only in a few cases. Our data imply that HCN-expressing GABAergic neurons provide rhythmic drive in all phases of the hippocampal theta activity. In most MS theta cells rhythm genesis is apparently determined by interactions at the level of the network rather than by the pacemaking property of HCN channels alone.

Phase segregation of medial septal GABAergic neurons during hippocampal theta activity.

Septo-hippocampal GABAergic neurons immunoreactive for parvalbumin are thought to play a crucial role in the generation of hippocampal theta oscillations associated with a specific stage of memory formation. Here we use in vivo juxtacellular recording and filling in the medial septum followed by immunocytochemical identification of the recorded cells containing parvalbumin to determine their firing pattern, phase relationship with hippocampal theta, morphology, and to thereby reveal their involvement in the generation of hippocampal theta activity. We have demonstrated that GABAergic medial septal neurons form two distinct populations exhibiting highly regular bursting activity that is tightly coupled to either the trough (178 degrees ) or the peak (330 degrees ) of hippocampal theta waves. Additionally, different types of bursting as well as nonbursting activity patterns were also observed. The morphological reconstruction of theta-bursting neurons revealed extensive axon arbors of these cells with numerous local collaterals establishing symmetrical synapses; thus, synchrony among the septal pacemaker units may be brought about by their recurrent collateral interactions. Long projecting axons could also be found running dorsally toward the hippocampus and ventrally in the direction of basal forebrain regions. We conclude that GABAergic neurons in the medial septum, which are known to selectively innervate hippocampal interneurons, are in a position to induce rhythmic disinhibition in the hippocampus and other theta-related subcortical areas at two different phases of hippocampal theta.