Theta-phase-specific modulation of dentate gyrus memory neurons.

The theta rhythm, a quasi-periodic 4-10 Hz oscillation, is observed during memory processing in the hippocampus, with different phases of theta hypothesized to separate independent streams of information related to the encoding and recall of memories. At the cellular level, the discovery of hippocampal memory cells (engram neurons), as well as the modulation of memory recall through optogenetic activation of these cells, has provided evidence that certain memories are stored, in part, in a sparse ensemble of neurons in the hippocampus. In previous research, however, engram reactivation has been carried out using open-loop stimulation at fixed frequencies; the relationship between engram neuron reactivation and ongoing network oscillations has not been taken into consideration. To address this concern, we implemented a closed-loop reactivation of engram neurons that enabled phase-specific stimulation relative to theta oscillations in the local field potential in CA1. Using this real-time approach, we tested the impact of activating dentate gyrus engram neurons during the peak (encoding phase) and trough (recall phase) of theta oscillations. Consistent with previously hypothesized functions of theta oscillations in memory function, we show that stimulating dentate gyrus engram neurons at the trough of theta is more effective in eliciting behavioral recall than either fixed-frequency stimulation or stimulation at the peak of theta. Moreover, phase-specific trough stimulation is accompanied by an increase in the coupling between gamma and theta oscillations in CA1 hippocampus. Our results provide a causal link between phase-specific activation of engram cells and the behavioral expression of memory.

Interneuronal network model of theta-nested fast oscillations predicts differential effects of heterogeneity, gap junctions and short term depression for hyperpolarizing versus shunting inhibition.

Theta and gamma oscillations in the hippocampus have been hypothesized to play a role in the encoding and retrieval of memories. Recently, it was shown that an intrinsic fast gamma mechanism in medial entorhinal cortex can be recruited by optogenetic stimulation at theta frequencies, which can persist with fast excitatory synaptic transmission blocked, suggesting a contribution of interneuronal network gamma (ING). We calibrated the passive and active properties of a 100-neuron model network to capture the range of passive properties and frequency/current relationships of experimentally recorded PV+ neurons in the medial entorhinal cortex (mEC). The strength and probabilities of chemical and electrical synapses were also calibrated using paired recordings, as were the kinetics and short-term depression (STD) of the chemical synapses. Gap junctions that contribute a noticeable fraction of the input resistance were required for synchrony with hyperpolarizing inhibition; these networks exhibited theta-nested high frequency oscillations similar to the putative ING observed experimentally in the optogenetically-driven PV-ChR2 mice. With STD included in the model, the network desynchronized at frequencies above ~200 Hz, so for sufficiently strong drive, fast oscillations were only observed before the peak of the theta. Because hyperpolarizing synapses provide a synchronizing drive that contributes to robustness in the presence of heterogeneity, synchronization decreases as the hyperpolarizing inhibition becomes weaker. In contrast, networks with shunting inhibition required non-physiological levels of gap junctions to synchronize using conduction delays within the measured range.

Hippocampal cells segregate positive and negative engrams.

The hippocampus is involved in processing a variety of mnemonic computations specifically the spatiotemporal components and emotional dimensions of contextual memory. Recent studies have demonstrated cellular heterogeneity along the hippocampal axis. The ventral hippocampus has been shown to be important in the processing of emotion and valence. Here, we combine transgenic and all-virus based activity-dependent tagging strategies to visualize multiple valence-specific engrams in the vHPC and demonstrate two partially segregated cell populations and projections that respond to appetitive and aversive experiences. Next, using RNA sequencing and DNA methylation sequencing approaches, we find that vHPC appetitive and aversive engram cells display different transcriptional programs and DNA methylation landscapes compared to a neutral engram population. Additionally, optogenetic manipulation of tagged cell bodies in vHPC is not sufficient to drive appetitive or aversive behavior in real-time place preference, stimulation of tagged vHPC terminals projecting to the amygdala and nucleus accumbens (NAc), but not the prefrontal cortex (PFC), showed the capacity drive preference and avoidance. These terminals also were able to change their capacity to drive behavior. We conclude that the vHPC contains genetically, cellularly, and behaviorally segregated populations of cells processing appetitive and aversive memory engrams.

Kinetics and Connectivity Properties of Parvalbumin- and Somatostatin-Positive Inhibition in Layer 2/3 Medial Entorhinal Cortex.

Parvalbumin-positive (Pvalb+) and somatostatin-positive (Sst+) cells are the two largest subgroups of inhibitory interneurons. Studies in visual cortex indicate that synaptic connections between Pvalb+ cells are common while connections between Sst+ interneurons have not been observed. The inhibitory connectivity and kinetics of these two interneuron subpopulations, however, have not been characterized in medial entorhinal cortex (mEC). Using fluorescence-guided paired recordings in mouse brain slices from interneurons and excitatory cells in layer 2/3 mEC, we found that, unlike neocortical measures, Sst+ cells inhibit each other, albeit with a lower probability than Pvalb+ cells (18% vs 36% for unidirectional connections). Gap junction connections were also more frequent between Pvalb+ cells than between Sst+ cells. Pvalb+ cells inhibited each other with larger conductances, smaller decay time constants, and shorter delays. Similarly, synaptic connections between Pvalb+ and excitatory cells were more likely and expressed faster decay times and shorter delays than those between Sst+ and excitatory cells. Inhibitory cells exhibited smaller synaptic decay time constants between interneurons than on their excitatory targets. Inhibition between interneurons also depressed faster, and to a greater extent. Finally, inhibition onto layer 2 pyramidal and stellate cells originating from Pvalb+ interneurons were very similar, with no significant differences in connection likelihood, inhibitory amplitude, and decay time. A model of short-term depression fitted to the data indicates that recovery time constants for refilling the available pool are in the range of 50-150 ms and that the fraction of the available pool released on each spike is in the range 0.2-0.5.

Modeling temperature- and Cav3 subtype-dependent alterations in T-type calcium channel mediated burst firing.

T-type calcium channels are important regulators of neuronal excitability. The mammalian brain expresses three T-type channel isoforms (Cav3.1, Cav3.2 and Cav3.3) with distinct biophysical properties that are critically regulated by temperature. Here, we test the effects of how temperature affects spike output in a reduced firing neuron model expressing specific Cav3 channel isoforms. The modeling data revealed only a minimal effect on baseline spontaneous firing near rest, but a dramatic increase in rebound burst discharge frequency for Cav3.1 compared to Cav3.2 or Cav3.3 due to differences in window current or activation/recovery time constants. The reduced response by Cav3.2 could optimize its activity where it is expressed in peripheral tissues more subject to temperature variations than Cav3.1 or Cav3.3 channels expressed prominently in the brain. These tests thus reveal that aspects of neuronal firing behavior are critically dependent on both temperature and T-type calcium channel subtype.

Non-genetic photoacoustic stimulation of single neurons by a tapered fiber optoacoustic emitter.

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments. Here, we developed a tapered fiber optoacoustic emitter (TFOE) generating an ultrasound field with a high spatial precision of 39.6 µm, enabling optoacoustic activation of single neurons or subcellular structures, such as axons and dendrites. Temporally, a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation. The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons. Direct measurements of the electrical response of single neurons to acoustic stimulation, which is difficult for conventional ultrasound stimulation, have been demonstrated. By coupling TFOE with ex vivo brain slice electrophysiology, we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation. These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology, which could shed new insights into the mechanism of ultrasound neurostimulation.

CaMKIIα-Positive Interneurons Identified via a microRNA-Based Viral Gene Targeting Strategy.

Single-cell analysis is revealing increasing diversity in gene expression profiles among brain cells. Traditional promotor-based viral gene expression techniques, however, cannot capture the growing variety among single cells. We demonstrate a novel viral gene expression strategy to target cells with specific miRNA expression using miRNA-guided neuron tags (mAGNET). We designed mAGNET viral vectors containing a CaMKIIα promoter and microRNA-128 (miR-128) binding sites, and labeled CaMKIIα+ cells with naturally low expression of miR-128 (Lm128C cells) in male and female mice. Although CaMKIIα has traditionally been considered as an excitatory neuron marker, our single-cell sequencing results reveal that Lm128C cells are CaMKIIα+ inhibitory neurons of parvalbumin or somatostatin subtypes. Further evaluation of the physiological properties of Lm128C cell in brain slices showed that Lm128C cells exhibit elevated membrane excitability, with biophysical properties closely resembling those of fast-spiking interneurons, consistent with previous transcriptomic findings of miR-128 in regulating gene networks that govern membrane excitability. To further demonstrate the utility of this new viral expression strategy, we expressed GCaMP6f in Lm128C cells in the superficial layers of the motor cortex and performed in vivo calcium imaging in mice during locomotion. We found that Lm128C cells exhibit elevated calcium event rates and greater intrapopulation correlation than the overall CaMKIIα+ cells during movement. In summary, the miRNA-based viral gene targeting strategy described here allows us to label a sparse population of CaMKIIα+ interneurons for functional studies, providing new capabilities to investigate the relationship between gene expression and physiological properties in the brain.SIGNIFICANCE STATEMENT We report the discovery of a class of CaMKIIα+ cortical interneurons, labeled via a novel miRNA-based viral gene targeting strategy, combinatorial to traditional promoter-based strategies. The fact that we found a small, yet distinct, population of cortical inhibitory neurons that express CaMKIIα demonstrates that CaMKIIα is not as specific for excitatory neurons as commonly believed. As single-cell sequencing tools are providing increasing insights into the gene expression diversity of neurons, including miRNA profile data, we expect that the miRNA-based gene targeting strategy presented here can help delineate many neuron populations whose physiological properties can be readily related to the miRNA gene regulatory networks.

Balanced synaptic currents underlie low-frequency oscillations in the subiculum.

Numerous synaptic and intrinsic membrane mechanisms have been proposed for generating oscillatory activity in the hippocampus. Few studies, however, have directly measured synaptic conductances and membrane properties during oscillations. The time course and relative contribution of excitatory and inhibitory synaptic conductances, as well as the role of intrinsic membrane properties in amplifying synaptic inputs, remains unclear. To address this issue, we used an isolated whole hippocampal preparation that generates autonomous low-frequency oscillations near the theta range. Using 2-photon microscopy and expression of genetically encoded fluorophores, we obtained on-cell and whole-cell patch recordings of pyramidal cells and fast-firing interneurons in the distal subiculum. Pyramidal cell and interneuron spiking shared similar phase-locking to local field potential oscillations. In pyramidal cells, spiking resulted from a concomitant and balanced increase in excitatory and inhibitory synaptic currents. In contrast, interneuron spiking was driven almost exclusively by excitatory synaptic current. Thus, similar to tightly balanced networks underlying hippocampal gamma oscillations and ripples, balanced synaptic inputs in the whole hippocampal preparation drive highly phase-locked spiking at the peak of slower network oscillations.

Voltage-Dependent Membrane Properties Shape the Size But Not the Frequency Content of Spontaneous Voltage Fluctuations in Layer 2/3 Somatosensory Cortex.

Under awake and idling conditions, spontaneous intracellular membrane voltage is characterized by large, synchronous, low-frequency fluctuations. Although these properties reflect correlations in synaptic inputs, intrinsic membrane properties often indicate voltage-dependent changes in membrane resistance and time constant values that can amplify and help to generate low-frequency voltage fluctuations. The specific contribution of intrinsic and synaptic factors to the generation of spontaneous fluctuations, however, remains poorly understood. Using visually guided intracellular recordings of somatosensory layer 2/3 pyramidal cells and interneurons in awake male and female mice, we measured the spectrum and size of voltage fluctuation and intrinsic cellular properties at different voltages. In both cell types, depolarizing neurons increased the size of voltage fluctuations. Amplitude changes scaled with voltage-dependent changes in membrane input resistance. Because of the small membrane time constants observed in both pyramidal cells and interneuron cell bodies, the low-frequency content of membrane fluctuations reflects correlations in the synaptic current inputs rather than significant filtering associated with membrane capacitance. Further, blocking synaptic inputs minimally altered somatic membrane resistance and time constant values. Overall, these results indicate that spontaneous synaptic inputs generate a low-conductance state in which the amplitude, but not frequency structure, is influenced by intrinsic membrane properties.SIGNIFICANCE STATEMENT In the absence of sensory drive, cortical activity in awake animals is associated with self-generated and seemingly random membrane voltage fluctuations characterized by large amplitude and low frequency. Partially, these properties reflect correlations in synaptic input. Nonetheless, neurons express voltage-dependent intrinsic properties that can potentially influence the amplitude and frequency of spontaneous activity. Using visually guided intracellular recordings of cortical neurons in awake mice, we measured the voltage dependence of spontaneous voltage fluctuations and intrinsic membrane properties. We show that voltage-dependent changes in membrane resistance amplify synaptic activity, whereas the frequency of voltage fluctuations reflects correlations in synaptic inputs. Last, synaptic activity has a small impact on intrinsic membrane properties in both pyramidal cells and interneurons.

A model of cholinergic suppression of hippocampal ripples through disruption of balanced excitation/inhibition.

Sharp wave-ripples (140-220 Hz) are patterns of brain activity observed in the local field potential of the hippocampus which are present during memory consolidation. As rodents switch from memory consolidation to memory encoding behaviors, cholinergic inputs to the hippocampus from neurons in the medial septum-diagonal band of Broca cause a marked reduction in ripple incidence. The mechanism for this disruption in ripple power is not fully understood. In isolated neurons, the major effect of cholinergic input on hippocampal neurons is depolarization of the membrane potential, which affects both hippocampal pyramidal neurons and inhibitory interneurons. Using an existing model of ripple-frequency oscillations that includes both pyramidal neurons and interneurons, we investigated the mechanism whereby depolarizing inputs to these neurons can affect ripple power and frequency. We observed that ripple power and frequency are maintained, as long as inputs to pyramidal neurons and interneurons are balanced. Preferential drive to pyramidal neurons or interneurons, however, affects ripple power and can disrupt ripple oscillations by pushing ripple frequency higher or lower. Thus, an imbalance in drive to pyramidal neurons and interneurons provides a means whereby cholinergic input can suppress hippocampal ripples.

Differences in the Electrophysiological Properties of Mouse Somatosensory Layer 2/3 Neurons In Vivo and Slice Stem from Intrinsic Sources Rather than a Network-Generated High Conductance State.

Synaptic activity in vivo can potentially alter the integration properties of neurons. Using recordings in awake mice, we targeted somatosensory layer 2/3 pyramidal neurons and compared neuronal properties with those from slices. Pyramidal cells in vivo had lower resistance and gain values, as well as broader spikes and increased spike frequency adaptation compared to the same cells in slices. Increasing conductance in neurons using dynamic clamp to levels observed in vivo, however, did not lessen the differences between in vivo and slice conditions. Further, local application of tetrodotoxin (TTX) in vivo blocked synaptic-mediated membrane voltage fluctuations but had little impact on pyramidal cell membrane input resistance and time constant values. Differences in electrophysiological properties of layer 2/3 neurons in mouse somatosensory cortex, therefore, stem from intrinsic sources separate from synaptic-mediated membrane voltage fluctuations.

Gain Modulation of Cholinergic Neurons in the Medial Septum-Diagonal Band of Broca Through Hyperpolarization.

Hippocampal network oscillations are important for learning and memory. Theta rhythms are involved in attention, navigation, and memory encoding, whereas sharp wave-ripple complexes are involved in memory consolidation. Cholinergic neurons in the medial septum-diagonal band of Broca (MS-DB) influence both types of hippocampal oscillations, promoting theta rhythms and suppressing sharp wave-ripples. They also receive frequency-dependent hyperpolarizing feedback from hippocamposeptal connections, potentially affecting their role as neuromodulators in the septohippocampal circuit. However, little is known about how the integration properties of cholinergic MS-DB neurons change with hyperpolarization. By potentially altering firing behavior in cholinergic neurons, hyperpolarizing feedback from the hippocampal neurons may, in turn, change hippocampal network activity. To study changes in membrane integration properties in cholinergic neurons in response to hyperpolarizing inputs, we used whole-cell patch-clamp recordings targeting genetically labeled, choline acetyltransferase-positive neurons in mouse brain slices. Hyperpolarization of cholinergic MS-DB neurons resulted in a long-lasting decrease in spike firing rate and input-output gain. Additionally, voltage-clamp measures implicated a slowly inactivating, 4-AP-insensitive, outward K+ conductance. Using a conductance-based model of cholinergic MS-DB neurons, we show that the ability of this conductance to modulate firing rate and gain depends on the expression of an experimentally verified shallow intrinsic spike frequency-voltage relationship. Together, these findings point to a means through which negative feedback from hippocampal neurons can influence the role of cholinergic MS-DB neurons. © 2016 Wiley Periodicals, Inc.

Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations.

The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na+ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances.

Imaging activity in neurons and glia with a Polr2a-based and cre-dependent GCaMP5G-IRES-tdTomato reporter mouse.

New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.

Entorhinal stellate cells show preferred spike phase-locking to theta inputs that is enhanced by correlations in synaptic activity.

In active networks, excitatory and inhibitory synaptic inputs generate membrane voltage fluctuations that drive spike activity in a probabilistic manner. Despite this, some cells in vivo show a strong propensity to precisely lock to the local field potential and maintain a specific spike-phase relationship relative to other cells. In recordings from rat medial entorhinal cortical stellate cells, we measured spike phase-locking in response to sinusoidal "test" inputs in the presence of different forms of background membrane voltage fluctuations, generated via dynamic clamp. We find that stellate cells show strong and robust spike phase-locking to theta (4-12 Hz) inputs. This response occurs under a wide variety of background membrane voltage fluctuation conditions that include a substantial increase in overall membrane conductance. Furthermore, the IH current present in stellate cells is critical to the enhanced spike phase-locking response at theta. Finally, we show that correlations between inhibitory and excitatory conductance fluctuations, which can arise through feedback and feedforward inhibition, can substantially enhance the spike phase-locking response. The enhancement in locking is a result of a selective reduction in the size of low-frequency membrane voltage fluctuations due to cancellation of inhibitory and excitatory current fluctuations with correlations. Hence, our results demonstrate that stellate cells have a strong preference for spike phase-locking to theta band inputs and that the absolute magnitude of locking to theta can be modulated by the properties of background membrane voltage fluctuations.

Bistability in Purkinje neurons: ups and downs in cerebellar research.

The output of cerebellar Purkinje cells has been characterized extensively and theories regarding the role of simple spike (SS) and complex spike (CS) patterns have evolved through many different studies. A bistable pattern of SS output can be observed in vitro; however, differing views exist regarding the occurrence of bistable SS output in vivo. Bistability in Purkinje cell output is characterized by abrupt transitions between tonic firing and quiescence, usually evoked by synaptic inputs to the neuron. This is in contrast to the trimodal pattern of activity which has been found in vitro and in vivo when climbing fiber input to Purkinje cells is removed. The mechanisms underlying bistable membrane properties in Purkinje cells have been determined through in vitro studies and computational analysis. In vitro studies have further established that Purkinje cells possess the ability to toggle between firing states, but in vivo studies in both awake and anesthetized animals have found conflicting results as to the presence of toggling in the intact circuit. Here, we provide an overview of the current state of research on bistability, examining the mechanisms underlying bistability and current findings from in vivo studies. We also suggest possible reasons for discrepancies between in vivo studies and propose future studies which would aid in clarifying the role of bistability in the cerebellar circuit.

Spike phase locking in CA1 pyramidal neurons depends on background conductance and firing rate.

Oscillatory activity in neuronal networks correlates with different behavioral states throughout the nervous system, and the frequency-response characteristics of individual neurons are believed to be critical for network oscillations. Recent in vivo studies suggest that neurons experience periods of high membrane conductance, and that action potentials are often driven by membrane potential fluctuations in the living animal. To investigate the frequency-response characteristics of CA1 pyramidal neurons in the presence of high conductance and voltage fluctuations, we performed dynamic-clamp experiments in rat hippocampal brain slices. We drove neurons with noisy stimuli that included a sinusoidal component ranging, in different trials, from 0.1 to 500 Hz. In subsequent data analysis, we determined action potential phase-locking profiles with respect to background conductance, average firing rate, and frequency of the sinusoidal component. We found that background conductance and firing rate qualitatively change the phase-locking profiles of CA1 pyramidal neurons versus frequency. In particular, higher average spiking rates promoted bandpass profiles, and the high-conductance state promoted phase-locking at frequencies well above what would be predicted from changes in the membrane time constant. Mechanistically, spike rate adaptation and frequency resonance in the spike-generating mechanism are implicated in shaping the different phase-locking profiles. Our results demonstrate that CA1 pyramidal cells can actively change their synchronization properties in response to global changes in activity associated with different behavioral states.

Spike resonance properties in hippocampal O-LM cells are dependent on refractory dynamics.

During a wide variety of behaviors, hippocampal field potentials show significant power in the theta (4-12 Hz) frequency range and individual neurons commonly phase-lock with the 4-12 Hz field potential. The underlying cellular and network mechanisms that generate the theta rhythm, however, are poorly understood. Oriens-lacunosum moleculare (O-LM) interneurons have been implicated as crucial contributors to generating theta in local hippocampal circuits because of their unique axonal projections, slow synaptic kinetics and the fact that spikes are phase-locked to theta field potentials in vivo. We performed experiments in brain slice preparations from Long-Evans rats to investigate the ability of O-LM cells to generate phase-locked spike output in response to artificial synaptic inputs. We find that O-LM cells do not respond with any preference in spike output at theta frequencies when injected with broadband artificial synaptic inputs. However, when presented with frequency-modulated inputs, O-LM spike output shows the ability to phase-lock well to theta-modulated inputs, despite their strong low-pass profiles of subthreshold membrane impedance. This result was dependent on spike refractory dynamics and could be controlled by real-time manipulation of the postspike afterhyperpolarization. Finally, we show that the ability of O-LM cells to phase-lock well to theta-rich inputs is independent of the h-current, a membrane mechanism often implicated in the generation of theta frequency activity.

Short conduction delays cause inhibition rather than excitation to favor synchrony in hybrid neuronal networks of the entorhinal cortex.

How stable synchrony in neuronal networks is sustained in the presence of conduction delays is an open question. The Dynamic Clamp was used to measure phase resetting curves (PRCs) for entorhinal cortical cells, and then to construct networks of two such neurons. PRCs were in general Type I (all advances or all delays) or weakly type II with a small region at early phases with the opposite type of resetting. We used previously developed theoretical methods based on PRCs under the assumption of pulsatile coupling to predict the delays that synchronize these hybrid circuits. For excitatory coupling, synchrony was predicted and observed only with no delay and for delays greater than half a network period that cause each neuron to receive an input late in its firing cycle and almost immediately fire an action potential. Synchronization for these long delays was surprisingly tight and robust to the noise and heterogeneity inherent in a biological system. In contrast to excitatory coupling, inhibitory coupling led to antiphase for no delay, very short delays and delays close to a network period, but to near-synchrony for a wide range of relatively short delays. PRC-based methods show that conduction delays can stabilize synchrony in several ways, including neutralizing a discontinuity introduced by strong inhibition, favoring synchrony in the case of noisy bistability, and avoiding an initial destabilizing region of a weakly type II PRC. PRCs can identify optimal conduction delays favoring synchronization at a given frequency, and also predict robustness to noise and heterogeneity.