Activity-Dependent Ectopic Spiking in Parvalbumin-Expressing Interneurons of the Neocortex.

Canonically, action potentials of most mammalian neurons initiate at the axon initial segment (AIS) and propagate bidirectionally: orthodromically along the distal axon and retrogradely into the soma and dendrites. Under some circumstances, action potentials may initiate ectopically, at sites distal to the AIS, and propagate antidromically along the axon. These "ectopic action potentials" (EAPs) have been observed in experimental models of seizures and chronic pain, and more rarely in nonpathological forebrain neurons. Here we report that a large majority of parvalbumin-expressing (PV+) interneurons in the upper layers of mouse neocortex, from both orbitofrontal and primary somatosensory areas, fire EAPs after sufficient activation of their somata. Somatostatin-expressing interneurons also fire EAPs, though less robustly. Ectopic firing in PV+ cells occurs in varying temporal patterns and can persist for several seconds. PV+ cells evoke strong synaptic inhibition in pyramidal neurons and interneurons and play critical roles in cortical function. Our results suggest that ectopic spiking of PV+ interneurons is common and may contribute to both normal and pathological network functions of the neocortex.

Activity-dependent ectopic action potentials in regular-spiking neurons of the neocortex.

Introduction: Action potentials usually travel orthodromically along a neuron's axon, from the axon initial segment (AIS) toward the presynaptic terminals. Under some circumstances action potentials also travel in the opposite direction, antidromically, after being initiated at a distal location. Given their initiation at an atypical site, we refer to these events as "ectopic action potentials." Ectopic action potentials (EAPs) were initially observed in pathological conditions including seizures and nerve injury. Several studies have described regular-spiking (RS) pyramidal neurons firing EAPs in seizure models. Under nonpathological conditions, EAPs were reported in a few populations of neurons, and our group has found that EAPs can be induced in a large proportion of parvalbumin-expressing interneurons in the neocortex. Nevertheless, to our knowledge there have been no prior reports of ectopic firing in the largest population of neurons in the neocortex, pyramidal neurons, under nonpathological conditions. Methods: We performed in vitro recordings utilizing the whole-cell patch clamp technique. To elicit EAPs, we triggered orthodromic action potentialswith either long, progressively increasing current steps, or with trains of brief pulses at 30, 60, or 100 Hz delivered in 3 different ways, varying in stimulus and resting period duration. Results: We found that a large proportion (72.7%) of neocortical RS cells from mice can fire EAPs after a specific stimulus in vitro, and that most RS cells (56.1%) are capable of firing EAPs across a broad range of stimulus conditions. Of the 37 RS neurons in which we were able to elicit EAPs, it took an average of 863.8 orthodromic action potentials delivered over the course of an average of ~81.4 s before the first EAP was seen. We observed that some cells responded to specific stimulus frequencies while less selective, suggesting frequency tuning in a subset of the cells. Discussion: Our findings suggest that pyramidal cells can integrate information over long time-scales before briefly entering a mode of self-generated firing that originates in distal axons. The surprising ubiquity of EAP generation in RS cells raises interesting questions about the potential roles of ectopic spiking in information processing, cortical oscillations, and seizure susceptibility.

Publisher Correction: Two dynamically distinct circuits drive inhibition in the sensory thalamus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Two dynamically distinct circuits drive inhibition in the sensory thalamus.

Most sensory information destined for the neocortex is relayed through the thalamus, where considerable transformation occurs1,2. One means of transformation involves interactions between excitatory thalamocortical neurons that carry data to the cortex and inhibitory neurons of the thalamic reticular nucleus (TRN) that regulate the flow of those data3-6. Although the importance of the TRN has long been recognised7-9, understanding of its cell types, their organization and their functional properties has lagged behind that of the thalamocortical systems they control. Here we address this by investigating the somatosensory and visual circuits of the TRN in mice. In the somatosensory TRN we observed two groups of genetically defined neurons that are topographically segregated and physiologically distinct, and that connect reciprocally with independent thalamocortical nuclei through dynamically divergent synapses. Calbindin-expressing cells-located in the central core-connect with the ventral posterior nucleus, the primary somatosensory thalamocortical relay. By contrast, somatostatin-expressing cells-which reside along the surrounding edges of the TRN-synapse with the posterior medial thalamic nucleus, a higher-order structure that carries both top-down and bottom-up information10-12. The two TRN cell groups process their inputs in pathway-specific ways. Synapses from the ventral posterior nucleus to central TRN cells transmit rapid excitatory currents that depress deeply during repetitive activity, driving phasic spike output. Synapses from the posterior medial thalamic nucleus to edge TRN cells evoke slower, less depressing excitatory currents that drive more persistent spiking. Differences in the intrinsic physiology of TRN cell types, including state-dependent bursting, contribute to these output dynamics. The processing specializations of these two somatosensory TRN subcircuits therefore appear to be tuned to the signals they carry-a primary central subcircuit tuned to discrete sensory events, and a higher-order edge subcircuit tuned to temporally distributed signals integrated from multiple sources. The structure and function of visual TRN subcircuits closely resemble those of the somatosensory TRN. These results provide insights into how subnetworks of TRN neurons may differentially process distinct classes of thalamic information.

Nitric Oxide-Mediated Plasticity of Interconnections Between T-Stellate cells of the Ventral Cochlear Nucleus Generate Positive Feedback and Constitute a Central Gain Control in the Auditory System.

T-stellate cells in the ventral cochlear nucleus (VCN) form an ascending pathway that conveys spectral information from the cochlea to brainstem nuclei, the inferior colliculi, and the thalamus. The tonotopic array of T-stellate cells enhances the encoding of spectral peaks relative to their auditory nerve fiber inputs. The alignment of local collaterals and T-stellate cell dendrites within the isofrequency lamina suggests that the cells make connections within the isofrequency lamina in which they reside. Recordings from pairs of T-stellate cells in mice of both sexes revealed that firing in the presynaptic cell evoked responses in the postsynaptic cell when presynaptic firing was paired with depolarization of the postsynaptic cell. After such experimental coactivation, presynaptic firing evoked EPSCs of uniform amplitude whose frequency depended on the duration of depolarization and diminished over minutes. Nitric oxide (NO) donors evoked EPSCs in T-stellate cells but not in the other types of principal cells. Blockers of neuronal nitric oxide synthase (nNOS) and of NMDA receptors blocked potentiation, indicating that NO mediates potentiation. nNOS and its receptor, guanylate cyclase (NO-GC), are expressed in somata of T-stellate cells. Excitatory interconnections were bidirectional and polysynaptic, indicating that T-stellate cells connect in networks. Positive feedback provided by temporarily potentiated interconnections between T-stellate cells could enhance the gain of auditory nerve excitation in proportion to the excitation, generating a form of short-term central gain control that could account for the ability of T-stellate cells to enhance the encoding of spectral peaks.SIGNIFICANCE STATEMENT T-stellate cells are interconnected through synapses that have a previously undescribed form of temporary, nitric oxide-mediated plasticity. Coactivation of neighboring cells enhances the activation of an excitatory network that feeds back on itself by enhancing the probability of EPSCs. Although there remain gaps in our understanding of how the interconnections revealed in slices contribute to hearing, our findings have interesting implications. Positive feedback through a network of interconnections could account for how T-stellate cells are able to encode spectral peaks over a wider range of intensities than many of their auditory nerve inputs (Blackburn and Sachs, 1990; May et al., 1998). The magnitude of the gain may itself be plastic because neuronal nitric oxide synthase increases when animals have tinnitus (Coomber et al., 2015).

Early Life Stress Drives Sex-Selective Impairment in Reversal Learning by Affecting Parvalbumin Interneurons in Orbitofrontal Cortex of Mice.

Poverty, displacement, and parental stress represent potent sources of early life stress (ELS). Stress disproportionately affects females, who are at increased risk for stress-related pathologies associated with cognitive impairment. Mechanisms underlying stress-associated cognitive impairment and enhanced risk of females remain unknown. Here, ELS is associated with impaired rule-reversal (RR) learning in females, but not males. Impaired performance was associated with decreased expression and density of interneurons expressing parvalbumin (PV+) in orbitofrontal cortex (OFC), but not other interneuron subtypes. Optogenetic silencing of PV+ interneuron activity in OFC of control mice phenocopied RR learning deficits observed in ELS females. Localization of reversal learning deficits to PV+ interneurons in OFC was confirmed by optogenetic studies in which neurons in medial prefrontal cortex (mPFC) were silenced and associated with select deficits in rule-shift learning. Sex-, cell-, and region-specific effects show altered PV+ interneuron development can be a driver of sex differences in cognitive dysfunction.

Infrabarrels Are Layer 6 Circuit Modules in the Barrel Cortex that Link Long-Range Inputs and Outputs.

The rodent somatosensory cortex includes well-defined examples of cortical columns-the barrel columns-that extend throughout the cortical depth and are defined by discrete clusters of neurons in layer 4 (L4) called barrels. Using the cell-type-specific Ntsr1-Cre mouse line, we found that L6 contains infrabarrels, readily identifiable units that align with the L4 barrels. Corticothalamic (CT) neurons and their local axons cluster within the infrabarrels, whereas corticocortical (CC) neurons are densest between infrabarrels. Optogenetic experiments showed that CC cells received robust input from somatosensory thalamic nuclei, whereas CT cells received much weaker thalamic inputs. We also found that CT neurons are intrinsically less excitable, revealing that both synaptic and intrinsic mechanisms contribute to the low firing rates of CT neurons often reported in vivo. In summary, infrabarrels are discrete cortical circuit modules containing two partially separated excitatory networks that link long-distance thalamic inputs with specific outputs.

Synchrony and so much more: Diverse roles for electrical synapses in neural circuits.

Electrical synapses are neuronal gap junctions that are ubiquitous across brain regions and species. The biophysical properties of most electrical synapses are relatively simple-transcellular channels allow nearly ohmic, bidirectional flow of ionic current. Yet these connections can play remarkably diverse roles in different neural circuit contexts. Recent findings illustrate how electrical synapses may excite or inhibit, synchronize or desynchronize, augment or diminish rhythms, phase-shift, detect coincidences, enhance signals relative to noise, adapt, and interact with nonlinear membrane and transmitter-release mechanisms. Most of these functions are likely to be widespread in central nervous systems. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 610-624, 2017.

Spontaneous dynamics of neural networks in deep layers of prefrontal cortex.

Cortical systems maintain and process information through the sustained activation of recurrent local networks of neurons. Layer 5 is known to have a major role in generating the recurrent activation associated with these functions, but relatively little is known about its intrinsic dynamics at the mesoscopic level of large numbers of neighboring neurons. Using calcium imaging, we measured the spontaneous activity of networks of deep-layer medial prefrontal cortical neurons in an acute slice model. Inferring the simultaneous activity of tens of neighboring neurons, we found that while the majority showed only sporadic activity, a subset of neurons engaged in sustained delta frequency rhythmic activity. Spontaneous activity under baseline conditions was weakly correlated between pairs of neurons, and rhythmic neurons showed little coherence in their oscillations. However, we consistently observed brief bouts of highly synchronous activity that must be attributed to network activity. NMDA-mediated stimulation enhanced rhythmicity, synchrony, and correlation within these local networks. These results characterize spontaneous prefrontal activity at a previously unexplored spatiotemporal scale and suggest that medial prefrontal cortex can act as an intrinsic generator of delta oscillations.NEW & NOTEWORTHY Using calcium imaging and a novel analytic framework, we characterized the spontaneous and NMDA-evoked activity of layer 5 prefrontal cortex at a largely unexplored spatiotemporal scale. Our results suggest that the mPFC microcircuitry is capable of intrinsically generating delta oscillations and sustaining synchronized network activity that is potentially relevant for understanding its contribution to cognitive processes.

Comment on "Principles of connectivity among morphologically defined cell types in adult neocortex".

Jiang et al (Research Article, 27 November 2015, aac9462) describe detailed experiments that substantially add to the knowledge of cortical microcircuitry and are unique in the number of connections reported and the quality of interneuron reconstruction. The work appeals to experts and laypersons because of the notion that it unveils new principles and provides a complete description of cortical circuits. We provide a counterbalance to the authors' claims to give those less familiar with the minutiae of cortical circuits a better sense of the contributions and the limitations of this study.

Distinct Roles of SOM and VIP Interneurons during Cortical Up States.

During cortical network activity, recurrent synaptic excitation among pyramidal neurons is approximately balanced by synaptic inhibition, which is provided by a vast diversity of inhibitory interneurons. The relative contributions of different interneuron subtypes to inhibitory tone during cortical network activity is not well-understood. We previously showed that many of the major interneuron subtypes in mouse barrel cortex are highly active during Up states (Neske et al., 2015); while fast-spiking (FS), parvalbumin (PV)-positive cells were the most active interneuron subtype, many non-fast-spiking (NFS), PV-negative interneurons were as active or more active than neighboring pyramidal cells. This suggests that the NFS cells could play a role in maintaining or modulating Up states. Here, using optogenetic techniques, we further dissected the functional roles during Up states of two major NFS, PV-negative interneuron subtypes: somatostatin (SOM)-positive cells and vasoactive intestinal peptide (VIP)-positive cells. We found that while pyramidal cell excitability during Up states significantly increased when SOM cells were optogenetically silenced, VIP cells did not influence pyramidal cell excitability either upon optogenetic silencing or activation. VIP cells failed to contribute to Up states despite their ability to inhibit SOM cells strongly. We suggest that the contribution of VIP cells to the excitability of pyramidal cells may vary with cortical state.

Diverse Ensembles of Inhibitory Interneurons.

In this issue of Neuron, Karnani et al. (2016) show that ensembles of specific types of inhibitory interneurons generate coordinated activity in mouse visual cortex. They also describe chemical and electrical synaptic mechanisms that may enable diverse interneuron ensembles to function as distinct operational units.

Synchronized gamma-frequency inhibition in neocortex depends on excitatory-inhibitory interactions but not electrical synapses.

Synaptic inhibition plays a crucial role in the precise timing of spiking activity in the cerebral cortex. Synchronized, rhythmic inhibitory activity in the gamma (30-80 Hz) range is thought to be especially important for the active, information-processing neocortex, but the circuit mechanisms that give rise to synchronized inhibition are uncertain. In particular, the relative contributions of reciprocal inhibitory connections, excitatory-inhibitory interactions, and electrical synapses to precise spike synchrony among inhibitory interneurons are not well understood. Here we describe experiments on mouse barrel cortex in vitro as it spontaneously generates slow (<1 Hz) oscillations (Up and Down states). During Up states, inhibitory postsynaptic currents (IPSCs) are generated at gamma frequencies and are more synchronized than excitatory postsynaptic currents (EPSCs) among neighboring pyramidal cells. Furthermore, spikes in homotypic pairs of interneurons are more synchronized than in pairs of pyramidal cells. Comparing connexin36 knockout and wild-type animals, we found that electrical synapses make a minimal contribution to synchronized inhibition during Up states. Estimations of the delays between EPSCs and IPSCs in single pyramidal cells showed that excitation often preceded inhibition by a few milliseconds. Finally, tonic optogenetic activation of different interneuron subtypes in the absence of excitation led to only weak synchrony of IPSCs in pairs of pyramidal neurons. Our results suggest that phasic excitatory inputs are indispensable for synchronized spiking in inhibitory interneurons during Up states and that electrical synapses play a minimal role.

Electrical synapses and the development of inhibitory circuits in the thalamus.

KEY POINTS: The thalamus is a structure critical for information processing and transfer to the cortex. Thalamic reticular neurons are inhibitory cells interconnected by electrical synapses, most of which require the gap junction protein connexin36 (Cx36). We investigated whether electrical synapses play a role in the maturation of thalamic networks by studying neurons in mice with and without Cx36. When Cx36 was deleted, inhibitory synapses were more numerous, although both divergent inhibitory connectivity and dendritic complexity were reduced. Surprisingly, we observed non-Cx36-dependent electrical synapses with unusual biophysical properties interconnecting some reticular neurons in mice lacking Cx36. The results of the present study suggest an important role for Cx36-dependent electrical synapses in the development of thalamic circuits. ABSTRACT: Neurons within the mature thalamic reticular nucleus (TRN) powerfully inhibit ventrobasal (VB) thalamic relay neurons via GABAergic synapses. TRN neurons are also coupled to one another by electrical synapses that depend strongly on the gap junction protein connexin36 (Cx36). Electrical synapses in the TRN precede the postnatal development of TRN-to-VB inhibition. We investigated how the deletion of Cx36 affects the maturation of TRN and VB neurons, electrical coupling and GABAergic synapses by studying wild-type (WT) and Cx36 knockout (KO) mice. The incidence and strength of electrical coupling in TRN was sharply reduced, but not abolished, in KO mice. Surprisingly, electrical synapses between Cx36-KO neurons had faster voltage-dependent decay kinetics and conductance asymmetry (rectification) than did electrical synapses between WT neurons. The properties of TRN-mediated inhibition in VB also depended on the Cx36 genotype. Deletion of Cx36 increased the frequency and shifted the amplitude distributions of miniature IPSCs, whereas the paired-pulse ratio of evoked IPSCs was unaffected, suggesting that the absence of Cx36 led to an increase in GABAergic synaptic contacts. VB neurons from Cx36-KO mice also tended to have simpler dendritic trees and fewer divergent inputs from the TRN compared to WT cells. The findings obtained in the present study suggest that proper development of thalamic inhibitory circuitry, neuronal morphology, TRN cell function and electrical coupling requires Cx36. In the absence of Cx36, some TRN neurons express asymmetric electrical coupling mediated by other unidentified connexin subtypes.

Bidirectional Modulation of Recognition Memory.

Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects. For example, animals and humans with perirhinal damage are unable to distinguish familiar from novel objects in recognition memory tasks. In the normal brain, perirhinal neurons respond to novelty and familiarity by increasing or decreasing firing rates. Recent work also implicates oscillatory activity in the low-beta and low-gamma frequency bands in sensory detection, perception, and recognition. Using optogenetic methods in a spontaneous object exploration (SOR) task, we altered recognition memory performance in rats. In the SOR task, normal rats preferentially explore novel images over familiar ones. We modulated exploratory behavior in this task by optically stimulating channelrhodopsin-expressing perirhinal neurons at various frequencies while rats looked at novel or familiar 2D images. Stimulation at 30-40 Hz during looking caused rats to treat a familiar image as if it were novel by increasing time looking at the image. Stimulation at 30-40 Hz was not effective in increasing exploration of novel images. Stimulation at 10-15 Hz caused animals to treat a novel image as familiar by decreasing time looking at the image, but did not affect looking times for images that were already familiar. We conclude that optical stimulation of PER at different frequencies can alter visual recognition memory bidirectionally. Significance statement: Recognition of novelty and familiarity are important for learning, memory, and decision making. Perirhinal cortex (PER) has a well established role in the familiarity-based recognition of individual items and objects, but how novelty and familiarity are encoded and transmitted in the brain is not known. Perirhinal neurons respond to novelty and familiarity by changing firing rates, but recent work suggests that brain oscillations may also be important for recognition. In this study, we showed that stimulation of the PER could increase or decrease exploration of novel and familiar images depending on the frequency of stimulation. Our findings suggest that optical stimulation of PER at specific frequencies can predictably alter recognition memory.

A corticothalamic switch: controlling the thalamus with dynamic synapses.

Corticothalamic neurons provide massive input to the thalamus. This top-down projection may allow the cortex to regulate sensory processing by modulating the excitability of thalamic cells. Layer 6 corticothalamic neurons monosynaptically excite thalamocortical cells, but also indirectly inhibit them by driving inhibitory cells of the thalamic reticular nucleus. Whether corticothalamic activity generally suppresses or excites the thalamus remains unclear. Here we show that the corticothalamic influence is dynamic, with the excitatory-inhibitory balance shifting in an activity-dependent fashion. During low-frequency activity, corticothalamic effects are mainly suppressive, whereas higher-frequency activity (even a short bout of gamma frequency oscillations) converts the corticothalamic influence to enhancement. The mechanism of this switching depends on distinct forms of short-term synaptic plasticity across multiple corticothalamic circuit components. Our results reveal an activity-dependent mechanism by which corticothalamic neurons can bidirectionally switch the excitability and sensory throughput of the thalamus, possibly to meet changing behavioral demands.

Contributions of diverse excitatory and inhibitory neurons to recurrent network activity in cerebral cortex.

The recurrent synaptic architecture of neocortex allows for self-generated network activity. One form of such activity is the Up state, in which neurons transiently receive barrages of excitatory and inhibitory synaptic inputs that depolarize many neurons to spike threshold before returning to a relatively quiescent Down state. The extent to which different cell types participate in Up states is still unclear. Inhibitory interneurons have particularly diverse intrinsic properties and synaptic connections with the local network, suggesting that different interneurons might play different roles in activated network states. We have studied the firing, subthreshold behavior, and synaptic conductances of identified cell types during Up and Down states in layers 5 and 2/3 in mouse barrel cortex in vitro. We recorded from pyramidal cells and interneurons expressing parvalbumin (PV), somatostatin (SOM), vasoactive intestinal peptide (VIP), or neuropeptide Y. PV cells were the most active interneuron subtype during the Up state, yet the other subtypes also received substantial synaptic conductances and often generated spikes. In all cell types except PV cells, the beginning of the Up state was dominated by synaptic inhibition, which decreased thereafter; excitation was more persistent, suggesting that inhibition is not the dominant force in terminating Up states. Compared with barrel cortex, SOM and VIP cells were much less active in entorhinal cortex during Up states. Our results provide a measure of functional connectivity of various neuron types in barrel cortex and suggest differential roles for interneuron types in the generation and control of persistent network activity.

Two functionally distinct networks of gap junction-coupled inhibitory neurons in the thalamic reticular nucleus.

Gap junctions (GJs) electrically couple GABAergic neurons of the forebrain. The spatial organization of neuron clusters coupled by GJs is an important determinant of network function, yet it is poorly described for nearly all mammalian brain regions. Here we used a novel dye-coupling technique to show that GABAergic neurons in the thalamic reticular nucleus (TRN) of mice and rats form two types of GJ-coupled clusters with distinctive patterns and axonal projections. Most clusters are elongated narrowly along functional modules within the plane of the TRN, with axons that selectively inhibit local groups of relay neurons. However, some coupled clusters have neurons arrayed across the thickness of the TRN and target their axons to both first- and higher-order relay nuclei. Dye coupling was reduced, but not abolished, among cells of connexin36 knock-out mice. Our results suggest that GJs form two distinct types of inhibitory networks that correlate activity either within or across functional modules of the thalamus.