A brief history of somatostatin interneuron taxonomy or: how many somatostatin subtypes are there, really?

We provide a brief (and unabashedly biased) overview of the pre-transcriptomic history of somatostatin interneuron taxonomy, followed by a chronological summary of the large-scale, NIH-supported effort over the last ten years to generate a comprehensive, single-cell RNA-seq-based taxonomy of cortical neurons. Focusing on somatostatin interneurons, we present the perspective of experimental neuroscientists trying to incorporate the new classification schemes into their own research while struggling to keep up with the ever-increasing number of proposed cell types, which seems to double every two years. We suggest that for experimental analysis, the most useful taxonomic level is the subdivision of somatostatin interneurons into ten or so "supertypes," which closely agrees with their more traditional classification by morphological, electrophysiological and neurochemical features. We argue that finer subdivisions ("t-types" or "clusters"), based on slight variations in gene expression profiles but lacking clear phenotypic differences, are less useful to researchers and may actually defeat the purpose of classifying neurons to begin with. We end by stressing the need for generating novel tools (mouse lines, viral vectors) for genetically targeting distinct supertypes for expression of fluorescent reporters, calcium sensors and excitatory or inhibitory opsins, allowing neuroscientists to chart the input and output synaptic connections of each proposed subtype, reveal the position they occupy in the cortical network and examine experimentally their roles in sensorimotor behaviors and cognitive brain functions.

Genetically Defined Subtypes of Somatostatin-Containing Cortical Interneurons.

Inhibitory interneurons play a crucial role in proper development and function of the mammalian cerebral cortex. Of the different inhibitory subclasses, dendritic-targeting, somatostatin-containing (SOM) interneurons may be the most diverse. Earlier studies used GFP-expressing and recombinase-expressing mouse lines to characterize genetically defined subtypes of SOM interneurons by morphologic, electrophysiological, and neurochemical properties. More recently, large-scale studies classified SOM interneurons into 13 morpho-electric transcriptomic (MET) types. It remains unclear, however, how these various classification schemes relate to each other, and experimental access to MET types has been limited by the scarcity of specific mouse driver lines. To address these issues, we crossed Flp and Cre driver lines with a dual-color intersectional reporter, allowing experimental access to several combinatorially defined SOM subsets. Brains from adult mice of both sexes were retrogradely dye labeled from the pial surface to identify layer 1-projecting neurons and immunostained against several marker proteins, revealing correlations between genetic label, axonal target, and marker protein expression in the same neurons. Lastly, using whole-cell recordings ex vivo, we analyzed and compared electrophysiological properties between different intersectional subsets. We identified two layer 1-targeting subtypes with nonoverlapping marker protein expression and electrophysiological properties, which, together with a previously characterized layer 4-targeting subtype, account for >50% of all layer 5 SOM cells and >40% of all SOM cells, and appear to map onto 5 of the 13 MET types. Genetic access to these subtypes will allow researchers to determine their synaptic inputs and outputs and uncover their roles in cortical computations and animal behavior.

Ultrafast (400 Hz) network oscillations induced in mouse barrel cortex by optogenetic activation of thalamocortical axons.

Oscillations of extracellular voltage, reflecting synchronous, rhythmic activity in large populations of neurons, are a ubiquitous feature in the mammalian brain, and are thought to subserve important, if not fully understood roles in normal and abnormal brain function. Oscillations at different frequency bands are hallmarks of specific brain and behavioral states. At the higher end of the spectrum, 150-200 Hz ripples occur in the hippocampus during slow-wave sleep, and ultrafast (400-600 Hz) oscillations arise in the somatosensory cortices of humans and several other mammalian species in response to peripheral nerve stimulation or punctate sensory stimuli. Here we report that brief optogenetic activation of thalamocortical axons, in brain slices from mouse somatosensory (barrel) cortex, elicited in the thalamorecipient layer local field potential (LFP) oscillations which we dubbed "ripplets". Ripplets originated in the postsynaptic cortical network and consisted of a precisely repeating sequence of 2­5 negative transients, closely resembling hippocampal ripples but, at ~400 Hz, over twice as fast. Fast-spiking (FS) inhibitory interneurons fired highly synchronous 400 Hz spike bursts entrained to the LFP oscillation, while regular-spiking (RS), excitatory neurons typically fired only 1-2 spikes per ripplet, in antiphase to FS spikes, and received synchronous sequences of alternating excitatory and inhibitory inputs. We suggest that ripplets are an intrinsically generated cortical response to a strong, synchronous thalamocortical volley, and could provide increased bandwidth for encoding and transmitting sensory information. Importantly, optogenetically induced ripplets are a uniquely accessible model system for studying synaptic mechanisms of fast and ultrafast cortical and hippocampal oscillations.

Genetically Defined Subtypes of Somatostatin-Containing Cortical Interneurons.

Inhibitory interneurons play a crucial role in proper development and function of the mammalian cerebral cortex. Of the different inhibitory subclasses, dendritic-targeting, somatostatin-containing (SOM) interneurons may be the most diverse. Earlier studies used transgenic mouse lines to identify and characterize subtypes of SOM interneurons by morphological, electrophysiological and neurochemical properties. More recently, large-scale studies classified SOM interneurons into 13 morpho-electro-transcriptomic (MET) types. It remains unclear, however, how these various classification schemes relate to each other, and experimental access to MET types has been limited by the scarcity of type-specific mouse driver lines. To begin to address these issues we crossed Flp and Cre driver mouse lines and a dual-color combinatorial reporter, allowing experimental access to genetically defined SOM subsets. Brains from adult mice of both sexes were retrogradely dye-labeled from the pial surface to identify layer 1-projecting neurons, and immunostained against several marker proteins, allowing correlation of genetic label, axonal target and marker protein expression in the same neurons. Using whole-cell recordings ex-vivo, we compared electrophysiological properties between intersectional and transgenic SOM subsets. We identified two layer 1-targeting intersectional subsets with non-overlapping marker protein expression and electrophysiological properties which, together with a previously characterized layer 4-targeting subtype, account for about half of all layer 5 SOM cells and >40% of all SOM cells, and appear to map onto 5 of the 13 MET types. Genetic access to these subtypes will allow researchers to determine their synaptic inputs and outputs and uncover their roles in cortical computations and animal behavior. SIGNIFICANCE STATEMENT: Inhibitory neurons are critically important for proper development and function of the cerebral cortex. Although a minority population, they are highly diverse, which poses a major challenge to investigating their contributions to cortical computations and animal and human behavior. As a step towards understanding this diversity we crossed genetically modified mouse lines to allow detailed examination of genetically-defined groups of the most diverse inhibitory subtype, somatostatin-containing interneurons. We identified and characterized three somatostatin subtypes in the deep cortical layers with distinct combinations of anatomical, neurochemical and electrophysiological properties. Future studies could now use these genetic tools to examine how these different subtypes are integrated into the cortical circuit and what roles they play during sensory, cognitive or motor behavior.

Recruitment of GABAergic Interneurons in the Barrel Cortex during Active Tactile Behavior.

Neural computation involves diverse types of GABAergic inhibitory interneurons that are integrated with excitatory (E) neurons into precisely structured circuits. To understand how each neuron type shapes sensory representations, we measured firing patterns of defined types of neurons in the barrel cortex while mice performed an active, whisker-dependent object localization task. Touch excited fast-spiking (FS) interneurons at short latency, followed by activation of E neurons and somatostatin-expressing (SST) interneurons. Touch only weakly modulated vasoactive intestinal polypeptide-expressing (VIP) interneurons. Voluntary whisker movement activated FS neurons in the ventral posteromedial nucleus (VPM) target layers, a subset of SST neurons and a majority of VIP neurons. Together, FS neurons track thalamic input, mediating feedforward inhibition. SST neurons monitor local excitation, providing feedback inhibition. VIP neurons are activated by non-sensory inputs, disinhibiting E and FS neurons. Our data reveal rules of recruitment for interneuron types during behavior, providing foundations for understanding computation in cortical microcircuits.

A Novel Method for Training Mice in Visuo-Tactile 3-D Object Discrimination and Recognition.

Perceiving, recognizing and remembering 3-dimensional (3-D) objects encountered in the environment has a very high survival value; unsurprisingly, this ability is shared among many animal species, including humans. The psychological, psychophysical and neural basis for object perception, discrimination, recognition and memory has been extensively studied in humans, monkeys, pigeons and rodents, but is still far from understood. Nearly all 3-D object recognition studies in the rodent used the "novel object recognition" paradigm, which relies on innate rather than learned behavior; however, this procedure has several important limitations. Recently, investigators have begun to recognize the power of behavioral tasks learned through reinforcement training (operant conditioning) to reveal the sensorimotor and cognitive abilities of mice and to elucidate their underlying neural mechanisms. Here, we describe a novel method for training and testing mice in visual and tactile object discrimination, recognition and memory, and use it to begin to examine the underlying sensory basis for these cognitive capacities. A custom-designed Y maze was used to train mice to associate one of two 3-D objects with a food reward. Out of nine mice trained in two cohorts, seven reached performance criterion in about 20-35 daily sessions of 20 trials each. The learned association was retained, or rapidly re-acquired, after a 6 weeks hiatus in training. When tested under low light conditions, individual animals differed in the degree to which they used tactile or visual cues to identify the objects. Switching to total darkness resulted only in a transient dip in performance, as did subsequent trimming of all large whiskers (macrovibrissae). Additional removal of the small whiskers (microvibrissae) did not degrade performance, but transiently increased the time spent inspecting the object. This novel method can be combined in future studies with the large arsenal of genetic tools available in the mouse, to elucidate the neural basis of object perception, recognition and memory.

Differential Excitation of Distally versus Proximally Targeting Cortical Interneurons by Unitary Thalamocortical Bursts.

UNLABELLED: Thalamocortical neurons relay sensory and motor information to the neocortex using both single spikes and bursts; bursts prevail during low-vigilance states but also occur during awake behavior. Bursts are suggested to provide an alerting signal to the cortex and enhance stimulus detection, but the synaptic mechanisms underlying these effects are not clear, because the postsynaptic responses of different subtypes of cortical neurons to unitary thalamocortical bursts are mostly unknown. Using optogenetically guided recordings in mouse thalamocortical slices, we achieved the first reported paired intracellular recordings from nine monosynaptically connected thalamic and cortical neurons, including principal cells and two subtypes of inhibitory interneurons, and compared between cortical responses to single thalamocortical spikes and bursts. In 18 additional cortical neurons, we elicited unitary burst responses optogenetically. Short-term dynamics and temporal summation of burst-evoked EPSPs were cell-type dependent: in principal cells and somatostatin-containing (SOM), but not fast-spiking (FS), interneurons, peak response during a burst was on average more than twofold larger than the response to the first spike. Thus, firing a burst instead of a single spike would more than double the probability of firing in postsynaptic excitatory neurons and in SOM, but not FS, interneurons. Consistent with this prediction, FS interneurons held near firing threshold fired most often on the first burst component, whereas SOM interneurons fired only on the second or later components. By increasing excitation of principal cells together with SOM-mediated, distally directed inhibition, thalamocortical bursts could momentarily enhance the saliency of the ascending sensory stimulus over less urgent, top-down inputs. SIGNIFICANCE STATEMENT: Thalamocortical neurons relay sensory and motor information to the cerebral cortex using both single spikes and high-frequency bursts, but the function of bursts is not fully understood. Using brain slices from mouse somatosensory thalamus and cortex, we achieved the first dual recordings of directly connected thalamic and cortical neurons and compared between cortical responses to single thalamic spikes and to bursts. We report that bursts enhanced the responses of excitatory neurons and of inhibitory interneurons that preferentially target dendrites. A potential consequence is that bursts will enhance the response to the immediate sensory event over responses to less urgent, modulatory inputs.

Properties of precise firing synchrony between synaptically coupled cortical interneurons depend on their mode of coupling.

Precise spike synchrony has been widely reported in the central nervous system, but its functional role in encoding, processing, and transmitting information is yet unresolved. Of particular interest is firing synchrony between inhibitory cortical interneurons, thought to drive various cortical rhythms such as gamma oscillations, the hallmark of cognitive states. Precise synchrony can arise between two interneurons connected electrically, through gap junctions, chemically, through fast inhibitory synapses, or dually, through both types of connections, but the properties of synchrony generated by these different modes of connectivity have never been compared in the same data set. In the present study we recorded in vitro from 152 homotypic pairs of two major subtypes of mouse neocortical interneurons: parvalbumin-containing, fast-spiking (FS) interneurons and somatostatin-containing (SOM) interneurons. We tested firing synchrony when the two neurons were driven to fire by long, depolarizing current steps and used a novel synchrony index to quantify the strength of synchrony, its temporal precision, and its dependence on firing rate. We found that SOM-SOM synchrony, driven solely by electrical coupling, was less precise than FS-FS synchrony, driven by inhibitory or dual coupling. Unlike SOM-SOM synchrony, FS-FS synchrony was strongly firing rate dependent and was not evident at the prototypical 40-Hz gamma frequency. Computer simulations reproduced these differences in synchrony without assuming any differences in intrinsic properties, suggesting that the mode of coupling is more important than the interneuron subtype. Our results provide novel insights into the mechanisms and properties of interneuron synchrony and point out important caveats in current models of cortical oscillations.

A novel, jitter-based method for detecting and measuring spike synchrony and quantifying temporal firing precision.

BACKGROUND: Precise spike synchrony, at the millisecond or even sub-millisecond time scale, has been reported in different brain areas, but its neurobiological meaning and its underlying mechanisms remain unknown or controversial. Studying these questions is complicated by the lack of a validated, well-normalized and robust index for quantifying synchrony. Previously used measures of synchrony are often improperly normalized and thereby are not comparable between different experimental conditions, are sensitive to variations in firing rate or to the firing rate differential between the two neurons, and/or rely on untenable assumptions of firing rate stationarity and Poisson statistics. I describe here a novel measure, the Jitter-Based Synchrony Index (JBSI), that overcomes these issues. RESULTS AND DISCUSSION: The JBSI method is based on the introduction of virtual spike jitter. While previous implementations of the jitter method used it only to detect synchrony, the JBSI method also quantifies synchrony. Previous implementations of the jitter method used computationally intensive Monte Carlo simulations to generate surrogate spike trains, whereas the JBSI is computed analytically. The JBSI method does not assume any specific firing model, and does not require that the spike trains be locked to a repeating external stimulus. The JBSI can assume values from 1 (maximal possible synchrony) to -1 (minimal possible synchrony) and is therefore properly normalized. Using simulated Poisson spike trains with introduced controlled spike coincidences, I demonstrate that the JBSI is a linear measure of the spike coincidence rate, is independent of the mean firing frequency or the firing frequency differential between the two neurons, and is not sensitive to co-modulations in the firing rates of the two neurons. In contrast, several commonly used synchrony indices fail under one or more of these scenarios. I also demonstrate how the JBSI can be used to estimate the spike timing precision in the system. CONCLUSIONS: The JBSI is a conceptually simple and computationally efficient method that can be used to compute the statistical significance of firing synchrony, to quantify synchrony as a well-normalized index, and to estimate the degree of temporal precision in the system.

Short-term plasticity of unitary inhibitory-to-inhibitory synapses depends on the presynaptic interneuron subtype.

Excitatory-to-inhibitory cortical synapses exhibit either short-term facilitation or depression, depending on the subtype identity of the postsynaptic interneuron, while the short-term plasticity (STP) of inhibitory-to-excitatory synapses depends on the presynaptic interneuron. However, the rules governing STP of inhibitory-to-inhibitory synapses have not yet been determined. We recorded 109 unitary connections made by the two major inhibitory interneuron subtypes in layer 4 of mouse somatosensory cortex, fast-spiking (FS) and somatostatin-containing (SOM) interneurons, on each other and on excitatory, regular-spiking (RS) neurons. In all pairs, we measured dynamic changes in the postsynaptic response to a 20 Hz train of presynaptic action potentials. In half of our dataset, we also measured kinetic properties of the unitary IPSC: latency, rise time, and decay time constant. We found a pronounced dependency of STP on the presynaptic, but not the postsynaptic, identity: FS interneurons made strongly depressing connections on FS, SOM, and RS targets, while in synapses made by SOM interneurons on FS and RS targets, weak early depression was followed by weak late facilitation. IPSC latency and rise time were also strongly dependent on the presynaptic interneuron subtype, being 1.5-2× slower in output synapses of SOM compared with FS interneurons. In contrast, the IPSC decay time constant depended only on the postsynaptic class, with 1.5× slower decay on excitatory compared with inhibitory targets. The properties of the inhibitory outputs of FS and SOM interneurons reciprocate the properties of their excitatory inputs and imply a dynamic spatiotemporal division of labor between these two major inhibitory subsystems.

Submillisecond firing synchrony between different subtypes of cortical interneurons connected chemically but not electrically.

Synchronous firing is commonly observed in the brain, but its underlying mechanisms and neurobiological meaning remain debated. Most commonly, synchrony is attributed either to electrical coupling by gap junctions or to shared excitatory inputs. In the cerebral cortex and hippocampus, fast-spiking (FS) or somatostatin-containing (SOM) inhibitory interneurons are electrically coupled to same-type neighbors, and each subtype-specific network tends to fire in synchrony. Electrical coupling across subtypes is weak or absent, but SOM-FS and FS-FS pairs are often connected by inhibitory synapses. Theoretical studies suggest that purely inhibitory coupling can also promote synchrony; however, this has not been confirmed experimentally. We recorded from 74 pairs of electrically noncoupled layer 4 interneurons in mouse somatosensory cortex in vitro, and found that tonically depolarized FS-FS and SOM-FS pairs connected by unidirectional or bidirectional inhibitory synapses often fired within 1 ms of each other. Using a novel, jitter-based measure of synchrony, we found that synchrony correlated with inhibitory coupling strength. Importantly, synchrony was resistant to ionotropic glutamate receptors antagonists but was strongly reduced when GABA(A) receptors were blocked, confirming that in our experimental system IPSPs were both necessary and sufficient for synchrony. Submillisecond firing lags emerged in a computer simulation of pairs of spiking neurons, in which the only assumed interaction between neurons was by inhibitory synapses. We conclude that cortical interneurons are capable of synchronizing both within and across subtypes, and that submillisecond coordination of firing can arise by mutual synaptic inhibition alone, with neither shared inputs nor electrical coupling.

Robust but delayed thalamocortical activation of dendritic-targeting inhibitory interneurons.

GABA-releasing cortical interneurons are crucial for the neural transformations underlying sensory perception, providing "feedforward" inhibition that constrains the temporal window for synaptic integration. To mediate feedforward inhibition, inhibitory interneurons need to fire in response to ascending thalamocortical inputs, and most previous studies concluded that ascending inputs activate mainly or solely proximally targeting, parvalbumin-containing "fast-spiking" interneurons. However, when thalamocortical axons fire at frequencies that are likely to occur during natural exploratory behavior, activation of fast-spiking interneurons is rapidly and strongly depressed, implying the paradoxical conclusion that feedforward inhibition is absent when it is most needed. To address this issue, we took advantage of lines of transgenic mice in which either parvalbumin- or somatostatin-containing interneurons express GFP and recorded the responses of interneurons from both subtypes to thalamocortical stimulation in vitro. We report that during thalamocortical activation at behaviorally expected frequencies, fast-spiking interneurons were indeed activated only transiently because of rapid depression of their thalamocortical inputs, but a subset of layer 5 somatostatin-containing interneurons were robustly and persistently activated after a delay, due to the facilitation and temporal summation of their thalamocortical excitatory postsynaptic potentials. Somatostatin-containing interneurons are considered distally targeting. Thus, they are likely to provide delayed dendritic inhibition during exploratory behavior, contributing to the maintenance of a balance between cortical excitation and inhibition while leaving a wide temporal window open for synaptic integration and plasticity in distal dendrites.

Distinct subtypes of somatostatin-containing neocortical interneurons revealed in transgenic mice.

GABA-releasing inhibitory interneurons in the cerebral cortex can be classified by their neurochemical content, firing patterns, or axonal targets, to name the most common criteria, but whether classifications using different criteria converge on the same neuronal subtypes, and how many such subtypes exist, is a matter of much current interest and considerable debate. To address these issues, we generated transgenic mice expressing green fluorescent protein (GFP) under control of the GAD67 promoter. In two of these lines, named X94 and X98, GFP expression in the barrel cortex was restricted to subsets of somatostatin-containing (SOM+) GABAergic interneurons, similar to the previously reported "GIN" line (Oliva et al., 2000), but the laminar distributions of GFP-expressing (GFP+) cell bodies in the X94, X98, and GIN lines were distinct and nearly complementary. We compared neurochemical content and axonal distribution patterns of GFP+ neurons among the three lines and analyzed in detail electrophysiological properties in a dataset of 150 neurons recorded in whole-cell, current-clamp mode. By all criteria, there was nearly perfect segregation of X94 and X98 GFP+ neurons, whereas GIN GFP+ neurons exhibited intermediate properties. In the X98 line, GFP expression was found in infragranular, calbindin-containing, layer 1-targeting ("Martinotti") cells that had a propensity to fire low-threshold calcium spikes, whereas X94 GFP+ cells were stuttering interneurons with quasi fast-spiking properties, residing in and targeting the thalamo-recipient neocortical layers. We conclude that much of the variability previously attributed to neocortical SOM+ interneurons can be accounted for by their natural grouping into distinct subtypes.

Brain-derived neurotrophic factor mediates activity-dependent dendritic growth in nonpyramidal neocortical interneurons in developing organotypic cultures.

Brain-derived neurotrophic factor (BDNF) promotes postnatal maturation of GABAergic inhibition in the cerebral and cerebellar cortices, and its expression and release are enhanced by neuronal activity, suggesting that it acts in a feedback manner to maintain a balance between excitation and inhibition during development. BDNF promotes differentiation of cerebellar, hippocampal, and neostriatal inhibitory neurons, but its effects on the dendritic development of neocortical inhibitory interneurons remain unknown. Here, we show that BDNF mediates depolarization-induced dendritic growth and branching in neocortical interneurons. To visualize inhibitory interneurons, we biolistically transfected organotypic cortical slice cultures from neonatal mice with green fluorescent protein (GFP) driven by the glutamic acid decarboxylase (GAD)67 promoter. Nearly all GAD67-GFP-expressing neurons were nonpyramidal, many contained GABA, and some expressed markers of neurochemically defined GABAergic subtypes, indicating that GAD67-GFP-expressing neurons were GABAergic. We traced dendritic trees from confocal images of the same GAD67-GFP-expressing neurons before and after a 5 d growth period, and quantified the change in total dendritic length (TDL) and total dendritic branch points (TDBPs) for each neuron. GAD67-GFP-expressing neurons growing in control medium exhibited a 20% increase in TDL, but in 200 ng/ml BDNF or 10 mm KCl, this increase nearly doubled and was accompanied by a significant increase in TDBPs. Blocking action potentials with TTX did not prevent the BDNF-induced growth, but antibodies against BDNF blocked the growth-promoting effect of KCl. We conclude that BDNF, released by neocortical pyramidal neurons in response to depolarization, enhances dendritic growth and branching in nearby inhibitory interneurons.

The role of the hyperpolarization-activated cationic current I(h) in the timing of interictal bursts in the neonatal hippocampus.

Under both pathological and experimental conditions, area CA3 of the adult or juvenile hippocampus generates periodic population discharges known as interictal bursts. Whereas the ionic and synaptic basis of individual bursts has been comprehensively studied experimentally and computationally, the pacemaker mechanisms underlying interictal rhythmicity remain conjectural. We showed previously that rhythmic population discharges resembling interictal bursts can be induced in hippocampal slices from first postnatal week mice, in Mg2+-free solution with GABA(A) receptor-mediated inhibition blocked. Here we show that these neonatal bursts occurred with high temporal precision and that their frequency and regularity were greatly reduced by the bradycardic agent ZD-7288 when applied at concentrations and durations that selectively block the hyperpolarization-activated, cationic current I(h). Augmenting I(h) by elevating intracellular cAMP dramatically increased burst frequency in a protein kinase A-independent manner. Burst amplitudes were strongly correlated with the preceding, but not the following, interburst intervals. The experimentally observed distribution of interburst intervals was modeled by assuming that a burst was triggered whenever the instantaneous rate of spontaneous EPSPs (sEPSPs) exceeded a threshold and that the mean sEPSP rate was minimal immediately after a burst and then relaxed exponentially to a steady-state level. The effect of blocking I(h) in any given slice could be modeled by decreasing only the steady-state sEPSP rate, suggesting that the instantaneous rate of sEPSPs is governed by the level of I(h) activation and raising the novel possibility that interburst intervals reflected the slow activation kinetics of I(h) in the neonatal CA3.