Multimodal cortical neuronal cell type classification.

Since more than a century, neuroscientists have distinguished excitatory (glutamatergic) neurons with long-distance projections from inhibitory (GABAergic) neurons with local projections and established layer-dependent schemes for the ~ 80% excitatory (principal) cells as well as the ~ 20% inhibitory neurons. Whereas, in the early days, mainly morphological criteria were used to define cell types, later supplemented by electrophysiological and neurochemical properties, nowadays. single-cell transcriptomics is the method of choice for cell type classification. Bringing recent insight together, we conclude that despite all established layer- and area-dependent differences, there is a set of reliably identifiable cortical cell types that were named (among others) intratelencephalic (IT), extratelencephalic (ET), and corticothalamic (CT) for the excitatory cells, which altogether comprise ~ 56 transcriptomic cell types (t-types). By the same means, inhibitory neurons were subdivided into parvalbumin (PV), somatostatin (SST), vasoactive intestinal polypeptide (VIP), and "other (i.e. Lamp5/Sncg)" subpopulations, which altogether comprise ~ 60 t-types. The coming years will show which t-types actually translate into "real" cell types that show a common set of multimodal features, including not only transcriptome but also physiology and morphology as well as connectivity and ultimately function. Only with the better knowledge of clear-cut cell types and experimental access to them, we will be able to reveal their specific functions, a task which turned out to be difficult in a part of the brain being so much specialized for cognition as the cerebral cortex.

Direction selectivity of inhibitory interneurons in mouse barrel cortex differs between interneuron subtypes.

GABAergic interneurons represent ∼15% to 20% of all cortical neurons, but their diversity grants them unique roles in cortical circuits. In the barrel cortex, responses of excitatory neurons to stimulation of facial whiskers are direction selective, whereby excitation is maximized over a narrow range of angular deflections. Whether GABAergic interneurons are also direction selective is unclear. Here, we use two-photon-guided whole-cell recordings in the barrel cortex of anesthetized mice and control whisker stimulation to measure direction selectivity in defined interneuron subtypes. Selectivity is ubiquitous in interneurons, but tuning sharpness varies across populations. Vasoactive intestinal polypeptide (VIP) interneurons are as selective as pyramidal neurons, but parvalbumin (PV) interneurons are more broadly tuned. Furthermore, a majority (2/3) of somatostatin (SST) interneurons receive direction-selective inhibition, with the rest receiving direction-selective excitation. Sensory evoked activity in the barrel cortex is thus cell-type specific, suggesting that interneuron subtypes make distinct contributions to cortical representations of stimuli.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling.

Fine-tuned gene expression is crucial for neurodevelopment. The gene expression program is tightly controlled at different levels, including RNA decay. N6-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for brain development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay contributes to brain development is largely unknown. Here, we show that Exosc10, a RNA exonuclease subunit of the RNA exosome complex, is indispensable for forebrain development. We report that cortical cells undergo overt apoptosis, culminating in cortical agenesis upon conditional deletion of Exosc10 in mouse cortex. Mechanistically, Exosc10 directly binds and degrades transcripts of the P53 signaling-related genes, such as Aen and Bbc3. Overall, our findings suggest a crucial role for Exosc10 in suppressing the P53 pathway, in which the rapid turnover of the apoptosis effectors Aen and Bbc3 mRNAs is essential for cell survival and normal cortical histogenesis.

Increased Callosal Connectivity in Reeler Mice Revealed by Brain-Wide Input Mapping of VIP Neurons in Barrel Cortex.

The neocortex is composed of layers. Whether layers constitute an essential framework for the formation of functional circuits is not well understood. We investigated the brain-wide input connectivity of vasoactive intestinal polypeptide (VIP) expressing neurons in the reeler mouse. This mutant is characterized by a migration deficit of cortical neurons so that no layers are formed. Still, neurons retain their properties and reeler mice show little cognitive impairment. We focused on VIP neurons because they are known to receive strong long-range inputs and have a typical laminar bias toward upper layers. In reeler, these neurons are more dispersed across the cortex. We mapped the brain-wide inputs of VIP neurons in barrel cortex of wild-type and reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical inputs were reduced in reeler, while contralateral inputs were strongly increased. Reeler mice had more callosal projection neurons. Hence, the corpus callosum was larger in reeler as shown by structural imaging. We argue that, in the absence of cortical layers, circuits with subcortical structures are maintained but cortical neurons establish a different network that largely preserves cognitive functions.

Neuromodulation Leads to a Burst-Tonic Switch in a Subset of VIP Neurons in Mouse Primary Somatosensory (Barrel) Cortex.

Neocortical GABAergic interneurons expressing vasoactive intestinal polypeptide (VIP) contribute to sensory processing, sensorimotor integration, and behavioral control. In contrast to other major subpopulations of GABAergic interneurons, VIP neurons show a remarkable diversity. Studying morphological and electrophysiological properties of VIP cells, we found a peculiar group of neurons in layer II/III of mouse primary somatosensory (barrel) cortex, which showed a highly dynamic burst firing behavior at resting membrane potential that switched to tonic mode at depolarized membrane potentials. Furthermore, we demonstrate that burst firing depends on T-type calcium channels. The burst-tonic switch could be induced by acetylcholine (ACh) and serotonin. ACh mediated a depolarization via nicotinic receptors whereas serotonin evoked a biphasic depolarization via ionotropic and metabotropic receptors in 48% of the population and a purely monophasic depolarization via metabotropic receptors in the remaining cells. These data disclose an electrophysiologically defined subpopulation of VIP neurons that via neuromodulator-induced changes in firing behavior is likely to regulate the state of cortical circuits in a profound manner.

Distribution Patterns of Three Molecularly Defined Classes of GABAergic Neurons Across Columnar Compartments in Mouse Barrel Cortex.

The mouse somatosensory cortex is an excellent model to study the structural basis of cortical information processing, since it possesses anatomically recognizable domains that receive different thalamic inputs, which indicates spatial segregation of different processing tasks. In this work we examined three genetically labeled, non-overlapping subpopulations of GABAergic neurons: parvalbumin- (PV+), somatostatin- (SST+), and vasoactive intestinal polypeptide-expressing (VIP+) cells. Each of these subpopulations displayed a unique cellular distribution pattern across layers. In terms of columnar localization, the distribution of these three populations was not quantitatively different between barrel-related versus septal compartments in most layers. However, in layer IV (LIV), SST+, and VIP+, but not PV+ neurons preferred the septal compartment over barrels. The examined cell types showed a tendency toward differential distribution in supragranular and infragranular barrel-related versus septal compartments, too. Our data suggests that the location of GABAergic neuron cell bodies correlates with the spatial pattern of cortical domains receiving different kinds of thalamic input. Thus, at least in LIV, lemniscal inputs present a close spatial relation preferentially to PV+ cells whereas paralemniscal inputs target compartments in which more SST+ and VIP+ cells are localized. Our findings suggest pathway-specific roles for neocortical GABAergic neurons.

RBM15 Modulates the Function of Chromatin Remodeling Factor BAF155 Through RNA Methylation in Developing Cortex.

Chromatin remodeling factor BAF155 is an important regulator of many biological processes. As a core and scaffold subunit of the BAF (SWI/SNF-like) complex, BAF155 is capable of regulating the stability and function of the BAF complex. The spatiotemporal expression of BAF155 during embryogenesis is essential for various aspects of organogenesis, particularly in the brain development. However, our understanding of the mechanisms that regulate the expression and function of BAF155 is limited. Here, we report that RBM15, a subunit of the m6A methyltransferase complex, interacts with BAF155 mRNA and mediates BAF155 mRNA degradation through the mRNA methylation machinery. Ablation of endogenous RBM15 expression in cultured neuronal cells and in the developing cortex augmented the expression of BAF155. Conversely, RBM15 overexpression decreased BAF155 mRNA and protein levels, and perturbed BAF155 functions in vivo, including repression of BAF155-dependent transcriptional activity and delamination of apical radial glial progenitors as a hallmark of basal radial glial progenitor genesis. Furthermore, we demonstrated that the regulation of BAF155 by RBM15 depends on the activity of the mRNA methylation complex core catalytic subunit METTL3. Altogether, our findings reveal a new regulatory avenue that elucidates how BAF complex subunit stoichiometry and functional modulation are achieved in mammalian cells.

Subcellular Targeting of VIP Boutons in Mouse Barrel Cortex is Layer-Dependent and not Restricted to Interneurons.

Neocortical vasoactive intestinal polypeptide (VIP) expressing cells are a diverse subpopulation of GABAergic interneurons issuing distinct axonal projections. They are known to inhibit other types of interneurons as well as excitatory principal neurons and possess a disinhibitory net effect in cortical circuits. In order to elucidate their targeting specificity, the output connectivity of VIP interneurons was studied at the subcellular level in barrel cortex of interneuron-specific Cre-driver mice, using pre- and postembedding electron microscopy. Systematically sampling VIP boutons across all layers, we found a substantial proportion of the innervated subcellular structures were dendrites (80%), with somata (13%), and spines (7%) being much less targeted. In layer VI, a high proportion of axosomatic synapses was found (39%). GABA-immunopositive ratio was quantified among the targets using statistically validated thresholds: only 37% of the dendrites, 7% of the spines, and 26% of the somata showed above-threshold immunogold labeling. For the main target structure "dendrite", a higher proportion of GABAergic subcellular profiles existed in deep than in superficial layers. In conclusion, VIP interneurons innervate non-GABAergic excitatory neurons and interneurons at their subcellular domains with layer-dependent specificity. This suggests a diverse output of VIP interneurons, which predicts multiple functionality in cortical circuitry beyond disinhibition.

TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus.

TRPV1 is an ion channel activated by heat and pungent agents including capsaicin, and has been extensively studied in nociception of sensory neurons. However, the location and function of TRPV1 in the hippocampus is debated. We found that TRPV1 is expressed in oriens-lacunosum-moleculare (OLM) interneurons in the hippocampus, and promotes excitatory innervation. TRPV1 knockout mice have reduced glutamatergic innervation of OLM neurons. When activated by capsaicin, TRPV1 recruits more glutamatergic, but not GABAergic, terminals to OLM neurons in vitro. When TRPV1 is blocked, glutamatergic input to OLM neurons is dramatically reduced. Heterologous expression of TRPV1 also increases excitatory innervation. Moreover, TRPV1 knockouts have reduced Schaffer collateral LTP, which is rescued by activating OLM neurons with nicotine-via α2ß2-containing nicotinic receptors-to bypass innervation defects. Our results reveal a synaptogenic function of TRPV1 in a specific interneuron population in the hippocampus, where it is important for gating hippocampal plasticity.

Thalamocortical Connections Drive Intracortical Activation of Functional Columns in the Mislaminated Reeler Somatosensory Cortex.

Neuronal wiring is key to proper neural information processing. Tactile information from the rodent's whiskers reaches the cortex via distinct anatomical pathways. The lemniscal pathway relays whisking and touch information from the ventral posteromedial thalamic nucleus to layer IV of the primary somatosensory "barrel" cortex. The disorganized neocortex of the reeler mouse is a model system that should severely compromise the ingrowth of thalamocortical axons (TCAs) into the cortex. Moreover, it could disrupt intracortical wiring. We found that neuronal intermingling within the reeler barrel cortex substantially exceeded previous descriptions, leading to the loss of layers. However, viral tracing revealed that TCAs still specifically targeted transgenically labeled spiny layer IV neurons. Slice electrophysiology and optogenetics proved that these connections represent functional synapses. In addition, we assessed intracortical activation via immediate-early-gene expression resulting from a behavioral exploration task. The cellular composition of activated neuronal ensembles suggests extensive similarities in intracolumnar information processing in the wild-type and reeler brains. We conclude that extensive ectopic positioning of neuronal partners can be compensated for by cell-autonomous mechanisms that allow for the establishment of proper connectivity. Thus, genetic neuronal fate seems to be of greater importance for correct cortical wiring than radial neuronal position.

Characterizing VIP Neurons in the Barrel Cortex of VIPcre/tdTomato Mice Reveals Layer-Specific Differences.

Neocortical GABAergic interneurons have a profound impact on cortical circuitry and its information processing capacity. Distinct subgroups of inhibitory interneurons can be distinguished by molecular markers, such as parvalbumin, somatostatin, and vasoactive intestinal polypeptide (VIP). Among these, VIP-expressing interneurons sparked a substantial interest since these neurons seem to operate disinhibitory circuit motifs found in all major neocortical areas. Several of these recent studies used transgenic Vip-ires-cre mice to specifically target the population of VIP-expressing interneurons. This makes it necessary to elucidate in detail the sensitivity and specificity of Cre expression for VIP neurons in these animals. Thus, we quantitatively compared endogenous tdTomato with Vip fluorescence in situ hybridization and αVIP immunohistochemistry in the barrel cortex of VIPcre/tdTomato mice in a layer-specific manner. We show that VIPcre/tdTomato mice are highly sensitive and specific for the entire population of VIP-expressing neurons. In the barrel cortex, approximately 13% of all GABAergic neurons are VIP expressing. Most VIP neurons are found in layer II/III (∼60%), whereas approximately 40% are found in the other layers of the barrel cortex. Layer II/III VIP neurons are significantly different from VIP neurons in layers IV-VI in several morphological and membrane properties, which suggest layer-dependent differences in functionality.

Persistence of Functional Sensory Maps in the Absence of Cortical Layers in the Somsatosensory Cortex of Reeler Mice.

In rodents, layer IV of the primary somatosensory cortex contains the barrel field, where individual, large facial whiskers are represented as a dense cluster of cells. In the reeler mouse, a model of disturbed cortical development characterized by a loss of cortical lamination, the barrel field exists in a distorted manner. Little is known about the consequences of such a highly disturbed lamination on cortical function in this model. We used in vivo intrinsic signal optical imaging together with piezo-controlled whisker stimulation to explore sensory map organization and stimulus representation in the barrel field. We found that the loss of cortical layers in reeler mice had surprisingly little incidence on these properties. The overall topological order of whisker representations is highly preserved and the functional activation of individual whisker representations is similar in size and strength to wild-type controls. Because intrinsic imaging measures hemodynamic signals, we furthermore investigated the cortical blood vessel pattern of both genotypes, where we also did not detect major differences. In summary, the loss of the reelin protein results in a widespread disturbance of cortical development which compromises neither the establishment nor the function of an ordered, somatotopic map of the facial whiskers.

Revisiting enigmatic cortical calretinin-expressing interneurons.

Cortical calretinin (CR)-expressing interneurons represent a heterogeneous subpopulation of about 10-30% of GABAergic interneurons, which altogether total ca. 12-20% of all cortical neurons. In the rodent neocortex, CR cells display different somatodendritic morphologies ranging from bipolar to multipolar but the bipolar cells and their variations dominate. They are also diverse at the molecular level as they were shown to express numerous neuropeptides in different combinations including vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), neurokinin B (NKB) corticotrophin releasing factor (CRF), enkephalin (Enk) but also neuropeptide Y (NPY) and somatostatin (SOM) to a lesser extent. CR-expressing interneurons exhibit different firing behaviors such as adapting, bursting or irregular. They mainly originate from the caudal ganglionic eminence (CGE) but a subpopulation also derives from the dorsal part of the medial ganglionic eminence (MGE). Cortical GABAergic CR-expressing interneurons can be divided in two main populations: VIP-bipolar interneurons deriving from the CGE and SOM-Martinotti-like interneurons originating in the dorsal MGE. Although bipolar cells account for the majority of CR-expressing interneurons, the roles they play in cortical neuronal circuits and in the more general metabolic physiology of the brain remained elusive and enigmatic. The aim of this review is, firstly, to provide a comprehensive view of the morphological, molecular and electrophysiological features defining this cell type. We will, secondly, also summarize what is known about their place in the cortical circuit, their modulation by subcortical afferents and the functional roles they might play in neuronal processing and energy metabolism.

The innervation of parvalbumin-containing interneurons by VIP-immunopositive interneurons in the primary somatosensory cortex of the adult rat.

gamma-Aminobutyric acid (GABA)ergic interneurons of neocortex consist of many subgroups with extremely heterogeneous morphological, physiological and molecular properties. To explore the putative effect of the vasoactive intestinal polypeptide-immunopositive (VIP +) neurons on neocortical circuitry, the number and distribution of VIP + boutons were analysed on somatodendritic domains of 272 parvalbumin immunopositive (PV +) 3D-reconstructed neurons. The synaptic nature of 91% of somatic and 76% of dendritic contacts was verified by electron microscopy. The target PV + neurons were separated in two significantly different groups by means of cluster analysis. The first group (Cluster 1, 26%) received on average five times more VIP + synapses than those of the second group. The second group (Cluster 2, 74%) contained cells that were poorly innervated by VIP + boutons or did not have either somatic or dendritic or any VIP innervation at all. The cells of Cluster 1 had a soma size and total dendritic length significantly smaller than that of Cluster 2, however, they received three times more dendritic synapses, which resulted in a five times higher VIP + synaptic density on dendrites. Our results showed that although most of the PV + cells are innervated by VIP + boutons at a varying degree, some 6% of PV + cells received no input from VIP + interneurons. This suggests a refined morphological basis to influence the majority of the PV + interneurons, which are very effectively controlling pyramidal cell firing. Together with metabolic and neuromodulatory effects of VIP, this would probably result in an enhanced responsiveness of the latter cell type to tactile stimuli.

Calbindin-containing interneurons are a target for VIP-immunoreactive synapses in rat primary somatosensory cortex.

Inhibitory interneurons in cerebral cortex are morphologically and physiologically extremely heterogeneous. This greatly interferes with an understanding of their functions. Progress has been made by classifying these neurons with the aid of molecular markers, e.g., neuropeptides or calcium-binding proteins, which are reliably expressed by certain subpopulations. We have used this approach to demonstrate an output of a subpopulation of cortical interneurons which express vasoactive intestinal polypeptide (VIP). By double immunostaining and correlated light and electron microscopy, we show that calbindin (CB)-containing interneurons located in layers II-VI of rat barrel cortex are targets of symmetric VIP-immunoreactive synapses. All CB-immunoreactive interneurons showed numerous contacts of VIP boutons on proximal and distal dendritic segments. A great majority of CB-immunoreactive interneurons (214/222) displayed such close appositions with VIP boutons on their soma as well. Quantification revealed that the number of VIP-immunoreactive boutons on CB-immunoreactive somata and dendrites of specified order is comparable for the different cortical layers. In conclusion, all calbindin-containing cortical interneurons seem to be under direct influence of other GABAergic interneurons expressing the peptide VIP. An indirect functional consequence of this may be disinhibition of pyramidal cells, which are considered the major target of calbindin interneurons. However, since the examined types of interneurons are intricately embedded in networks of yet different interneurons, the outcome of these multiple inhibitory interactions is likely to be less simplistic. It may be related to the timing of pyramidal cell discharge within and across layers of cortical columns.

Innervation of interneurons immunoreactive for VIP by intrinsically bursting pyramidal cells and fast-spiking interneurons in infragranular layers of juvenile rat neocortex.

Cortical columns contain specific neuronal populations with characteristic sets of connections. This wiring forms the structural basis of dynamic information processing. However, at the single-cell level little is known about specific connectivity patterns. We performed experiments in infragranular layers (V and VI) of rat somatosensory cortex, to clarify further the input patterns of inhibitory interneurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP). Neurons in acute slices were electrophysiologically characterized using whole-cell recordings and filled with biocytin. This allowed us to determine their firing pattern as regular-spiking, intrinsically bursting and fast-spiking, respectively. Biocytin was revealed histochemically and VIP immunohistochemically. Sections were examined for contacts between the axons of the filled neurons and the VIP-ir targets. Twenty pyramidal cells and five nonpyramidal (inter)neurons were recovered and sufficiently stained for further analysis. Regular-spiking pyramidal cells displayed no axonal boutons in contact with VIP-ir targets. In contrast, intrinsically bursting layer V pyramidal cells showed four putative single contacts with a proximal dendrite of VIP neurons. Fast-spiking interneurons formed contacts with two to six VIP neurons, preferentially at their somata. Single as well as multiple contacts on individual target cells were found. Electron microscopic examinations showed that light-microscopically determined contacts represent sites of synaptic interactions. Our results suggest that, within infragranular local cortical circuits, (i) fast-spiking interneurons are more likely to influence VIP cells than are pyramidal cells and (ii) pyramidal cell input probably needs to be highly convergent to fire VIP target cells.

Expression of c-Fos, ICER, Krox-24 and JunB in the whisker-to-barrel pathway of rats: time course of induction upon whisker stimulation by tactile exploration of an enriched environment.

Modified tactile information has been shown to induce adaptive plasticity in the somatosensory cortex of rat. The cellular mechanisms resulting in plastic neuronal responses, however, are largely unknown. Inducible transcription factors have been proposed as one major link in the cascade from modified input to altered neuronal structure and function. We investigated the spatial and temporal patterns of transcription factor induction in the rat whisker-to-barrel pathway by placing the animals in a novel, enriched environment while having clipped sets of whiskers on one side of the face. Such stimulation resulted not only in a specific c-Fos induction in brainstem barrelettes and thalamic barreloids, but also in the barrel-related cortical columns, each with different time courses. In the barrel cortex, c-Fos and Krox-24 immunostaining showed a rapid induction with peak levels at 1 h and a return to basal levels after 14 h. JunB was induced after 1 h of exploration, declined at 6 h and returned to basal levels after this time point. The inducible cyclic AMP early repressor (ICER), a transcription factor of the cAMP signaling pathway, showed a maximum after 6 h, decreased slowly, but elevated levels were still detectable after 5 days. Our data demonstrate that upon whisker stimulation by exploration of a novel, enriched environment, (i) subcortical relay stations in the whisker-to-barrel pathway are able to express elevated levels of c-Fos and (ii) in the barrel cortex c-Fos, JunB, Krox-24 and ICER are differentially regulated in the temporal domain.