Intersectional strategy to study cortical inhibitory parvalbumin-expressing interneurons.

Parvalbumin-expressing (PV) interneurons are key neuronal elements to a global excitatory-inhibitory balance in normal cortical functioning. To better understand the circuit functions of PV interneurons, reliable animal models are needed. This study investigated the sensitivity and specificity of the most frequently used PV-Cre/tdTomato mouse line in this regard. The colocalization of the transgene (tdTomato) with the parvalbumin protein, with GAD1 (a conclusive inhibitory cell marker) and Vglut1 (a conclusive excitatory cell marker) as well as with a marker for perineuronal nets (WFA) was assessed and a substantial proportion of layer 5 PV neurons was found to be excitatory and not inhibitory in the PV-Cre/tdTomato mouse. The intersectional transgenic mouse line Vgat-Cre/PV-Flp/tdTomato provided a solution, since no colocalization of tdTomato with the Vglut1 probe was found there. In conclusion, the Vgat-Cre/PV-Flp/tdTomato mouse line seems to be a more reliable animal model for functional studies of GABAergic PV interneurons.

Repetitively burst-spiking neurons in reeler mice show conserved but also highly variable morphological features of layer Vb-fated "thick-tufted" pyramidal cells.

Reelin is a large extracellular glycoprotein that is secreted by Cajal-Retzius cells during embryonic development to regulate neuronal migration and cell proliferation but it also seems to regulate ion channel distribution and synaptic vesicle release properties of excitatory neurons well into adulthood. Mouse mutants with a compromised reelin signaling cascade show a highly disorganized neocortex but the basic connectional features of the displaced excitatory principal cells seem to be relatively intact. Very little is known, however, about the intrinsic electrophysiological and morphological properties of individual cells in the reeler cortex. Repetitive burst-spiking (RB) is a unique property of large, thick-tufted pyramidal cells of wild-type layer Vb exclusively, which project to several subcortical targets. In addition, they are known to possess sparse but far-reaching intracortical recurrent collaterals. Here, we compared the electrophysiological properties and morphological features of neurons in the reeler primary somatosensory cortex with those of wild-type controls. Whereas in wild-type mice, RB pyramidal cells were only detected in layer Vb, and the vast majority of reeler RB pyramidal cells were found in the superficial third of the cortical depth. There were no obvious differences in the intrinsic electrophysiological properties and basic morphological features (such as soma size or the number of dendrites) were also well preserved. However, the spatial orientation of the entire dendritic tree was highly variable in the reeler neocortex, whereas it was completely stereotyped in wild-type mice. It seems that basic quantitative features of layer Vb-fated RB pyramidal cells are well conserved in the highly disorganized mutant neocortex, whereas qualitative morphological features vary, possibly to properly orient toward the appropriate input pathways, which are known to show an atypical oblique path through the reeler cortex. The oblique dendritic orientation thus presumably reflects a re-orientation of dendritic input domains toward spatially highly disorganized afferent projections.

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.

Characterizing the morphology of somatostatin-expressing interneurons and their synaptic innervation pattern in the barrel cortex of the GFP-expressing inhibitory neurons mouse.

Somatostatin-expressing (SST+) cells form the second largest subpopulation of neocortical GABAergic neurons that contain diverse subtypes, which participate in layer-specific cortical circuits. Martinotti cells, as the most abundant subtype of SST+ interneurons, are mainly located in layers II/III and V/VI, and are characterized by dense axonal arborizations in layer I. GFP-expressing inhibitory neurons (GIN), representing a fraction of mainly upper layer SST+ interneurons in various cortical areas, were recently claimed to include both Martinotti cells and non-Martinotti cells. This makes it necessary to examine in detail the morphology and synaptic innervation pattern of the GIN cells, in order to better predict their functional implications. In our study, we characterized the neurochemical specificity, somatodendritic morphology, synaptic ultrastructure as well as synaptic innervation pattern of GIN cells in the barrel cortex in a layer-specific manner. We showed that GIN cells account for 44% of the SST+ interneurons in layer II/III and around 35% in layers IV and Va. There are 29% of GIN cells coexpressing calretinin with 54% in layer II/III, 8% in layer IV, and 13% in layer V. They have diverse somatodendritic configurations and form relatively small synapses across all examined layers. They almost exclusively innervate dendrites of excitatory cells, preferentially targeting distal apical dendrites and apical dendritic tufts of pyramidal neurons in layer I, and rarely target other inhibitory neurons. In summary, our study reveals unique features in terms of the morphology and output of GIN cells, which can help to better understand their diversity and structure-function relationships.

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.

Mapping Brain-Wide Afferent Inputs of Parvalbumin-Expressing GABAergic Neurons in Barrel Cortex Reveals Local and Long-Range Circuit Motifs.

Parvalbumin (PV)-expressing GABAergic neurons are the largest class of inhibitory neocortical cells. We visualize brain-wide, monosynaptic inputs to PV neurons in mouse barrel cortex. We develop intersectional rabies virus tracing to specifically target GABAergic PV cells and exclude a small fraction of excitatory PV cells from our starter population. Local inputs are mainly from layer (L) IV and excitatory cells. A small number of inhibitory inputs originate from LI neurons, which connect to LII/III PV neurons. Long-range inputs originate mainly from other sensory cortices and the thalamus. In visual cortex, most transsynaptically labeled neurons are located in LIV, which contains a molecularly mixed population of projection neurons with putative functional similarity to LIII neurons. This study expands our knowledge of the brain-wide circuits in which PV neurons are embedded and introduces intersectional rabies virus tracing as an applicable tool to dissect the circuitry of more clearly defined cell types.

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.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by nicotinic acetylcholine receptor (nAChR) activation by cholinergic inputs. PFC spontaneous default activity is altered in neuropsychiatric disorders, including schizophrenia-a disorder that can be accompanied by heavy smoking. Recently, genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, encoding the α5 nAChR subunit, that increase the risks for both smoking and schizophrenia. Mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but it is unknown how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying behavior. Here we show that mice expressing a human α5 SNP exhibit neurocognitive behavioral deficits in social interaction and sensorimotor gating tasks. Two-photon calcium imaging in awake mouse models showed that nicotine can differentially influence PFC pyramidal cell activity by nAChR modulation of layer II/III hierarchical inhibitory circuits. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive over layer II/III pyramidal neurons. The decreased activity observed in α5-SNP-expressing mice resembles the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine administration reversed this hypofrontality, suggesting that administration of nicotine may represent a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking.

Intracortical Network Effects Preserve Thalamocortical Input Efficacy in a Cortex Without Layers.

Layer IV (LIV) of the rodent somatosensory cortex contains the somatotopic barrel field. Barrels receive much of the sensory input to the cortex through innervation by thalamocortical axons from the ventral posteromedial nucleus. In the reeler mouse, the absence of cortical layers results in the formation of mispositioned barrel-equivalent clusters of LIV fated neurons. Although functional imaging suggests that sensory input activates the cortex, little is known about the cellular and synaptic properties of identified excitatory neurons of the reeler cortex. We examined the properties of thalamic input to spiny stellate (SpS) neurons in the reeler cortex with in vitro electrophysiology, optogenetics, and subcellular channelrhodopsin-2-assisted circuit mapping (sCRACM). Our results indicate that reeler SpS neurons receive direct but weakened input from the thalamus, with a dispersed spatial distribution along the somatodendritic arbor. These results further document subtle alterations in functional connectivity concomitant of absent layering in the reeler mutant. We suggest that intracortical amplification mechanisms compensate for this weakening in order to allow reliable sensory transmission to the mutant neocortex.

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.

Presynaptic and postsynaptic origin of multicomponent extracellular waveforms at the endbulb of Held-spherical bushy cell synapse.

Extracellular signals from the endbulb of Held-spherical bushy cell (SBC) synapse exhibit up to three component waves ('P', 'A' and 'B'). Signals lacking the third component (B) are frequently observed but as the origin of each of the components is uncertain, interpretation of this lack of B has been controversial: is it a failure to release transmitter or a failure to generate or propagate an action potential? Our aim was to determine the origin of each component. We combined single- and multiunit in vitro methods in Mongolian gerbils and Wistar rats and used pharmacological tools to modulate glutamate receptors or voltage-gated sodium channels. Simultaneous extra- and intracellular recordings from single SBCs demonstrated a presynaptic origin of the P-component, consistent with data obtained with multielectrode array recordings of local field potentials. The later components (A and B) correspond to the excitatory postsynaptic potential (EPSP) and action potential of the SBC, respectively. These results allow a clear interpretation of in vivo extracellular signals. We conclude that action potential failures occurring at the endbulb-SBC synaptic junction largely reflect failures of the EPSP to trigger an action potential and not failures of synaptic transmission. The data provide the basis for future investigation of convergence of excitatory and inhibitory inputs in modulating transmission at a fully functional neuronal system using physiological stimulation.

P2 receptor-mediated signaling in spherical bushy cells of the mammalian cochlear nucleus.

Purinoreceptors of the P2 family contribute strongly to signaling in the cochlea, but little is known about the effects of purinergic neurotransmission in the central auditory system. Here we examine P2 receptor-mediated signaling in the large spherical bushy cells (SBCs) of Mongolian gerbils around the onset of acoustically evoked signal processing (P9-P14). Brief adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) application evoked inward current, membrane depolarization, and somatic Ca2+ signals. Moreover, ATPgammaS changed the SBCs firing pattern from phasic to tonic, when the application was synchronized with depolarizing current injection. This bursting discharge activity was dependent on [Ca2+]i and Ca2+-dependent protein kinase (PKC) activity and is presumably caused by modulation of low-threshold K+ conductance. Activation of P2Y1 receptors could not evoke these changes per se, thus it was concluded that the involvement of P2X receptors seems to be necessary. Ca2+ imaging data showed that both P2X and P2Y1 receptors mediate Ca2+ signals in SBCs where P2Y1 receptors most likely activate the PLC-IP3 (inositol trisphosphate) pathway and release Ca2+ from internal stores. Immunohistochemical staining confirmed the expression of P2X2 and P2Y1 receptor proteins in SBCs, providing additional evidence for the involvement of both receptors in signal transduction in these neurons. Purinergic signaling might modulate excitability of SBCs and thereby contribute to regulation of synaptic strength. Functionally, the increase in firing rate mediated by P2 receptors could reduce temporal precision of the postsynaptic firing, e.g., phase locking, which has an immediate effect on signal processing related to sound localization. This might provide a mechanism for adaptation to the ambient acoustic environment.

Development of chloride-mediated inhibition in neurons of the anteroventral cochlear nucleus of gerbil (Meriones unguiculatus).

At the initial stages in neuronal development, GABAergic and glycinergic neurotransmission exert depolarizing responses, assumed to be of importance for maturation, which in turn shift to hyperpolarizing in early postnatal life due to development of the chloride homeostasis system. Spherical bushy cells (SBC) of the mammalian cochlear nucleus integrate excitatory glutamatergic inputs with inhibitory (GABAergic and glycinergic) inputs to compute signals that contribute to sound localization based on interaural time differences. To provide a fundamental understanding of the properties of GABAergic neurotransmission in mammalian cochlear nucleus, we investigated the reversal potential of the GABA-evoked currents (E GABA) by means of gramicidin-perforated-patch recordings in developing SBC. The action of GABA switches from depolarizing to hyperpolarizing by the postnatal day 7 due to the negative shift in E GABA. Furthermore, we studied the expression pattern of the K+-Cl(-)-extruding cotransporter KCC2, previously shown to induce a switch from neonatal Cl(-) efflux to the mature Cl(-) influx in various neuron types, thereby causing a shift from depolarizing to hyperpolarizing GABA action. The KCC2 protein is expressed in SBC already at birth, yet its activity is attained toward the end of the first postnatal week as indicated by pharmacological inhibition. Interruption of the Cl(-) extrusion by [(dihydroindenyl)oxy] alkanoic acid or furosemide gradually shifted E(GABA) in positive direction with increasing maturity, suggesting that KCC2 could be involved in maintaining low [Cl(-)]i after the postnatal day 7 thereby providing the hyperpolarizing Cl(-)-mediated inhibition in SBC.