Association cortical areas in the mouse contain a large population of fast-spiking GABAergic neurons that do not express parvalbumin.

GABAergic neurons represent 10-15% of the neuronal population of the cortex but exert a powerful control over information flow in cortical circuits. The largest GABAergic class in the neocortex is represented by the parvalbumin-expressing fast-spiking neurons, which provide powerful somatic inhibition to their postsynaptic targets. Recently, the density of parvalbumin interneurons has been shown to be lower in associative areas of the mouse cortex as compared with sensory and motor areas. Modelling work based on these quantifications linked the low-density of parvalbumin interneurons with specific computations of associative cortices. However, it is still unknown whether the total GABAergic population of association cortices is smaller or whether another GABAergic type can compensate for the low density of parvalbumin interneurons. In the present study, we investigated these hypotheses using a combination of neuroanatomy, mouse genetics and neurophysiology. We found that the GABAergic population of association areas is comparable with that of primary sensory areas, and it is enriched of fast-spiking neurons that do not express parvalbumin and were not accounted for by previous quantifications. We developed an intersectional viral strategy to demonstrate that the population of fast-spiking neurons is comparable across cortical regions. Our results provide quantifications of the density of fast-spiking GABAergic neurons and offers new biological constrains to refine current models of cortical computations.

Postnatal persistence of hippocampal Cajal-Retzius cells has a crucial role in the establishment of the hippocampal circuit.

Cajal-Retzius (CR) cells are a transient neuron type that populate the postnatal hippocampus. To understand how the persistence of CR cells influences the maturation of hippocampal circuits, we combined a specific transgenic mouse line with viral vector injection to selectively ablate CR cells from the postnatal hippocampus. We observed layer-specific changes in the dendritic complexity and spine density of CA1 pyramidal cells. In addition, transcriptomic analysis highlighted significant changes in the expression of synapse-related genes across development. Finally, we were able to identify significant changes in the expression levels of latrophilin 2, a postsynaptic guidance molecule known for its role in the entorhinal-hippocampal connectivity. These findings were supported by changes in the synaptic proteomic content in CA1 stratum lacunosum-moleculare. Our results reveal a crucial role for CR cells in the establishment of the hippocampal network.

Not All That Is Gold Glitters: PV-IRES-Cre Mouse Line Shows Low Efficiency of Labeling of Parvalbumin Interneurons in the Perirhinal Cortex.

The wide diversity of cortical inhibitory neuron types populating the cortex allows the assembly of diverse microcircuits and endows these circuits with different computational properties. Thus, characterizing neuronal diversity is fundamental to describe the building blocks of cortical microcircuits and probe their function. To this purpose, the mouse has emerged as a powerful tool to genetically label and manipulate specific inhibitory cell-types in the mammalian brain. Among these cell-types, the parvalbumin-expressing interneuron type (PV-INs) is perhaps the most characterized. Several mouse lines have been generated to target PV-INs. Among these mouse lines, the PV-IRES-Cre lines is the most widely used and demonstrated a high specificity and efficiency in targeting PV-INs in different cortical areas. However, a characterization of the performance across cortical regions is still missing. Here we show that the PV-IRES-Cre mouse line labels only a fraction of PV immunoreactive neurons in perirhinal cortex and other association areas. Our results point to a yet uncharacterized diversity within the PV-INs and emphasize the need to characterize these tools in specific cortical areas.

Resurgent Sodium Current in Neurons of the Cerebral Cortex.

In the late '90, Dr. Indira Raman, at the time a postdoctoral fellow with Dr. Bruce Bean, at Harvard University, identified a new type of sodium current, flowing through the channels that reopens when the membrane is repolarized. This current, called "resurgent Sodium current," was originally identified in cerebellar Purkinje neurons and has now been confirmed in around 20 different neuronal types. Since moving to Northwestern University in 1999 to establish her own research group, Dr. Raman has dedicated great efforts in identifying the mechanisms supporting the resurgent Sodium current and how its biophysical properties shape the firing of the different cell types. Her work has impacted greatly the field of cellular neurophysiology, from basic research to translation neuroscience. In fact, alterations in the resurgent sodium currents have been observed in several neuropathologies, from Huntington's disease to epilepsy. In this Perspective we will focus on the current knowledge on the expression and function of the resurgent Sodium current in neurons of the cerebral cortex and hippocampus. We will also briefly highlight the role of Dr. Raman's as teacher and mentor, not only for her pupils, but for the whole scientific community.

The Electrophysiological Determinants of Corticospinal Motor Neuron Vulnerability in ALS.

The brain is complex and heterogeneous. Even though numerous independent studies indicate cortical hyperexcitability as a potential contributor to amyotrophic lateral sclerosis (ALS) pathology, the mechanisms that are responsible for upper motor neuron (UMN) vulnerability remain elusive. To reveal the electrophysiological determinants of corticospinal motor neuron (CSMN, a.k.a UMN in mice) vulnerability, we investigated the motor cortex of hSOD1G93A mice at P30 (postnatal day 30), a presymptomatic time point. Glutamate uncaging by laser scanning photostimulation (LSPS) revealed altered dynamics especially within the inhibitory circuitry and more specifically in L2/3 of the motor cortex, whereas the excitatory microcircuits were unchanged. Observed microcircuitry changes were specific to CSMN in the motor column. Electrophysiological evaluation of the intrinsic properties in response to the microcircuit changes, as well as the exon microarray expression profiles of CSMN isolated from hSOD1G93A and healthy mice at P30, revealed the presence of a very dynamic set of events, ultimately directed to establish, maintain and retain the balance at this early stage. Also, the expression profile of key voltage-gated potassium and sodium channel subunits as well as of the inhibitory GABA receptor subunits and modulatory proteins began to suggest the challenges CSMN face at this early age. Since neurodegeneration is initiated when neurons can no longer maintain balance, the complex cellular events that occur at this critical time point help reveal how CSMN try to cope with the challenges of disease manifestation. This information is critically important for the proper modulation of UMNs and for developing effective treatment strategies.

Rbfox1 Mediates Cell-type-Specific Splicing in Cortical Interneurons.

Cortical interneurons display a remarkable diversity in their morphology, physiological properties, and connectivity. Elucidating the molecular determinants underlying this heterogeneity is essential for understanding interneuron development and function. We discovered that alternative splicing differentially regulates the integration of somatostatin- and parvalbumin-expressing interneurons into nascent cortical circuits through the cell-type-specific tailoring of mRNAs. Specifically, we identified a role for the activity-dependent splicing regulator Rbfox1 in the development of cortical interneuron-subtype-specific efferent connectivity. Our work demonstrates that Rbfox1 mediates largely non-overlapping alternative splicing programs within two distinct but related classes of interneurons.

Diversity and Connectivity of Layer 5 Somatostatin-Expressing Interneurons in the Mouse Barrel Cortex.

Inhibitory interneurons represent 10-15% of the neurons in the somatosensory cortex, and their activity powerfully shapes sensory processing. Three major groups of GABAergic interneurons have been defined according to developmental, molecular, morphological, electrophysiological, and synaptic features. Dendritic-targeting somatostatin-expressing interneurons (SST-INs) have been shown to display diverse morphological, electrophysiological, and molecular properties and activity patterns in vivo However, the correlation between these properties and SST-IN subtype is unclear. In this study, we aimed to correlate the morphological diversity of layer 5 (L5) SST-INs with their electrophysiological and molecular diversity in mice of either sex. Our morphological analysis demonstrated the existence of three subtypes of L5 SST-INs with distinct electrophysiological properties: T-shaped Martinotti cells innervate L1, and are low-threshold spiking; fanning-out Martinotti cells innervate L2/3 and the lower half of L1, and show adapting firing patterns; non-Martinotti cells innervate L4, and show a quasi-fast spiking firing pattern. We estimated the proportion of each subtype in L5 and found that T-shaped Martinotti, fanning-out Martinotti, and Non-Martinotti cells represent ∼10, ∼50, and ∼40% of L5 SST-INs, respectively. Last, we examined the connectivity between the three SST-IN subtypes and L5 pyramidal cells (PCs). We found that L5 T-shaped Martinotti cells inhibit the L1 apical tuft of nearby PCs; L5 fanning-out Martinotti cells also inhibit nearby PCs but they target the dendrite mainly in L2/3. On the other hand, non-Martinotti cells inhibit the dendrites of L4 neurons while avoiding L5 PCs. Our data suggest that morphologically distinct SST-INs gate different excitatory inputs in the barrel cortex.SIGNIFICANCE STATEMENT Morphologically diverse layer 5 SST-INs show different patterns of activity in behaving animals. However, little is known about the abundance and connectivity of each morphological type and the correlation between morphological subtype and spiking properties. We demonstrate a correlation between the morphological and electrophysiological diversity of layer 5 SST-INs. Based on these findings we built a classifier to infer the abundance of each morphological subtype. Last, using paired recordings combined with morphological analysis, we investigated the connectivity of each morphological subtype. Our data suggest that, by targeting different cell types and cellular compartments, morphologically diverse SST-INs might gate different excitatory inputs in the mouse barrel cortex.

Strategies and Tools for Combinatorial Targeting of GABAergic Neurons in Mouse Cerebral Cortex.

Systematic genetic access to GABAergic cell types will facilitate studying the function and development of inhibitory circuitry. However, single gene-driven recombinase lines mark relatively broad and heterogeneous cell populations. Although intersectional approaches improve precision, it remains unclear whether they can capture cell types defined by multiple features. Here we demonstrate that combinatorial genetic and viral approaches target restricted GABAergic subpopulations and cell types characterized by distinct laminar location, morphology, axonal projection, and electrophysiological properties. Intersectional embryonic transcription factor drivers allow finer fate mapping of progenitor pools that give rise to distinct GABAergic populations, including laminar cohorts. Conversion of progenitor fate restriction signals to constitutive recombinase expression enables viral targeting of cell types based on their lineage and birth time. Properly designed intersection, subtraction, conversion, and multi-color reporters enhance the precision and versatility of drivers and viral vectors. These strategies and tools will facilitate studying GABAergic neurons throughout the mouse brain.

Expression and functional roles of Kv7/KCNQ/M-channels in rat medial entorhinal cortex layer II stellate cells.

The medial entorhinal cortex (MEC) is important for spatial navigation and memory. Stellate cells (SCs) of MEC layer II provide major input to the hippocampus, and are thought to be the neuronal correlate of the grid cells. Their electrophysiological properties have been used to explain grid field formation. However, little is known about the functional roles of potassium channels in SCs. M-current is a slowly activating potassium current, active at subthreshold potentials. Although some studies have suggested that Kv7/M-channels may affect subthreshold resonance in SCs, others have found no Kv7/M-current in these cells, so the expression and roles of Kv7/M-channels in SCs are still debated. Using whole-cell voltage-clamp, we have identified a typical M-current with pharmacological properties characteristic of Kv7/M-channels in rat MEC SCs. Current-clamp experiments showed that the specific Kv7/M-channel blocker XE991 increased SCs excitability, and reduced spike frequency adaptation. Our results demonstrate that Kv7/M-channels are expressed in SCs and contribute substantially to regulation of excitability in these cells.

Distinct developmental patterns in the expression of transient, persistent, and resurgent Na+ currents in entorhinal cortex layer-II neurons.

Sub- and near-threshold voltage-dependent Na+ currents (VDSCs) are of major importance in determining the electrical properties of medial entorhinal cortex (mEC) layer-II neurons. Developmental changes in the ability of mEC layer-II stellate cells (SCs) to generate Na+ -dependent, subthreshold electrical events have been reported between P14 and P18. In this study we examined the modifications occurring in the various components of VDSCs during postnatal development of mEC SCs. The transient, resurgent, and persistent Na+ currents (I(NaT), I(NaR), and I(NaP), respectively) showed distinct patterns of developmental expression in the time window considered (P5 to P24-27). All three currents prominently and steeply increased in absolute amplitude and conductance from P5 to at least P16. However, capacitive charge accumulation, an index of membrane surface area, also markedly increased in the same time window, and in the case of I(NaT) the specific conductance per unit of accumulated capacitive charge remained relatively constant. By contrast, specific I(NaR) and I(NaP) conductances showed a significant tendency to increase, especially from P5 to P18. Neither I(NaR) nor I(NaP) represented a constant fraction of the total Na+ current at all developmental ages. Indeed, detectable levels of I(NaR) and I(NaP) were present in only ~20% and ~70%, respectively, of the cells on P5, and were observed in all cells only from P10 onwards. Moreover, the average I(NaR)-to-I(NaT) conductance ratio increased steadily from ~0.004 (P5) up to a plateau level of ~0.05 (P22+), whereas the I(NaP)-to-I(NaT) conductance ratio increased only from ~0.009 on P5 to ~0.02 on P22+. The relative increase in conductance ratio from P5 to P22 was significantly greater for I(NaR) than for I(NaP), indicating that I(NaR) expression starts later than that of I(NaP). These findings show that in mEC layer-II SCs the single functional components of the VDSC are regulated differentially from each other as far as their developmental expression is concerned.

Differential effects of Zn2+ on activation, deactivation, and inactivation kinetics in neuronal voltage-gated Na+ channels.

Whole-cell, patch-clamp recordings were carried out in acutely dissociated neurons from entorhinal cortex (EC) layer II to study the effects of Zn(2+) on Na(+) current kinetics and voltage dependence. In the presence of 200 µM extracellular Cd(2+) to abolish voltage-dependent Ca(2+) currents, and 100 mM extracellular Na(+), 1 mM Zn(2+) inhibited the transient Na(+) current, I (NaT), only to a modest degree (~17% on average). A more pronounced inhibition (~36%) was induced by Zn(2+) when extracellular Na(+) was lowered to 40 mM. Zn(2+) also proved to modify I (NaT) voltage-dependent and kinetic properties in multiple ways. Zn(2+) (1 mM) shifted the voltage dependence of I (NaT) activation and that of I (NaT) onset speed in the positive direction by ~5 mV. The voltage dependence of I (NaT) steady-state inactivation and that of I (NaT) inactivation kinetics were markedly less affected by Zn(2+). By contrast, I (NaT) deactivation speed was prominently accelerated, and its voltage dependence was shifted by a significantly greater amount (~8 mV on average) than that of I (NaT) activation. In addition, the kinetics of I (NaT) recovery from inactivation were significantly slowed by Zn(2+). Zn(2+) inhibition of I (NaT) showed no signs of voltage dependence over the explored membrane-voltage window, indicating that the above effects cannot be explained by voltage dependence of Zn(2+)-induced channel-pore block. These findings suggest that the multiple, voltage-dependent state transitions that the Na(+) channel undergoes through its activation path are differentially sensitive to the gating-modifying effects of Zn(2+), thus resulting in differential modifications of the macroscopic current's activation, inactivation, and deactivation. Computer modeling provided support to this hypothesis.