Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Kv2.1 Potassium Channels Regulate Repetitive Burst Firing in Extratelencephalic Neocortical Pyramidal Neurons.

Coincidence detection and cortical rhythmicity are both greatly influenced by neurons' propensity to fire bursts of action potentials. In the neocortex, repetitive burst firing can also initiate abnormal neocortical rhythmicity (including epilepsy). Bursts are generated by inward currents that underlie a fast afterdepolarization (fADP) but less is known about outward currents that regulate bursting. We tested whether Kv2 channels regulate the fADP and burst firing in labeled layer 5 PNs from motor cortex of the Thy1-h mouse. Kv2 block with guangxitoxin-1E (GTx) converted single spike responses evoked by dendritic stimulation into multispike bursts riding on an enhanced fADP. Immunohistochemistry revealed that Thy1-h PNs expressed Kv2.1 (not Kv2.2) channels perisomatically (not in the dendrites). In somatic macropatches, GTx-sensitive current was the largest component of outward current with biophysical properties well-suited for regulating bursting. GTx drove ~40% of Thy1 PNs stimulated with noisy somatic current steps to repetitive burst firing and shifted the maximal frequency-dependent gain. A network model showed that reduction of Kv2-like conductance in a small subset of neurons resulted in repetitive bursting and entrainment of the circuit to seizure-like rhythmic activity. Kv2 channels play a dominant role in regulating onset bursts and preventing repetitive bursting in Thy1 PNs.

Specialized Subpopulations of Deep-Layer Pyramidal Neurons in the Neocortex: Bridging Cellular Properties to Functional Consequences.

Neocortical pyramidal neurons with somata in layers 5 and 6 are among the most visually striking and enigmatic neurons in the brain. These deep-layer pyramidal neurons (DLPNs) integrate a plethora of cortical and extracortical synaptic inputs along their impressive dendritic arbors. The pattern of cortical output to both local and long-distance targets is sculpted by the unique physiological properties of specific DLPN subpopulations. Here we revisit two broad DLPN subpopulations: those that send their axons within the telencephalon (intratelencephalic neurons) and those that project to additional target areas outside the telencephalon (extratelencephalic neurons). While neuroscientists across many subdisciplines have characterized the intrinsic and synaptic physiological properties of DLPN subpopulations, our increasing ability to selectively target and manipulate these output neuron subtypes advances our understanding of their distinct functional contributions. This Viewpoints article summarizes our current knowledge about DLPNs and highlights recent work elucidating the functional differences between DLPN subpopulations.

Temporal dynamics of L5 dendrites in medial prefrontal cortex regulate integration versus coincidence detection of afferent inputs.

Distinct brain regions are highly interconnected via long-range projections. How this inter-regional communication occurs depends not only upon which subsets of postsynaptic neurons receive input, but also, and equally importantly, upon what cellular subcompartments the projections target. Neocortical pyramidal neurons receive input onto their apical dendrites. However, physiological characterization of these inputs thus far has been exclusively somatocentric, leaving how the dendrites respond to spatial and temporal patterns of input unexplored. Here we used a combination of optogenetics with multisite electrode recordings to simultaneously measure dendritic and somatic responses to afferent fiber activation in two different populations of layer 5 (L5) pyramidal neurons in the rat medial prefrontal cortex (mPFC). We found that commissural inputs evoked monosynaptic responses in both intratelencephalic (IT) and pyramidal tract (PT) dendrites, whereas monosynaptic hippocampal input primarily targeted IT, but not PT, dendrites. To understand the role of dendritic integration in the processing of long-range inputs, we used dynamic clamp to simulate synaptic currents in the dendrites. IT dendrites functioned as temporal integrators that were particularly responsive to dendritic inputs within the gamma frequency range (40-140 Hz). In contrast, PT dendrites acted as coincidence detectors by responding to spatially distributed signals within a narrow time window. Thus, the PFC extracts information from different brain regions through the combination of selective dendritic targeting and the distinct dendritic physiological properties of L5 pyramidal dendrites.

Dendritic generation of mGluR-mediated slow afterdepolarization in layer 5 neurons of prefrontal cortex.

Many prefrontal cortex (PFC)-dependent tasks require individual neurons to fire persistently in response to brief stimuli. Persistent activity is proposed to involve changes in intrinsic properties, resulting in an increased sensitivity to inputs. The dendrite is particularly relevant to this hypothesis because it receives the majority of synaptic inputs and is enriched for conductances implicated in persistent firing. We provide evidence that dendritic conductances contribute to persistent activity-related changes in intrinsic properties. The effects of Group 1 metabotropic glutamate receptor (mGluR) activation on persistent activity-related properties were tested in two classes of rat L5 neurons with distinct membrane properties: those projecting to the pons (CPn) and those projecting across the commissure to the contralateral cortex (COM). mGluR activation produced long-term changes in the subthreshold properties of CPn, but not COM neurons. These changes were indicative of a decrease in hyperpolarization-activated cation nonselective current (I(h)) at the soma and dendrite. mGluR activation also transiently increased the amplitude of the postburst slow afterdepolarization potential (sADP) at the soma of both neuron types. Interestingly, the sADP occurred along the extent of the apical dendrite in CPn and COM neurons. Simultaneous somatic/dendritic recordings revealed that the dendritic sADP does not result solely from passive propagation of the somatic sADP. Focal mGluR activation in L5, near the soma or at the border of L1/L2, near the tuft, generates a local sADP. This dendritic depolarization may act synergistically with synaptic input to regulate mnemonic activity in PFC.

Areal specializations in the morpho-electric and transcriptomic properties of primate layer 5 extratelencephalic projection neurons.

Large-scale analysis of single-cell gene expression has revealed transcriptomically defined cell subclasses present throughout the primate neocortex with gene expression profiles that differ depending upon neocortical region. Here, we test whether the interareal differences in gene expression translate to regional specializations in the physiology and morphology of infragranular glutamatergic neurons by performing Patch-seq experiments in brain slices from the temporal cortex (TCx) and motor cortex (MCx) of the macaque. We confirm that transcriptomically defined extratelencephalically projecting neurons of layer 5 (L5 ET neurons) include retrogradely labeled corticospinal neurons in the MCx and find multiple physiological properties and ion channel genes that distinguish L5 ET from non-ET neurons in both areas. Additionally, while infragranular ET and non-ET neurons retain distinct neuronal properties across multiple regions, there are regional morpho-electric and gene expression specializations in the L5 ET subclass, providing mechanistic insights into the specialized functional architecture of the primate neocortex.

Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues.

Recent advances in tissue processing, labeling, and fluorescence microscopy are providing unprecedented views of the structure of cells and tissues at sub-diffraction resolutions and near single molecule sensitivity, driving discoveries in diverse fields of biology, including neuroscience. Biological tissue is organized over scales of nanometers to centimeters. Harnessing molecular imaging across three-dimensional samples on this scale requires new types of microscopes with larger fields of view and working distance, as well as higher imaging throughput. We present a new expansion-assisted selective plane illumination microscope (ExA-SPIM) with diffraction-limited and aberration-free performance over a large field of view (85 mm 2 ) and working distance (35 mm). Combined with new tissue clearing and expansion methods, the microscope allows nanoscale imaging of centimeter-scale samples, including entire mouse brains, with diffraction-limited resolutions and high contrast without sectioning. We illustrate ExA-SPIM by reconstructing individual neurons across the mouse brain, imaging cortico-spinal neurons in the macaque motor cortex, and tracing axons in human white matter.

Input rate encoding and gain control in dendrites of neocortical pyramidal neurons.

Elucidating how neurons encode network activity is essential to understanding how the brain processes information. Neocortical pyramidal cells receive excitatory input onto spines distributed along dendritic branches. Local dendritic branch nonlinearities can boost the response to spatially clustered and synchronous input, but how this translates into the integration of patterns of ongoing activity remains unclear. To examine dendritic integration under naturalistic stimulus regimes, we use two-photon glutamate uncaging to repeatedly activate multiple dendritic spines at random intervals. In the proximal dendrites of two populations of layer 5 pyramidal neurons in the mouse motor cortex, spatially restricted synchrony is not a prerequisite for dendritic boosting. Branches encode afferent inputs with distinct rate sensitivities depending upon cell and branch type. Thus, inputs distributed along a dendritic branch can recruit supralinear boosting and the window of this nonlinearity may provide a mechanism by which dendrites can preferentially amplify slow-frequency network oscillations.

Projection-specific neuromodulation of medial prefrontal cortex neurons.

Mnemonic persistent activity in the prefrontal cortex (PFC) constitutes the neural basis of working memory. To understand how neuromodulators contribute to the generation of persistent activity, it is necessary to identify the intrinsic properties of the layer V pyramidal neurons that transfer this information to downstream networks. Here we show that the somatic dynamic and integrative properties of layer V pyramidal neurons in the rat medial PFC depend on whether they project subcortically to the pons [corticopontine (CPn)] or to the contralateral cortex [commissural (COM)]. CPn neurons display low temporal summation and accelerate in firing frequency when depolarized, whereas COM neurons have high temporal summation and display spike frequency accommodation. In response to dynamic stimuli, COM neurons act as low-pass filters, whereas CPn neurons act as bandpass filters, resonating in the theta frequency range (3-6 Hz). The disparate subthreshold properties of COM and CPn neurons can be accounted for by differences in the hyperpolarization-activated cyclic nucleotide gated cation h-current. Interestingly, neuromodulators hypothesized to enhance mnemonic persistent activity affect COM and CPn neurons distinctly. Adrenergic modulation shifts the dynamic properties of CPn but not COM neurons and increases the excitability of CPn neurons significantly more than COM neurons. In response to cholinergic modulation, CPn neurons were much more likely to display activity-dependent intrinsic persistent firing than COM neurons. Together, these data suggest that the two categories of projection neurons may subserve separate functions in PFC and may be engaged differently during working memory processes.

Feedforward compensation mediated by the central and peripheral actions of a single neuropeptide discovered using representational difference analysis.

Compensatory mechanisms are often used to achieve stability by reducing variance, which can be accomplished via negative feedback during homeostatic regulation. In principle, compensation can also be implemented through feedforward mechanisms where a regulator acts to offset the anticipated output variation; however, few such neural mechanisms have been demonstrated. We provide evidence that an Aplysia neuropeptide, identified using an enhanced representational difference analysis procedure, implements feedforward compensation within the feeding network. We named the novel peptide "allatotropin-related peptide" (ATRP) because of its similarity to insect allatotropin. Mass spectrometry confirmed the peptide's identity, and in situ hybridization and immunostaining mapped its distribution in the Aplysia CNS. ATRP is present in the higher-order cerebral-buccal interneuron (CBI) CBI-4, but not in CBI-2. Previous work showed that CBI-4-elicited motor programs have a shorter protraction duration than those elicited by CBI-2. Here we show that ATRP shortens protraction duration of CBI-2-elicited ingestive programs, suggesting a contribution of ATRP to the parametric differences between CBI-4-evoked and CBI-2-evoked programs. Importantly, because Aplysia muscle contractions are a graded function of motoneuronal activity, one consequence of the shortening of protraction is that it can weaken protraction movements. However, this potential weakening is offset by feedforward compensatory actions exerted by ATRP. Centrally, ATRP increases the activity of protraction motoneurons. Moreover, ATRP is present in peripheral varicosities of protraction motoneurons and enhances peripheral motoneuron-elicited protraction muscle contractions. Therefore, feedforward compensatory mechanisms mediated by ATRP make it possible to generate a faster movement with an amplitude that is not greatly reduced, thereby producing stability.

A "sample-and-hold" pulse-counting integrator as a mechanism for graded memory underlying sensorimotor adaptation.

The mechanisms behind the induction of cellular correlates of memory by sensory input and their contribution to meaningful behavioral changes are largely unknown. We previously reported a graded memory in the form of sensorimotor adaptation in the electromotor output of electric fish. Here we show that the mechanism for this adaptation is a synaptically induced long-lasting shift in intrinsic neuronal excitability. This mechanism rapidly integrates hundreds of spikes in a second, or gradually integrates the same number of spikes delivered over tens of minutes. Thus, this mechanism appears immune to frequency-dependent fluctuations in input and operates as a simple pulse counter over a wide range of time scales, enabling it to transduce graded sensory information into a graded memory and a corresponding change in the behavioral output. This adaptation is based on an NMDA receptor-mediated change in intrinsic excitability of the postsynaptic neurons involving the Ca2+-dependent activation of TRP channels.

Calcium dynamics encode the magnitude of a graded memory underlying sensorimotor adaptation.

The role of Ca(2+) in the induction of neural correlates of memory has frequently been described in binary terms despite the fact that many forms of memory are graded in their strength and/or persistence. We find that Ca(2+) dynamics encode the magnitude of sensorimotor adaptation of the electromotor output in a weakly electric fish. The neural correlate of this memory is a synaptically induced Ca(2+)-dependent enhancement of intrinsic excitability of neurons responsible for setting the electromotor output. Changes in Ca(2+) during induction accurately predict the magnitude of this graded memory over a wide range of stimuli. Thus despite operating over a range from seconds to tens of minutes, the encoding of graded memory can be mediated by a relatively simple cellular mechanism.

A specific synaptic pathway activates a conditional plateau potential underlying protraction phase in the Aplysia feeding central pattern generator.

A common feature in the architecture of neuronal networks is a high degree of seemingly redundant synaptic connectivity. In many cases, the synaptic inputs converging on any particular neuron all use the same neurotransmitter and appear to be fundamentally equivalent. Here, we analyze a striking counterexample in which such inputs are not equivalent and, as a result, play very different roles in the generation of the pattern of activity produced by the network. In the feeding central pattern generator of Aplysia, the pattern-initiating neuron B50 elicits motor programs by exciting the plateauing neuron B31/B32 in two ways: directly and indirectly through neuron B63. All of the synaptic connections use ACh. Despite the direct input of B50 to B31/B32, the indirect pathway of exciting B31/B32 through B63 is required for B50 to elicit the B31/B32 plateau potential and the motor program. We dissect this requirement using the muscarinic cholinergic antagonist pirenzepine. Pirenzepine blocks the B50-elicited motor program, the plateau potential in B31/B32, and, notably, a slow component of the EPSP elicited in B31/B32 by B63 but not that elicited by B50. The muscarinic agonist oxotremorine restores the plateau potential in B31/B32 and eliminates the necessity for B63 in B50-elicited motor programs. Together, our analysis shows that the plateau potential in B31/B32 is not endogenous but conditional, furthermore conditional on one particular synaptic input, that from B63. Thus, among several inputs to B31/B32 that use the same transmitter, the input from B63 is functionally distinct in its preferential access to the plateau potential that represents the committed step toward the initiation of a motor program.

Subcircuit-specific neuromodulation in the prefrontal cortex.

During goal-directed behavior, the prefrontal cortex (PFC) exerts top-down control over numerous cortical and subcortical regions. PFC dysfunction has been linked to many disorders that involve deficits in cognitive performance, attention, motivation, and/or impulse control. A common theme among these disorders is that neuromodulation of the PFC is disrupted. Anatomically, the PFC is reciprocally connected with virtually all neuromodulatory centers. Recent studies of PFC neurons, both in vivo and ex vivo, have found that subpopulations of prefrontal projection neurons can be segregated into distinct subcircuits based on their long-range projection targets. These subpopulations differ in their connectivity, intrinsic properties, and responses to neuromodulators. In this review we outline the evidence for subcircuit-specific neuromodulation in the PFC, and describe some of the functional consequences of selective neuromodulation on behavioral states during goal-directed behavior.

Species-specific differences in the medial prefrontal projections to the pons between rat and rabbit.

The medial prefrontal cortex (mPFC) of both rats and rabbits has been shown to support trace eyeblink conditioning, presumably by providing an input to the cerebellum via the pons that bridges the temporal gap between conditioning stimuli. The pons of rats and rabbits, however, shows divergence in gross anatomical organization, leaving open the question of whether the topography of prefrontal inputs to the pons is similar in rats and rabbits. To investigate this question, we injected anterograde tracer into the mPFC of rats and rabbits to visualize and map in 3D the distribution of labeled terminals in the pons. Effective mPFC injections showed labeled axons in the ipsilateral descending pyramidal tract in both species. In rats, discrete clusters of densely labeled terminals were observed primarily in the rostromedial pons. Clusters of labeled terminals were also observed contralateral to mPFC injection sites in rats, appearing as a less dense "mirror-image" of ipsilateral labeling. In rabbits, mPFC labeled corticopontine terminals were absent in the rostral pons, and instead were restricted to the intermediate pons. The densest terminal fields were typically observed in association with the ipsilateral pyramidal tract as it descended ventromedially through the rabbit pons. No contralateral terminal labeling was observed for any injections made in the rabbit mPFC. The results suggest the possibility that mPFC inputs to the pons may be integrated with different sources of cortical inputs between rats and rabbits. The resulting implications for mPFC or pons manipulations for studies of trace eyeblink in each species are discussed.