Intratelencephalic Research Articles

Pyramidal tract neurons drive amplification of excitatory inputs to striatum through cholinergic interneurons.

Corticostriatal connectivity is central for many cognitive and motor processes, such as reinforcement or action initiation and invigoration. The cortical input to the striatum arises from two main cortical populations: intratelencephalic (IT) and pyramidal tract (PT) neurons. We report a previously unknown excitatory circuit, supported by a polysynaptic motif from PT neurons to cholinergic interneurons (ChIs) to glutamate-releasing axons, which runs in parallel to the canonical monosynaptic corticostriatal connection. This motif conveys a delayed second phase of excitation to striatal spiny projection neurons, through an acetylcholine-dependent glutamate release mechanism mediated by α4-containing nicotinic receptors, resulting in biphasic corticostriatal signals. These biphasic signals are a hallmark of PT, but not IT, corticostriatal inputs, due to a stronger relative input from PT neurons to ChIs. These results describe a previously unidentified circuit mechanism by which PT activity amplifies excitatory inputs to the striatum, with potential implications for behavior, plasticity, and learning.

Robust cell-type-specific associations with Alzheimer's disease within excitatory and inhibitory neuronal subclasses.

Cortical neuron loss is a pathological hallmark of late-onset Alzheimer's disease (AD). However, despite recent single-cell transcriptomic analyses of human AD brain tissue, it remains unclear which neuronal subtypes are most vulnerable to degeneration. Here we applied cell-type proportion estimation methods to multiple RNA sequencing (RNAseq) datasets derived from human postmortem brain to identify the neuronal subtypes most strongly associated with AD. Postmortem bulk-brain RNAseq data collected from three AD studies were analyzed (n=953), comprising six datasets of different neocortical regions. Relative cell type proportions were estimated for each brain sample using the markerGeneProfile package and marker genes for specific neuronal subtypes derived from ultra-high depth single-nucleus RNAseq (Hodge et al., 2019). Linear models were used to test associations of cell-type proportion estimations (CTPs) with AD diagnosis in each dataset separately, followed by random-effects meta-analysis. Pseudo-bulk investigation of the Mathys et al. (2019) single-nucleus RNAseq dataset was used to further validate findings. Follow-up in the Religious Orders Study and Memory and Aging Project (ROS/MAP) cohorts (n=593) permitted the identification of specific neuropathologies associated with each neuronal subtype. Meta-analysis revealed several neuronal subtypes significantly associated with AD, with the strongest signals from intra-telencephalic (IT) excitatory neurons (meta p=4.82x10-13 ) and somatostatin (SST) inhibitory neurons (meta p= 1.55x10-10 ). Pseudo-bulk single-nucleus analysis suggested that these signals are most likely due to differences in cell proportions rather than differences in expression of any predominant marker gene for each class. In-depth analysis of CTPs in ROS/MAP consistently implicated a decrease in IT and SST CTPs with increasing amyloid and tau neuropathology, as well as more severe cognitive decline prior to death. Decreases in SST and IT neuron subtypes, estimated from bulk RNAseq data, were significantly associated with AD diagnosis across multiple cortical regions. The SST signal in particular is robust to the exclusion of the SST mRNA from CTP calculations, suggesting the pathological relevance of a loss of SST-expressing neurons rather than a downregulation of the SST gene specifically. These findings support seminal work implicating somatostatin in the pathogenesis of AD and helps to re-focus our current understanding of neuronal vulnerability in AD.

How does TMS “engage” the human cortex? Insights from structural and functional mapping of the precentral motor hand knob

Abstract Background: TMS of the primary motor hand area (M1-HAND) can probe the function of fast corticomotor connections to the contralateral hand. TMS gives rise to indirect (transsynaptic) excitation of large pyramidal tract (PT) neurons in M1-HAND that make monosynaptic connections with spinal motoneurons in the cervical cord. Other cell types are effectively excited by TMS as well. Yet, it is still unclear which neuronal elements (e.g. axon terminals or axon hillock), which cell types (e.g. PT neurons, intra-telencephalic (IT) neurons, or inhibitory interneurons), and which sites in the precentral hand knob (e.g. crown, lip, or sulcus region) are preferentially depolarized by the TMS-induced electrical field in the precentral gyrus. Objective: To address whether non-invasive neuroimaging in human volunteers can help to pinpoint the primary targets for TMS of M1-HAND. Methods: Structural and functional neuroimaging (MRI and PET) were employed in a series of experiments to target the M1-HAND. Structural MRI was used for shaped-based TMS mapping, electrical field modelling, and mapping of cortical myelin content. Functional MRI and PET were used to localize clusters in the precentral hand knob that are functionally activated by focal TMS of M1-HAND. Results: Inter-individual differences in precentral cortical myelin content account for interindividual differences in rostro-caudal “motor hotspot” location, linking cortical myelination to TMS target engagement. Functional brain mapping reveals different peak target sites in the precentral hand knob and indicates that PT and IT neurons as well as inhibitory interneurons are effectively excited by TMS. Discussion: Structural and functional neuroimaging can help to elucidate how TMS engages its local cortical targets in the precentral gyrus. Causal interpretation of the results needs to consider the potential and pitfalls inherent to the neuroimaging method and the physiological implications of the TMS protocol (e.g. intensity, number of stimuli, online-offline TMS). Keywords: Transcranial magnetic stimulation, Motor cortex, Magnetic resonance imaging, Positron emission tomography

Variations in Commissural Input Processing Across Different Types of Cortical Projection Neurons.

To understand how incoming cortical inputs are processed by different types of cortical projection neurons in the medial prefrontal cortex, we compared intrinsic physiological properties of and commissural excitatory/inhibitory influences on layer 5 intratelencephalic (IT), layer 5 pyramidal tract (PT), and layers 2/3 IT projection neurons. We found that intrinsic physiological properties and commissural synaptic transmission varied across the three types of projection neurons. The rank order of intrinsic excitability was layer 5 PT > layer 5 IT > layers 2/3 IT neurons. Commissural connectivity was higher in layers 2/3 than layer 5 projection neurons, but commissural excitatory influence was stronger on layer 5 than layers 2/3 pyramidal neurons. Paired-pulse ratio was also greater in PT than IT neurons. These results indicate that commissural inputs activate deep layer PT neurons most preferentially and superficial layer IT neurons least preferentially. Deep layer PT neurons might faithfully transmit cortical input signals to downstream subcortical structures for reliable control of behavior, whereas superficial layer IT neurons might integrate cortical input signals from diverse sources in support of higher-order cognitive functions.

Parallel processing of working memory and temporal information by distinct types of cortical projection neurons

It is unclear how different types of cortical projection neurons work together to support diverse cortical functions. We examined the discharge characteristics and inactivation effects of intratelencephalic (IT) and pyramidal tract (PT) neurons—two major types of cortical excitatory neurons that project to cortical and subcortical structures, respectively—in the deep layer of the medial prefrontal cortex in mice performing a delayed response task. We found stronger target-dependent firing of IT than PT neurons during the delay period. We also found the inactivation of IT neurons, but not PT neurons, impairs behavioral performance. In contrast, PT neurons carry more temporal information than IT neurons during the delay period. Our results indicate a division of labor between IT and PT projection neurons in the prefrontal cortex for the maintenance of working memory and for tracking the passage of time, respectively.

Distinct populations of cortical pyramidal neurons mediate drug reward and aversion

Processing within the anterior cingulate cortex (ACC) is crucial for the patterning of appropriate behavior, and ACC dysfunction following chronic drug use is thought to play a major role in drug addiction. However, cortical pyramidal projection neurons can be subdivided into two major types (intratelencephalic (IT) and pyramidal tract (PT)), with distinct inputs and projection targets, molecular and receptor profiles, morphologies and electrophysiological properties. Yet, how each of these cell populations modulate behavior related to addiction is unknown. We demonstrate that PT neurons regulate the positive features of a drug experience whereas IT neurons regulate the negative features. These findings support a revised theory of cortical function in addiction, with distinct cells and circuits mediating reward and aversion.

Hippocampal inputs engage CCK+ interneurons to mediate endocannabinoid-modulated feed-forward inhibition in the prefrontal cortex.

Connections from the ventral hippocampus (vHPC) to the prefrontal cortex (PFC) regulate cognition, emotion, and memory. These functions are also tightly controlled by inhibitory networks in the PFC, whose disruption is thought to contribute to mental health disorders. However, relatively little is known about how the vHPC engages different populations of interneurons in the PFC. Here we use slice physiology and optogenetics to study vHPC-evoked feed-forward inhibition in the mouse PFC. We first show that cholecystokinin (CCK+), parvalbumin (PV+), and somatostatin (SOM+) expressing interneurons are prominent in layer 5 (L5) of infralimbic PFC. We then show that vHPC inputs primarily activate CCK+ and PV+ interneurons, with weaker connections onto SOM+ interneurons. CCK+ interneurons make stronger synapses onto pyramidal tract (PT) cells over nearby intratelencephalic (IT) cells. However, CCK+ inputs undergo depolarization-induced suppression of inhibition (DSI) and CB1 receptor modulation only at IT cells. Moreover, vHPC-evoked feed-forward inhibition undergoes DSI only at IT cells, confirming a central role for CCK+ interneurons. Together, our findings show how vHPC directly engages multiple populations of inhibitory cells in deep layers of the infralimbic PFC, highlighting unexpected roles for both CCK+ interneurons and endocannabinoid modulation in hippocampal-prefrontal communication.

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington's disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

Differential Striatal Axonal Arborizations of the Intratelencephalic and Pyramidal-Tract Neurons: Analysis of the Data in the MouseLight Database.

There exist two major types of striatum-targeting neocortical neurons, specifically, intratelencephalic (IT) neurons and pyramidal-tract (PT) neurons. Regarding their striatal projections, it was once suggested that IT axons are extended whereas PT axons are primarily focal. However, subsequent study with an increased number of well-stained extended axons concluded that such an apparent distinction was spurious due to limited sample size. Recent work using genetically labeled neurons reintroduced the differential spatial extent of the striatal projections of IT and PT neurons through population-level analyses, complemented by observations of single axons. However, quantitative IT vs. PT comparison of a large number of axons remained to be conducted. We analyzed the data of axonal end-points of 161 IT neurons and 33 PT neurons in the MouseLight database (http://ml-neuronbrowser.janelia.org/). The number of axonal end-points in the ipsilateral striatum exhibits roughly monotonically decreasing distributions in both neuron types. Excluding neurons with no ipsilateral end-point, the distributions of the logarithm of the number of ipsilateral end-points are considerably overlapped between IT and PT neurons, although the proportion of neurons having more than 50 ipsilateral end-points is somewhat larger in IT neurons than in PT neurons. Looking at more details, among IT subpopulations in the secondary motor area (MOs), layer 5 neurons and bilateral striatum-targeting layer 2/3 neurons, but not contralateral striatum-non-targeting layer 2/3 neurons, have a larger number of ipsilateral end-points than MOs PT neurons. We also found that IT ipsilateral striatal axonal end-points are on average more widely distributed than PT end-points, especially in the medial-lateral direction. These results indicate that IT and PT striatal axons differ in the frequencies and spatial extent of end-points while there are wide varieties within each neuron type.

Dopaminergic D1 receptor effects on commissural inputs targeting layer V pyramidal subtypes of the mouse medial prefrontal cortex.

In humans, prefrontal cortical areas are known to support goal‐directed behaviors, mediating a variety of functions that render behavior more flexible in the face of changing environmental demands. In mice, these functions are mediated by homologous regions within medial prefrontal cortex (mPFC) and rely heavily on proper dopaminergic tone. Comprised of two major subtypes, pyramidal tract (PT) and intratelencephalic (IT), layer V pyramidal cells serve as the major outputs of the mPFC, targeting brainstem nuclei and the contralateral hemisphere, respectively. However, it remains relatively unknown how cortical inputs targeting these subtypes are integrated. We explored how layer V pyramidal cell subtypes integrate commissural inputs, which integrate information flow between the hemispheres. An optogenetic approach was used to elicit commissural fiber activation onto PT and IT cells and the effects of D1 receptor activation on elicited EPSPs were explored. We showed that commissural inputs into PT and IT cells elicit facilitating and depressing EPSP patterns, respectively. D1 receptor activation increased the initial EPSP amplitude, enhanced EPSP facilitation, and prolonged EPSP decay time constant in PT cells. In IT cells, D1 receptor activation increased commissural‐evoked initial EPSP amplitude but did not affect facilitation or EPSP shape. Furthermore, D1 receptor activation elicited burst firing in a subset of PT cells in response to commissural fiber activation. Combined, these results lend insight into the role of dopamine in promoting persistent firing and temporal integration in PT and IT cells, respectively, that in turn may contribute to working memory functions.

Neocortical Projection Neurons Instruct Inhibitory Interneuron Circuit Development in a Lineage-Dependent Manner

Neocortical circuits consist of stereotypical motifs that must self-assemble during development. Recent evidence suggests that the subtype identity of both excitatory projection neurons (PNs) and inhibitory interneurons (INs) is important for this process. We knocked out the transcription factor Satb2 in PNs to induce those of the intratelencephalic (IT) type to adopt a pyramidal tract (PT)-type identity. Loss of IT-type PNs selectively disrupted the lamination and circuit integration of INs derived from the caudal ganglionic eminence (CGE). Strikingly, reprogrammed PNs demonstrated reduced synaptic targeting of CGE-derived INs relative to controls. In control mice, IT-type PNs targeted neighboring CGE INs, while PT-type PNs did not in deep layers, confirming this lineage-dependent motif. Finally, single-cell RNA sequencing revealed that major CGE IN subtypes were conserved after loss of IT PNs, but with differential transcription of synaptic proteins and signaling molecules. Thus, IT-type PNs influence CGE-derived INs in a non-cell-autonomous manner during cortical development.

Single Synapse Indicators of Impaired Glutamate Clearance Derived from Fast iGlu u Imaging of Cortical Afferents in the Striatum of Normal and Huntington (Q175) Mice.

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single-synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGlu u We report findings from individual corticostriatal synapses in acute slices prepared from mice of either sex that were >1 year of age. Contrasting patterns of short-term plasticity and a size criterion identified two classes of terminals, presumably corresponding to the previously defined IT (intratelencephalic) and PT (pyramidal tract) synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGlu u single-synapse study in Q175 mice, a model of Huntington's disease (HD). In HD mice, the decay time constant of the perisynaptic Glu concentration (TauD), as an indicator of uptake, and the peak iGlu u amplitude, as an indicator of release, were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nm) mimicked the TauD changes in homozygotes. Considering the largest TauD values encountered in WT, ∼40% of PT synapses tested in Q175 heterozygotes can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGlu u Finally, EAAT2 (excitatory amino acid transport protein 2) immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention.SIGNIFICANCE STATEMENT Alterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, the sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor extracellular glutamate concentration ([Glu]) at individual corticostriatal synapses, we can now quantify the time constant of perisynaptic [Glu] decay (as an indicator of uptake) and the maximal [Glu] elevation next to the active zone (as an indicator of Glu release). The results provide a positive answer to the hitherto unresolved question of whether neurodegeneration (e.g., Huntington's disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

A Dual Role Hypothesis of the Cortico-Basal-Ganglia Pathways: Opponency and Temporal Difference Through Dopamine and Adenosine.

The hypothesis that the basal-ganglia direct and indirect pathways represent goodness (or benefit) and badness (or cost) of options, respectively, explains a wide range of phenomena. However, this hypothesis, named the Opponent Actor Learning (OpAL), still has limitations. Structurally, the OpAL model does not incorporate differentiation of the two types of cortical inputs to the basal-ganglia pathways received from intratelencephalic (IT) and pyramidal-tract (PT) neurons. Functionally, the OpAL model does not describe the temporal-difference (TD)-type reward-prediction-error (RPE), nor explains how RPE is calculated in the circuitry connecting to the DA neurons. In fact, there is a different hypothesis on the basal-ganglia pathways and DA, named the Cortico-Striatal-Temporal-Difference (CS-TD) model. The CS-TD model differentiates the IT and PT inputs, describes the TD-type RPE, and explains how TD-RPE is calculated. However, a critical difficulty in this model lies in its assumption that DA induces the same direction of plasticity in both direct and indirect pathways, which apparently contradicts the experimentally observed opposite effects of DA on these pathways. Here, we propose a new hypothesis that integrates the OpAL and CS-TD models. Specifically, we propose that the IT-basal-ganglia pathways represent goodness/badness of current options while the PT-indirect pathway represents the overall value of the previously chosen option, and both of these have influence on the DA neurons, through the basal-ganglia output, so that a variant of TD-RPE is calculated. A key assumption is that opposite directions of plasticity are induced upon phasic activation of DA neurons in the IT-indirect pathway and PT-indirect pathway because of different profiles of IT and PT inputs. Specifically, at PT→indirect-pathway-medium-spiny-neuron (iMSN) synapses, sustained glutamatergic inputs generate rich adenosine, which allosterically prevents DA-D2 receptor signaling and instead favors adenosine-A2A receptor signaling. Then, phasic DA-induced phasic adenosine, which reflects TD-RPE, causes long-term synaptic potentiation. In contrast, at IT→iMSN synapses where adenosine is scarce, phasic DA causes long-term synaptic depression via D2 receptor signaling. This new Opponency and Temporal-Difference (OTD) model provides unique predictions, part of which is potentially in line with recently reported activity patterns of neurons in the globus pallidus externus on the indirect pathway.

Neocortical Lamination: Insights from Neuron Types and Evolutionary Precursors.

The neocortex is characterized by lamination of its neuron cell bodies in six layers, but there are few clues as to how this comes about and what is its function. Recent studies provide evidence that evolution from simple three-layer cortex may give insight into this problem. Three-layer cortex arose in the olfactory, hippocampal and dorsal cortex of the early amniote forebrain based on a cortical module of excitatory and inhibitory inputs to an intratelencephalic (IT) type of pyramidal neuron with feedback excitation and inhibition and related interneurons. We summarize recent evidence suggesting the hypothesis that the developmental program of three-layer olfactory cortex was co-opted to form six-layer mammalian neocortex, elaborating IT cortical units in layers 2–6 while adding layer 4 stellate cells, layer 5B pyramidal tract (PT) cells and layer 6 corticothalamic (CT) cells.

Distinct Laterality in Forelimb-Movement Representations of Rat Primary and Secondary Motor Cortical Neurons with Intratelencephalic and Pyramidal Tract Projections.

Two distinct motor areas, the primary and secondary motor cortices (M1 and M2), play crucial roles in voluntary movement in rodents. The aim of this study was to characterize the laterality in motor cortical representations of right and left forelimb movements. To achieve this goal, we developed a novel behavioral task, the Right-Left Pedal task, in which a head-restrained male rat manipulates a right or left pedal with the corresponding forelimb. This task enabled us to monitor independent movements of both forelimbs with high spatiotemporal resolution. We observed phasic movement-related neuronal activity (Go-type) and tonic hold-related activity (Hold-type) in isolated unilateral movements. In both M1 and M2, Go-type neurons exhibited bias toward contralateral preference, whereas Hold-type neurons exhibited no bias. The contralateral bias was weaker in M2 than M1. Moreover, we differentiated between intratelencephalic (IT) and pyramidal tract (PT) neurons using optogenetically evoked spike collision in rats expressing channelrhodopsin-2. Even in identified PT and IT neurons, Hold-type neurons exhibited no lateral bias. Go-type PT neurons exhibited bias toward contralateral preference, whereas IT neurons exhibited no bias. Our findings suggest a different laterality of movement representations of M1 and M2, in each of which IT neurons are involved in cooperation of bilateral movements, whereas PT neurons control contralateral movements.SIGNIFICANCE STATEMENT In rodents, the primary and secondary motor cortices (M1 and M2) are involved in voluntary movements via distinct projection neurons: intratelencephalic (IT) neurons and pyramidal tract (PT) neurons. However, it remains unclear whether the two motor cortices (M1 vs M2) and the two classes of projection neurons (IT vs PT) have different laterality of movement representations. We optogenetically identified these neurons and analyzed their functional activity using a novel behavioral task to monitor movements of the right and left forelimbs separately. We found that contralateral bias was reduced in M2 relative to M1, and in IT relative to PT neurons. Our findings suggest that the motor information processing that controls forelimb movement is coordinated by a distinct cell population.

D3 Receptors Regulate Excitability in a Unique Class of Prefrontal Pyramidal Cells.

The D3 dopamine receptor, a member of the Gi-coupled D2 family of dopamine receptors, is expressed throughout limbic circuits affected in neuropsychiatric disorders, including prefrontal cortex (PFC). These receptors are important for prefrontal executive function because pharmacological and genetic manipulations that affect prefrontal D3 receptors alter anxiety, social interaction, and reversal learning. However, the mechanisms by which D3 receptors regulate prefrontal circuits and whether D3 receptors regulate specific prefrontal subnetworks remains unknown. Here, we combine dopamine receptor reporter lines, anatomical tracing techniques, and electrophysiology to show that D3 receptor expression defines a novel subclass of layer 5 glutamatergic pyramidal cell in mouse PFC (either sex). D3-receptor-expressing pyramidal neurons are electrophysiologically and anatomically separable from neighboring neurons expressing D1 or D2 receptors based on their dendritic morphology and subthreshold and suprathreshold intrinsic excitability. D3-receptor-expressing neurons send axonal projections to intratelencephalic (IT) targets, including contralateral cortex, nucleus accumbens, and basolateral amygdala. Within these neurons, D3 receptor activation was found to regulate low-voltage-activated CaV3.2 calcium channels localized to the axon initial segment, which suppressed action potential (AP) excitability, particularly when APs occurred at high frequency. Therefore, these data indicate that D3 receptors regulate the excitability of a unique, IT prefrontal cell population, thereby defining novel circuitry and cellular actions for D3 receptors in PFC.SIGNIFICANCE STATEMENT The D3 dopamine receptor, a member of the Gi-coupled D2 family of dopamine receptors, are expressed throughout limbic circuits, including prefrontal cortex (PFC). They are of broad interest as a site for therapeutic intervention in serious mental illness, yet we know very little about their distribution or function within PFC. Here, we show that D3 receptors define a unique population of glutamatergic principal cells in mouse PFC that largely lack expression of D1 or D2 receptors. Within these cells, we find that D3 receptors regulate the ability to generate high-frequency action potential bursts through mechanisms not supported by other dopamine receptors. These results define unique circuitry and cellular actions for D3 receptors in regulating PFC networks.

In Vivo Spiking Dynamics of Intra- and Extratelencephalic Projection Neurons in Rat Motor Cortex

In motor cortex, 2 types of deep layer pyramidal cells send their axons to other areas: intratelencephalic (IT)-type neurons specifically project bilaterally to the cerebral cortex and striatum, whereas neurons of the extratelencephalic (ET)-type, termed conventionally pyramidal tract-type, project ipsilaterally to the thalamus and other areas. Although they have totally different synaptic and membrane potential properties in vitro, little is known about the differences between them in ongoing spiking dynamics in vivo. We identified IT-type and ET-type neurons, as well as fast-spiking-type interneurons, using novel multineuronal analysis based on optogenetically evoked spike collision along their axons in behaving/resting rats expressing channelrhodopsin-2 (Multi-Linc method). We found "postspike suppression" (~100 ms) as a characteristic of ET-type neurons in spike auto-correlograms, and it remained constant independent of behavioral conditions in functionally different ET-type neurons. Postspike suppression followed even solitary spikes, and spike bursts significantly extended its duration. We also observed relatively strong spike synchrony in pairs containing IT-type neurons. Thus, spiking dynamics in IT-type and ET-type neurons may be optimized differently for precise and coordinated motor control.

Synaptic circuit organization of motor corticothalamic neurons.

Corticothalamic (CT) neurons in layer 6 constitute a large but enigmatic class of cortical projection neurons. How they are integrated into intracortical and thalamo-cortico-thalamic circuits is incompletely understood, especially outside of sensory cortex. Here, we investigated CT circuits in mouse forelimb motor cortex (M1) using multiple circuit-analysis methods. Stimulating and recording from CT, intratelencephalic (IT), and pyramidal tract (PT) projection neurons, we found strong CT↔ CT and CT↔ IT connections; however, CT→IT connections were limited to IT neurons in layer 6, not 5B. There was strikingly little CT↔ PT excitatory connectivity. Disynaptic inhibition systematically accompanied excitation in these pathways, scaling with the amplitude of excitation according to both presynaptic (class-specific) and postsynaptic (cell-by-cell) factors. In particular, CT neurons evoked proportionally more inhibition relative to excitation (I/E ratio) than IT neurons. Furthermore, the amplitude of inhibition was tuned to match the amount of excitation at the level of individual neurons; in the extreme, neurons receiving no excitation received no inhibition either. Extending these studies to dissect the connectivity between cortex and thalamus, we found that M1-CT neurons and thalamocortical neurons in the ventrolateral (VL) nucleus were remarkably unconnected in either direction. Instead, VL axons in the cortex excited both IT and PT neurons, and CT axons in the thalamus excited other thalamic neurons, including those in the posterior nucleus, which additionally received PT excitation. These findings, which contrast in several ways with previous observations in sensory areas, illuminate the basic circuit organization of CT neurons within M1 and between M1 and thalamus.

Differential cortical activation of the striatal direct and indirect pathway cells: reconciling the anatomical and optogenetic results by using a computational method

The corticostriatal system is considered to be crucially involved in learning and action selection. Anatomical studies have shown that two types of corticostriatal neurons, intratelencephalic (IT) and pyramidal tract (PT) cells, preferentially project to dopamine D1 or D2 receptor-expressing striatal projection neurons, respectively. In contrast, an optogenetic study has shown that stimulation of IT axons evokes comparable responses in D1 and D2 cells and that stimulation of PT axons evokes larger responses in D1 cells. Since the optogenetic study applied brief stimulation only, however, the overall impacts of repetitive inputs remain unclear. Moreover, the apparent contradiction between the anatomical and optogenetic results remains to be resolved. I addressed these issues by using a computational approach. Specifically, I constructed a model of striatal response to cortical inputs, with parameters regarding short-term synaptic plasticity and anatomical connection strength for each connection type. Under the constraint of the optogenetic results, I then explored the parameters that best explain the previously reported paired-pulse ratio of response in D1 and D2 cells to cortical and intrastriatal stimulations, which presumably recruit different compositions of IT and PT fibers. The results indicate that 1) IT→D1 and PT→D2 connections are anatomically stronger than IT→D2 and PT→D1 connections, respectively, consistent with the previous findings, and that 2) IT→D1 and PT→D2 synapses entail short-term facilitation, whereas IT→D2 and PT→D1 synapses would basically show depression, and thereby 3) repetitive IT or PT inputs have larger overall impacts on D1 or D2 cells, respectively, supporting a recently proposed hypothesis on the roles of corticostriatal circuits in reinforcement learning.

Morphological and electrophysiological changes in intratelencephalic-type pyramidal neurons in the motor cortex of a rat model of levodopa-induced dyskinesia

Levodopa-induced dyskinesia (LID) is a major complication of long-term dopamine replacement therapy for Parkinson's disease, and becomes increasingly problematic in the advanced stage of the disease. Although the cause of LID still remains unclear, there is accumulating evidence from animal experiments that it results from maladaptive plasticity, resulting in supersensitive excitatory transmission at corticostriatal synapses. Recent work using transcranial magnetic stimulation suggests that the motor cortex displays the same supersensitivity in Parkinson's disease patients with LID. To date, the cellular mechanisms underlying the abnormal cortical plasticity have not been examined. The morphology of the dendritic spines has a strong relationship to synaptic plasticity. Therefore, we explored the spine morphology of pyramidal neurons in the motor cortex in a rat model of LID. We used control rats, 6-hydroxydopamine-lesioned rats (a model of Parkinson's disease), 6-hydroxydopamine-lesioned rats chronically treated with levodopa (a model of LID), and control rats chronically treated with levodopa. Because the direct pathway of the basal ganglia plays a central role in the development of LID, we quantified the density and size of dendritic spines in intratelencephalic (IT)-type pyramidal neurons in M1 cortex that project to the striatal medium spiny neurons in the direct pathway. The spine density was not different among the four groups. In contrast, spine size became enlarged in the Parkinson's disease and LID rat models. The enlargement was significantly greater in the LID model than in the Parkinson's disease model. This enlargement of the spines suggests that IT-type pyramidal neurons acquire supersensitivity to excitatory stimuli. To confirm this possibility, we monitored miniature excitatory postsynaptic currents (mEPSCs) in the IT-type pyramidal neurons in M1 cortex using whole-cell patch clamp. The amplitude of the mEPSCs was significantly increased in the LID model compared with the control. This indicates that the IT-type pyramidal neurons become hyperexcited in the LID model, paralleling the enlargement of spines. Thus, spine enlargement and the resultant hyperexcitability of IT-type pyramidal neurons in M1 cortex might contribute to the abnormal cortical neuronal plasticity in LID.