Convergence of inputs from the basal ganglia with layer 5 of motor cortex and cerebellum in mouse motor thalamus.

A key to motor control is the motor thalamus, where several inputs converge. One excitatory input originates from layer 5 of primary motor cortex (M1L5), while another arises from the deep cerebellar nuclei (Cb). M1L5 terminals distribute throughout the motor thalamus and overlap with GABAergic inputs from the basal ganglia output nuclei, the internal segment of the globus pallidus (GPi), and substantia nigra pars reticulata (SNr). In contrast, it is thought that Cb and basal ganglia inputs are segregated. Therefore, we hypothesized that one potential function of the GABAergic inputs from basal ganglia is to selectively inhibit, or gate, excitatory signals from M1L5 in the motor thalamus. Here, we tested this possibility and determined the circuit organization of mouse (both sexes) motor thalamus using an optogenetic strategy in acute slices. First, we demonstrated the presence of a feedforward transthalamic pathway from M1L5 through motor thalamus. Importantly, we discovered that GABAergic inputs from the GPi and SNr converge onto single motor thalamic cells with excitatory synapses from M1L5. Separately, we also demonstrate that, perhaps unexpectedly, GABAergic GPi and SNr inputs converge with those from the Cb. We interpret these results to indicate that a role of the basal ganglia is to gate the thalamic transmission of M1L5 and Cb information to cortex.

A Reconsideration of the Core and Matrix Classification of Thalamocortical Projections.

In 1998, Jones suggested a classification of thalamocortical projections into core and matrix divisions (Jones, 1998). In this classification, core projections are specific, topographical, innervate middle cortical layers, and serve to transmit specific information to the cortex for further analysis; matrix projections, in contrast, are diffuse, much less topographic, innervate upper layers, especially Layer 1, and serve a more global, modulatory function, such as affecting levels of arousal. This classification has proven especially influential in studies of thalamocortical relationships. Whereas it may be the case that a clear subset of thalamocortical connections fit the core motif, since they are specific, topographic, and innervate middle layers, we argue that there is no clear evidence for any single class that encompasses the remainder of thalamocortical connections as is claimed for matrix. Instead, there is great morphological variation in connections made by thalamocortical projections fitting neither a core nor matrix classification. We thus conclude that the core/matrix classification should be abandoned, because its application is not helpful in providing insights into thalamocortical interactions and can even be misleading. As one example of the latter, recent suggestions indicate that core projections are equivalent to first-order thalamic relays (i.e., those that relay subcortical information to the cortex) and matrix to higher-order relays (i.e., those that relay information from one cortical area to another), but available evidence does not support this relationship. All of this points to a need to replace the core/matrix grouping with a more complete classification of thalamocortical projections.

Convergence of inputs from the basal ganglia with layer 5 of motor cortex and cerebellum in mouse motor thalamus.

A key to motor control is the motor thalamus, where several inputs converge. One excitatory input originates from layer 5 of primary motor cortex (M1L5), while another arises from the deep cerebellar nuclei (Cb). M1L5 terminals distribute throughout the motor thalamus and overlap with GABAergic inputs from the basal ganglia output nuclei, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr). In contrast, it is thought that Cb and basal ganglia inputs are segregated. Therefore, we hypothesized that one potential function of the GABAergic inputs from basal ganglia is to selectively inhibit, or gate, excitatory signals from M1L5 in the motor thalamus. Here, we tested this possibility and determined the circuit organization of mouse (both sexes) motor thalamus using an optogenetic strategy in acute slices. First, we demonstrated the presence of a feedforward transthalamic pathway from M1L5 through motor thalamus. Importantly, we discovered that GABAergic inputs from the GPi and SNr converge onto single motor thalamic cells with excitatory synapses from M1L5 and, unexpectedly, Cb as well. We interpret these results to indicate that a role of the basal ganglia is to gate the thalamic transmission of M1L5 and Cb information to cortex.

Layer 5 of cortex innervates the thalamic reticular nucleus in mice.

Neurons in the thalamic reticular nucleus (TRN) are a primary source of inhibition to the dorsal thalamus and, as they are innervated in part by the cortex, are a means of corticothalamic regulation. Previously, cortical inputs to the TRN were thought to originate solely from layer 6 (L6), but we recently reported the presence of putative synaptic terminals from layer 5 (L5) neurons in multiple cortical areas in the TRN [J. A. Prasad, B. J. Carroll, S. M. Sherman, J. Neurosci. 40, 5785-5796 (2020)]. Here, we demonstrate with electron microscopy that L5 terminals from multiple cortical regions make bona fide synapses in the TRN. We further use light microscopy to localize these synapses relative to recently described TRN subdivisions and show that L5 terminals target the edges of the somatosensory TRN, where neurons reciprocally connect to higher-order thalamus, and that L5 terminals are scarce in the core of the TRN, where neurons reciprocally connect to first-order thalamus. In contrast, L6 terminals densely innervate both edge and core subregions and are smaller than those from L5. These data suggest that a sparse but potent input from L5 neurons of multiple cortical regions to the TRN may yield transreticular inhibition targeted to higher-order thalamus.

Conserved patterns of functional organization between cortex and thalamus in mice.

Higher-order thalamic nuclei contribute to sensory processing via projections to primary and higher cerebral cortical areas, but it is unknown which of their cortical and subcortical inputs contribute to their distinct output pathways. We used subpopulation specific viral strategies in mice to anatomically and physiologically dissect pathways of the higher-order thalamic nuclei of the somatosensory and visual systems (the posterior medial nucleus and pulvinar). Employing a complementary optogenetics and electrical stimulation strategy, we show that synapses in cortex from higher-order thalamus have functionally divergent properties in primary vs. higher cortical areas. Higher-order thalamic projections onto excitatory targets in S1 and V1 were weakly modulatory, while projections to S2 and higher visual areas were strong drivers of postsynaptic targets. Then, using transsynaptic tracing verified by optogenetics to map inputs to higher-order thalamus, we show that posterior medial nucleus cells projecting to S1 are driven by neurons in layer 5 of S1, S2, and M1 and that pulvinar cells projecting to V1 are driven by neurons in layer 5 of V1 and higher visual areas. Therefore, in both systems, layer 5 of primary and higher cortical areas drives transthalamic feedback modulation of primary sensory cortex through higher-order thalamus. These results highlight conserved organization that may be shared by other thalamocortical circuitry. They also support the hypothesis that direct corticocortical projections in the brain are paralleled by transthalamic pathways, even in the feedback direction, with feedforward transthalamic pathways acting as drivers, while feedback through thalamus is modulatory.

Integration of signals from different cortical areas in higher order thalamic neurons.

Higher order thalamic neurons receive driving inputs from cortical layer 5 and project back to the cortex, reflecting a transthalamic route for corticocortical communication. To determine whether or not individual neurons integrate signals from different cortical populations, we combined electron microscopy "connectomics" in mice with genetic labeling to disambiguate layer 5 synapses from somatosensory and motor cortices to the higher order thalamic posterior medial nucleus. A significant convergence of these inputs was found on 19 of 33 reconstructed thalamic cells, and as a population, the layer 5 synapses were larger and located more proximally on dendrites than were unlabeled synapses. Thus, many or most of these thalamic neurons do not simply relay afferent information but instead integrate signals as disparate in this case as those emanating from sensory and motor cortices. These findings add further depth and complexity to the role of the higher order thalamus in overall cortical functioning.

Cortical control of behavior and attention from an evolutionary perspective.

For animals to survive, they must interact with their environment, taking in sensory information and making appropriate motor responses. Early on during vertebrate evolution, this was accomplished with neural circuits located mostly within the spinal cord and brainstem. As the cerebral cortex evolved, it provided additional and powerful advantages for assessing environmental cues and guiding appropriate responses. Importantly, the cerebral cortex was added onto an already functional nervous system. Moreover, every cortical area, including areas traditionally considered sensory, provides input to the subcortical motor structures that are bottlenecks for driving action. These facts have important ramifications for cognitive aspects of motor control. Here we consider the evolution of cortical mechanisms for attention from the perspective of having to work through these subcortical bottlenecks. From this perspective, many features of attention can be explained, including the preferential engagement of some cortical areas at the cost of disengagement from others to improve appropriate behavioral responses.

Layer 5 Corticofugal Projections from Diverse Cortical Areas: Variations on a Pattern of Thalamic and Extrathalamic Targets.

The cerebral cortex, with all its computational power, can only influence behavior via corticofugal connections originating from layer 5 (L5) cells (Sherman and Guillery, 2013). To begin to establish the global pattern of these outputs, we examined L5 efferents originating from four cortical areas: somatosensory, visual, motor, and prefrontal (i.e., ventromedial orbitofrontal) cortex. We injected Cre-dependent adeno-associated virus in an Rbp4-Cre transgenic mouse line (both sexes) to label these L5 efferents selectively. Our study reveals that, across this diverse series of cortical regions, L5 commonly projects to multiple thalamic and extrathalamic sites. We also identified several novel corticofugal targets (i.e., the lateral dorsal nucleus, submedial nucleus) previously unidentified as L5 targets. We identified common patterns for these projections: all areas innervated both thalamus and the midbrain, and all areas innervated multiple thalamic targets, including those with core and matrix cell types (Jones, 1998). An examination of the terminal size within each of these targets suggests that terminal populations of L5 efferents are not consistently large but vary with cortical area and target; and in some cases, these include small terminals only. Overall, our data reveal more widespread and diverse L5 efferents than previously appreciated, suggesting a generalizable role for this cortical layer in influencing motor commands and cognitive processes.SIGNIFICANCE STATEMENT While the neocortex is responsible for coordination of complex behavior, it requires communication with subcortical regions to do so. It is specifically cortical layer 5 (L5) that is thought to underlie these behaviors, although it is unknown whether this holds true across functionally different cortical areas. Using a selective viral tracing method and transgenic mice, we examined the connectivity of four cortical regions (somatosensory, visual, motor and prefrontal cortex) to assess the generalizability of these L5 projections. All areas of cortex projected to overlapping as well as distinct thalamic and brainstem structures. Terminals within these regions varied in size, implicating that L5 has a broad and diverse impact on behavior.

Thalamocortical Circuit Motifs: A General Framework.

The role of the thalamus in cortical sensory transmission is well known, but its broader role in cognition is less appreciated. Recent studies have shown thalamic engagement in dynamic regulation of cortical activity in attention, executive control, and perceptual decision-making, but the circuit mechanisms underlying such functionality are unknown. Because the thalamus is composed of excitatory neurons that are devoid of local recurrent excitatory connectivity, delineating long-range, input-output connectivity patterns of single thalamic neurons is critical for building functional models. We discuss this need in relation to existing organizational schemes such as core versus matrix and first-order versus higher-order relay nuclei. We propose that a new classification is needed based on thalamocortical motifs, where structure naturally informs function. Overall, our synthesis puts understanding thalamic organization at the forefront of existing research in systems and computational neuroscience, with both basic and translational applications.

A Sensorimotor Pathway via Higher-Order Thalamus.

We now know that sensory processing in cortex occurs not only via direct communication between primary to secondary areas, but also via their parallel cortico-thalamo-cortical (i.e., trans-thalamic) pathways. Both corticocortical and trans-thalamic pathways mainly signal through glutamatergic class 1 (driver) synapses, which have robust and efficient synaptic dynamics suited for the transfer of information such as receptive field properties, suggesting the importance of class 1 synapses in feedforward, hierarchical processing. However, such a parallel arrangement has only been identified in sensory cortical areas: visual, somatosensory, and auditory. To test the generality of trans-thalamic pathways, we sought to establish its presence beyond purely sensory cortices to determine whether there is a trans-thalamic pathway parallel to the established primary somatosensory (S1) to primary motor (M1) pathway. We used trans-synaptic viral tracing, optogenetics in slice preparations, and bouton size analysis in the mouse (both sexes) to document that a circuit exists from layer 5 of S1 through the posterior medial nucleus of the thalamus to M1 with glutamatergic class 1 properties. This represents a hitherto unknown, robust sensorimotor linkage and suggests that the arrangement of parallel direct and trans-thalamic corticocortical circuits may be present as a general feature of cortical functioning.SIGNIFICANCE STATEMENT During sensory processing, feedforward pathways carry information such as receptive field properties via glutamatergic class 1 synapses, which have robust and efficient synaptic dynamics. As expected, class 1 synapses subserve the feedforward projection from primary to secondary sensory cortex, but also a route through specific higher-order thalamic nuclei, creating a parallel feedforward trans-thalamic pathway. We now extend the concept of cortical areas being connected via parallel, direct, and trans-thalamic circuits from purely sensory cortices to a sensorimotor cortical circuit (i.e., primary sensory cortex to primary motor cortex). This suggests a generalized arrangement for corticocortical communication.

Convergent synaptic inputs to layer 1 cells of mouse cortex.

We used whole cell recordings from slice preparations of mouse cortex to identify various inputs to neurons of layer 1. Two sensory cortical areas were targeted: a primary somatosensory area, namely, the barrel cortex of S1, and a higher order visual area, namely, V2M. Results were similar from both areas. By activating local inputs using photostimulation with caged glutamate, we also identified glutamatergic (and possibly GABAergic) inputs from all lower layers plus GABAergic inputs from nearby layer 1 neurons. However, the patterns of such inputs to layer 1 neurons showed great variation among cells. In separate experiments, we found that electrical stimulation of axons running parallel to the cortical surface in layer 1 also evoked a variety of convergent input types to layer 1 neurons, including glutamatergic "drivers" and "modulators" plus classic modulatory inputs, including serotonergic, nicotinic, α- and ß-adrenergic, from subcortical sites. Given that these layer 1 cells significantly affect the responses of other cortical neurons, especially via affecting the apical dendrites of pyramidal cells so important to cortical functioning, their role in cortical processing is significant. We believe that the data presented here lead to better understanding of the functioning of layer 1 neurons in their role of influencing cortical processing.

My prolonged collaboration with Ray Guillery.

My active collaboration with Ray Guillery started in 1968, when he was a Full Professor at the University of Wisconsin and I was a graduate student at the University of Pennsylvania. The collaboration lasted almost 50 years with virtually no breaks. Among the ideas we proposed are that glutamatergic pathways in thalamus and cortex can be classified into drivers and modulators; that many thalamic nuclei could be classified as higher order, meaning that they receive driving input from layer 5 of cortex and participate in cortico-thalamocortical circuits; and that much of the information relayed by thalamus serves as an efference copy for motor commands initiated by cortex.

Corticofugal circuits: Communication lines from the cortex to the rest of the brain.

Pyramidal cells in cortical Layers 5 and 6 are the only cells in the cerebral cortex with axons that leave the cortex to influence the thalamus. Layer 6 cells provide modulatory feedback input to all thalamic nuclei. Layer 5 cells provide driving input to higher-order thalamic nuclei and do not innervate first-order nuclei, which get their driving inputs from subcortical sources. Higher-order nuclei innervated by Layer 5 cells thus seem to be involved with cortico-thalamo-cortical communication. The Layer 5 axons branch to also target additional subcortical structures that mediate interactions with the external environment. These corticofugal pathways represent the only means by which the cortex influences the rest of the neuraxis and thus are essential for proper cortical function and species survival. Here we review current understanding of the corticofugal pathways from Layers 5 and 6 and speculate on their functional contributions to neural processing and behavior.

Synaptic properties of the lemniscal and paralemniscal pathways to the mouse somatosensory thalamus.

Somatosensory information is thought to arrive in thalamus through two glutamatergic routes called the lemniscal and paralemniscal pathways via the ventral posterior medial (VPm) and posterior medial (POm) nuclei. Here we challenge the view that these pathways functionally represent parallel information routes. Using electrical stimulation and an optogenetic approach in brain slices from the mouse, we investigated the synaptic properties of the lemniscal and paralemniscal input to VPm and POm. Stimulation of the lemniscal pathway produced class 1, or "driver," responses in VPm relay cells, which is consistent with this being an information-bearing channel. However, stimulation of the paralemniscal pathway produced two distinct types of responses in POm relay cells: class 1 (driver) responses in 29% of the cells, and class 2, or "modulator," responses in the rest. Our data suggest that, unlike the lemniscal pathway, the paralemniscal one is not homogenous and that it is primarily modulatory. This finding requires major rethinking regarding the routes of somatosensory information to cortex and suggests that the paralemniscal route is chiefly involved in modulatory functions rather than simply being an information route parallel to the lemniscal channel.

Functioning of Circuits Connecting Thalamus and Cortex.

Glutamatergic pathways in thalamus and cortex are divided into two distinct classes: driver, which carries the main information between cells, and modulator, which modifies how driver inputs function. Identifying driver inputs helps to reveal functional computational circuits, and one set of such circuits identified by this approach are cortico-thalamo-cortical (or transthalamic corticocortical) circuits. This, in turn, leads to the conclusion that there are two types of thalamic relay: first order nuclei (such as the lateral geniculate nucleus) that relay driver input from a subcortical source (i.e., retina), and higher order nuclei (such as the pulvinar) which are involved in these transthalamic pathways by relaying driver input from layer 5 of one cortical area to another. This thalamic division is also seen in other sensory pathways and beyond these so that most of thalamus by volume consists of higher-order relays. Many, and perhaps all, direct driver connections between cortical areas are paralleled by an indirect cortico-thalamo-cortical (transthalamic) driver route involving higher order thalamic relays. Such thalamic relays represent a heretofore unappreciated role in cortical functioning, and this assessment challenges and extends conventional views regarding both the role of thalamus and mechanisms of corticocortical communication. Finally, many and perhaps the vast majority of driver inputs relayed through thalamus arrive via branching axons, with extrathalamic targets often being subcortical motor centers. This raises the possibility that inputs relayed by thalamus to cortex also serve as efference copies, and this may represent an important feature of information relayed up the cortical hierarchy via transthalamic circuits. © 2017 American Physiological Society. Compr Physiol 7:713-739, 2017.

Neuroscience Training for the 21st Century.

The field of neuroscience is enjoying a rapid expansion in scope, coupled with a remarkable broadening of conceptual approaches, scientific tools, and clinical applications. This growth poses new challenges for academic training programs as they prepare young neuroscientists for a more complex, competitive, and diverse career landscape. Multiple stakeholders, including academia, federal funding agencies, industry, scientific societies, private foundations, and other public and private sector contributors, need to be actively engaged in supporting this broad training effort. A renewed commitment to a more forward-looking, flexible yet integrative training vision offers opportunities for a bright future for young neuroscientists as they assume the role of vanguard of the enterprise that enriches our understanding of the brain.

Thalamus plays a central role in ongoing cortical functioning.

Several challenges to current views of thalamocortical processing are offered here. Glutamatergic pathways in thalamus and cortex are divided into two distinct classes: driver and modulator. We suggest that driver inputs are the main conduits of information and that modulator inputs modify how driver inputs are processed. Different driver sources reveal two types of thalamic relays: first order relays receive subcortical driver input (for example, retinal input to the lateral geniculate nucleus), whereas higher order relays (for example, pulvinar) receive driver input from layer 5 of cortex and participate in cortico-thalamo-cortical (or transthalamic) circuits. These transthalamic circuits represent an unappreciated aspect of cortical functioning, which I discuss here. Direct corticocortical connections are often paralleled by transthalamic ones. Furthermore, driver inputs to thalamus, both first and higher order, typically arrive via branching axons, and the transthalamic branch often innervates subcortical motor centers, leading to the suggestion that these inputs to thalamus serve as efference copies.

Modulatory effects of activation of metabotropic glutamate receptors on GABAergic circuits in the mouse thalamus.

Metabotropic glutamate receptors (mGluRs) are widely distributed in the central nervous system and modulate the release of neurotransmitters in different ways. We have previously shown that activation of presynaptic group II mGluRs reduces the gain of GABAergic inputs in both primary visual and auditory cortices (V1 and A1). In the present study, we sought to determine whether activation of mGluRs can also affect the inhibitory inputs in thalamus. Using whole cell recordings in a mouse slice preparation, we studied two GABAergic inputs to thalamic relay cells: that of the thalamic reticular nucleus (TRN) to cells of the ventral posteromedial nucleus (VPM) and that of interneurons to cells of the lateral geniculate nucleus (LGN). We found that activation of mGluRs significantly reduced the amplitudes of inhibitory postsynaptic currents (IPSCs) evoked from TRN inputs to VPM cells, and further experiments indicated that this was due to activation of presynaptic group I and group II mGluRs. Similar results were found in the interneuronal inputs to LGN cells. Activation of presynaptic group I (type 1 but not type 5) and group II mGluRs significantly reduced the amplitudes of evoked IPSCs of the axonal inputs to relay cells, and additional experiments were consistent with previous observations that activation of type 5 mGluRs on the dendritic terminals of interneurons enhanced postsynaptic IPSCs. We concluded that group I and II mGluRs may generally reduce the amplitude of evoked GABAergic IPSCs of axonal inputs to thalamic relay cells, operating through presynaptic mechanisms, and this extends our previous findings in cortex.

Functional topographic organization of the motor reticulothalamic pathway.

The thalamic reticular nucleus (TRN) is a thin layer of GABAergic cells lying rostral and lateral to the dorsal thalamus, and its projection to thalamic relay cells (i.e., the reticulothalamic pathway) strongly inhibits these cells. In an attempt to extend earlier studies of reticulothalamic connections to sensory thalamic nuclei, we used laser-scanning photostimulation to study the reticulothalamic projections to the main motor thalamic relays, the ventral anterior and lateral (VA and VL) nuclei, as well as to the nearby central lateral (CL) thalamic nucleus. VA/VL and the earlier studied somatosensory thalamic nuclei are considered "core" nuclei with topographic thalamocortical projections, whereas CL is thought to be a "matrix" nucleus with diffuse thalamocortical projections. We found that the TRN input footprints to VA/VL and CL are spatially localized and topographic and generally conform to the patterns established earlier for the TRN projections to sensory thalamic relays. These remarkable similarities suggest similar organization of reticulothalamic pathways and TRN regulation of thalamocortical communication for motor and sensory systems and perhaps also for core and matrix thalamus. Furthermore, we found that VA/VL and CL shared overlapping TRN input regions, suggesting that CL may also be involved in the relay of motor information.