Cell-type specific binocular interactions in mouse visual thalamus.

Projections from each eye are segregated in separate domains within the dorsal lateral geniculate nucleus (dLGN). Yet, in vivo studies indicate that the activity of single dLGN neurons can be influenced by visual stimuli presented to either eye. In this study we explored whether intrinsic circuits mediate binocular interactions in the mouse dLGN. We employed dual color optogenetics in vitro to selectively activate input from each eye and recorded synaptic responses in thalamocortical (relay) cells as well as inhibitory interneurons, which have extensive dendritic arbors that are not confined to eye specific domains. While most relay cells received monocular retinal input, most interneurons received binocular retinal input; consequently, the majority of dLGN relay cells received binocular retinogeniculate-evoked inhibition. Moreover, in recordings from adjacent pairs of relay cells and interneurons, the most common relationship observed was binocular excitation of interneurons paired with binocular inhibition of adjacent relay cells. Finally, we found that dLGN interneurons are interconnected, displaying both monocular and binocular inhibition in response to retinal activation. In sum, our results indicate that geniculate interneurons provide one of the first locations where signals from the two eyes can be compared, integrated, and adjusted before being transmitted to cortex, shedding new light on the role of the thalamus in binocular vision. Highlights: In vitro dual color optogenetics examined convergence of eye-specific retinal inputs to thalamocortical (relay) cells and interneurons in the dLGNThe majority of relay cells receive monocular excitatory retinogeniculate input while the majority of interneurons receive binocular inputBinocular relay cells are located in and around the ipsilateral patch whereas binocular interneurons are distributed throughout the dLGNThe majority of relay cells receive binocular retinogeniculate-evoked inhibitiondLGN interneurons are interconnected, receiving both monocular and binocular retinogeniculate-evoked inhibition.

Synaptic properties of mouse tecto-parabigeminal pathways.

The superior colliculus (SC) is a critical hub for the generation of visually-evoked orienting and defensive behaviors. Among the SC's myriad downstream targets is the parabigeminal nucleus (PBG), the mammalian homolog of the nucleus isthmi, which has been implicated in motion processing and the production of defensive behaviors. The inputs to the PBG are thought to arise exclusively from the SC but little is known regarding the precise synaptic relationships linking the SC to the PBG. In the current study, we use optogenetics as well as viral tracing and electron microscopy in mice to better characterize the anatomical and functional properties of the SC-PBG circuit, as well as the morphological and ultrastructural characteristics of neurons residing in the PBG. We characterized GABAergic SC-PBG projections (that do not contain parvalbumin) and glutamatergic SC-PBG projections (which include neurons that contain parvalbumin). These two terminal populations were found to converge on different morphological populations of PBG neurons and elicit opposing postsynaptic effects. Additionally, we identified a population of non-tectal GABAergic terminals in the PBG that partially arise from neurons in the surrounding tegmentum, as well as several organizing principles that divide the nucleus into anatomically distinct regions and preserve a coarse retinotopy inherited from its SC-derived inputs. These studies provide an essential first step toward understanding how PBG circuits contribute to the initiation of behavior in response to visual signals.

The parabigeminal nucleus is a source of "retinogeniculate replacement terminals" in mice that lack retinofugal input.

In the dorsal lateral geniculate nucleus (LGN) of mice that lack retinal input, a population of large terminals supplants the synaptic arrangements normally made by the missing retinogeniculate terminals. To identify potential sources of these "retinogeniculate replacement terminals," we used mutant mice (math5-/- ) which lack retinofugal projections due to the failure of retinal ganglion cells to develop. In this line, we labeled LGN terminals that originate from the primary visual cortex (V1) or the parabigeminal nucleus (PBG), and compared their ultrastructure to retinogeniculate, V1 or PBG terminals in the dLGN of C57Blk6 (WT) mice (schematically depicted above graph). Corticogeniculate terminals labeled in WT and math5-/- mice were similar in size and both groups were significantly smaller than WT retinogeniculate terminals. In contrast, the PBG projection in math5-/- mice was extensive and there was considerable overlap in the sizes of retinogeniculate terminals in WT mice and PBG terminals in math5-/- mice (summarized in histogram). The data indicate that V1 is not a source of "retinogeniculate replacement terminals" and suggests that large PBG terminals expand their innervation territory to replace retinogeniculate terminals in their absence.

The organization of cholinergic projections in the visual thalamus of the mouse.

Cholinergic projections from the brainstem serve as important modulators of activity in visual thalamic nuclei such as the dorsal lateral geniculate nucleus (dLGN). While these projections have been studied in several mammals, a comprehensive examination of their organization in the mouse is lacking. We used the retrograde transport of viruses or cholera toxin subunit B (CTB) injected in the dLGN, immunocytochemical labeling with antibodies against choline acetyltransferase (ChAT), brain nitric oxide synthase (BNOS), and vesicular acetylcholine transporter (VAChT), ChAT-Cre mice crossed with a reporter line (Ai9), as well as brainstem virus injections in ChAT-Cre mice to examine the pattern of thalamic innervation from cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg), laterodorsal tegmental nucleus (LDTg), and the parabigeminal nucleus (PBG). Retrograde tracing demonstrated that the dLGN receives input from the PPTg, LDTg, and PBG. Viral tracing in ChAT-Cre mice and retrograde tracing combined with immunocytochemistry revealed that many of these inputs originate from cholinergic neurons in the PBG and PPTg. Most notable was an extensive cholinergic projection from the PBG which innervated most of the contralateral dLGN, with an especially dense concentration in the dorsolateral shell, as well as a small region in the dorsomedial pole of the ipsilateral dLGN. The PPTg was found to provide a sparse somewhat diffuse innervation of the ipsilateral dLGN. Neurons in the PPTg co-expressed ChAT, BNOS, and VAChT, whereas PBG neurons expressed ChAT, but not BNOS or VAChT. These results highlight the presence of distinct cholinergic populations that innervate the mouse dLGN.

Unraveling circuits of visual perception and cognition through the superior colliculus.

The superior colliculus is a conserved sensorimotor structure that integrates visual and other sensory information to drive reflexive behaviors. Although the evidence for this is strong and compelling, a number of experiments reveal a role for the superior colliculus in behaviors usually associated with the cerebral cortex, such as attention and decision-making. Indeed, in addition to collicular outputs targeting brainstem regions controlling movements, the superior colliculus also has ascending projections linking it to forebrain structures including the basal ganglia and amygdala, highlighting the fact that the superior colliculus, with its vast inputs and outputs, can influence processing throughout the neuraxis. Today, modern molecular and genetic methods combined with sophisticated behavioral assessments have the potential to make significant breakthroughs in our understanding of the evolution and conservation of neuronal cell types and circuits in the superior colliculus that give rise to simple and complex behaviors.

Synaptic properties of the feedback connections from the thalamic reticular nucleus to the dorsal lateral geniculate nucleus.

The thalamic reticular nucleus (TRN) is a shell-like structure comprised of GABAergic neurons that surrounds the dorsal thalamus. While playing a key role in modulating thalamocortical interactions, TRN inhibition of thalamic activity is often thought of as having an all-or-none impact. Although TRN neurons have a dynamic firing range, it remains unclear how variable rates of TRN activity gate thalamocortical transmission. To address this, we examined the ultrastructural features and functional synaptic properties of the feedback connections in the mouse thalamus between TRN and the dorsal lateral geniculate nucleus (dLGN), the principal relay of retinal signals to visual cortex. Using electron microscopy to identify TRN input to dLGN, we found that TRN terminals formed synapses with non-GABAergic postsynaptic profiles. Compared with other nonretinal terminals in dLGN, those from TRN were relatively large and tended to contact proximal regions of relay cell dendrites. To evoke TRN activity in dLGN, we adopted an optogenetic approach by expressing ChR2, or a variant (ChIEF) in TRN terminals. Both in vitro and in vivo recordings revealed that repetitive stimulation of TRN terminals led to a frequency-dependent inhibition of dLGN activity, with higher rates of stimulation resulting in increasing levels of membrane hyperpolarization and corresponding decreases in spike firing. This relationship suggests that alterations in TRN activity lead to graded changes in relay cell spike firing.NEW & NOTEWORTHY The thalamic reticular nucleus (TRN) modulates thalamocortical transmission through inhibition. In mouse, TRN terminals in the dorsal lateral geniculate nucleus (dLGN) form synapses with relay neurons but not interneurons. Stimulation of TRN terminals in dLGN leads to a frequency-dependent form of inhibition, with higher rates of stimulation leading to a greater suppression of spike firing. Thus, TRN inhibition appears more dynamic than previously recognized, having a graded rather than an all-or-none impact on thalamocortical transmission.

GABAergic cell types in the superficial layers of the mouse superior colliculus.

To begin to unravel the complexities of GABAergic circuits in the superior colliculus (SC), we utilized mouse lines that express green fluorescent protein (GFP) in cells that contain the 67 kDa isoform of glutamic acid decarboxylase (GAD67-GFP), or Cre-recombinase in cells that contain glutamic acid decarboxylase (GAD; GAD2-cre). We used Cre-dependent virus injections in GAD2-Cre mice and tracer injections in GAD67-GFP mice, as well as immunocytochemical staining for gamma amino butyric acid (GABA) and parvalbumin (PV) to characterize GABAergic cells that project to the pretectum (PT), ventral lateral geniculate nucleus (vLGN) or parabigeminal nucleus (PBG), and interneurons in the stratum griseum superficiale (SGS) that do not project outside the SC. We found that approximately 30% of SGS neurons in the mouse are GABAergic. Of these GABAergic neurons, we identified three categories of potential interneurons in the GAD67-GFP line (GABA+GFP ~45%, GABA+GFP + PV ~15%, and GABA+PV ~10%). GABAergic cells that did not contain GFP or PV were identified as potential projection neurons (GABA only ~30%). We found that GABAergic neurons that project to the PBG are primarily located in the SGS and exhibit narrow field vertical, stellate, and horizontal dendritic morphologies, while GABAergic neurons that project to the PT and vLGN are primarily located in layers ventral to the SGS. In addition, we examined GABA and GAD67-containing elements of the mouse SGS using electron microscopy to further delineate the relationship between GABAergic circuits and retinotectal input. Approximately 30% of retinotectal synaptic targets are the presynaptic dendrites of GABAergic interneurons, and GAD67-GFP interneurons are a source of these presynaptic dendrites.

The Second Visual System of The Tree Shrew.

This review provides a historical account of the discovery of secondary visual pathways (from retina to the superior colliculus to the dorsal thalamus and extrastriate cortex), and Vivien Casagrande's pioneering studies of this system using the tree shrew as a model. Subsequent studies of visual pathways in the tree shrew are also reviewed, beginning with a description of the organization and central projections of the tree shrew retina. The organization and connectivity of second visual system components that include the retino-recipient superior colliculus, tecto-recipient pulvinar nucleus and its projections, and the tecto-recipient dorsal lateral geniculate nucleus and its projections are detailed. Potential functions of the second visual system are discussed in the context of this work and in the context of the behavioral studies that initially inspired the secondary visual system concept.

Synaptic organization of the dorsal lateral geniculate nucleus.

A half century after Ray Guillery's classic descriptions of cell types, axon types, and synaptic architecture of the dorsal lateral geniculate nucleus, the functional organization of this nucleus, as well as all other thalamic nuclei, is still of enormous interest. This review will focus on two classic papers written by Ray Guillery: 'A study of Golgi preparations from the dorsal lateral geniculate nucleus of the adult cat', and 'The organization of synaptic interconnections in the laminae of the dorsal lateral geniculate nucleus of the cat', as well as the studies that most directly followed from the insights these landmark manuscripts provided. It is hoped that this review will honor Ray Guillery by encouraging further investigations of the synaptic organization of the dorsal thalamus.

The Mouse Pulvinar Nucleus Links the Lateral Extrastriate Cortex, Striatum, and Amygdala.

The pulvinar nucleus is a large thalamic structure involved in the integration of visual and motor signals. The pulvinar forms extensive connections with striate and extrastriate cortical areas, but the impact of these connections on cortical circuits has not previously been directly tested. Using a variety of anatomical, optogenetic, and in vitro physiological techniques in male and female mice, we show that pulvinocortical terminals are densely distributed in the extrastriate cortex where they form synaptic connections with spines and small-diameter dendrites. Optogenetic activation of these synapses in vitro evoked large excitatory postsynaptic responses in the majority of pyramidal cells, spiny stellate cells, and interneurons within the extrastriate cortex. However, specificity in pulvinar targeting was revealed when recordings were targeted to projection neuron subtypes. The neurons most responsive to pulvinar input were those that project to the striatum and amygdala (76% responsive) or V1 (55%), whereas neurons that project to the superior colliculus were rarely responsive (6%). Because the pulvinar also projects directly to the striatum and amygdala, these results establish the pulvinar nucleus as a hub linking the visual cortex with subcortical regions involved in the initiation and control of movement. We suggest that these circuits may be particularly important for coordinating body movements and visual perception.SIGNIFICANCE STATEMENT We found that the pulvinar nucleus can strongly influence extrastriate cortical circuits and exerts a particularly strong impact on the activity of extrastriate neurons that project to the striatum and amygdala. Our results suggest that the conventional hierarchical view of visual cortical processing may not apply to the mouse visual cortex. Instead, our results establish the pulvinar nucleus as a hub linking the visual cortex with subcortical regions involved in the initiation and control of movement, and predict that the execution of visually guided movements relies on this network.

New Breakthroughs in Understanding the Role of Functional Interactions between the Neocortex and the Claustrum.

Almost all areas of the neocortex are connected with the claustrum, a nucleus located between the neocortex and the striatum, yet the functions of corticoclaustral and claustrocortical connections remain largely obscure. As major efforts to model the neocortex are currently underway, it has become increasingly important to incorporate the corticoclaustral system into theories of cortical function. This Mini-Symposium was motivated by a series of recent studies which have sparked new hypotheses regarding the function of claustral circuits. Anatomical, ultrastructural, and functional studies indicate that the claustrum is most highly interconnected with prefrontal cortex, suggesting important roles in higher cognitive processing, and that the organization of the corticoclaustral system is distinct from the driver/modulator framework often used to describe the corticothalamic system. Recent findings supporting roles in detecting novel sensory stimuli, directing attention and setting behavioral states, were the subject of the Mini-Symposium at the 2017 Society for Neuroscience Annual Meeting.

The mouse pulvinar nucleus: Organization of the tectorecipient zones.

Comparative studies have greatly contributed to our understanding of the organization and function of visual pathways of the brain, including that of humans. This comparative approach is a particularly useful tactic for studying the pulvinar nucleus, an enigmatic structure which comprises the largest territory of the human thalamus. This review focuses on the regions of the mouse pulvinar that receive input from the superior colliculus, and highlights similarities of the tectorecipient pulvinar identified across species. Open questions are discussed, as well as the potential contributions of the mouse model for endeavors to elucidate the function of the pulvinar nucleus.

Synaptic organization of striate cortex projections in the tree shrew: A comparison of the claustrum and dorsal thalamus.

The tree shrew (Tupaia belangeri) striate cortex is reciprocally connected with the dorsal lateral geniculate nucleus (dLGN), the ventral pulvinar nucleus (Pv), and the claustrum. In the Pv or the dLGN, striate cortex projections are thought to either strongly "drive", or more subtly "modulate" activity patterns respectively. To provide clues to the function of the claustrum, we compare the synaptic arrangements of striate cortex projections to the dLGN, Pv, and claustrum, using anterograde tracing and electron microscopy. Tissue was additionally stained with antibodies against γ-aminobutyric acid (GABA) to identify GABAergic interneurons and non-GABAergic projection cells. The striate cortex terminals were largest in the Pv (0.94 ± 0.08 µm2 ), intermediate in the claustrum (0.34 ± 0.02 µm2 ), and smallest in the dLGN (0.24 ± 0.01 µm2 ). Contacts on interneurons were most common in the Pv (39%), intermediate in the claustrum (15%), and least common in the dLGN (12%). In the claustrum, non-GABAergic terminals (0.34 ± 0.01 µm2 ) and striate cortex terminals were not significantly different in size. The largest terminals in the claustrum were GABAergic (0.51 ± 0.02 µm2 ), and these terminals contacted dendrites and somata that were significantly larger (1.90 ± 0.30 µm2 ) than those contacted by cortex or non-GABAergic terminals (0.28 ± 0.02 µm2 and 0.25 ± 0.02 µm2 , respectively). Our results indicate that the synaptic organization of the claustrum does not correspond to a driver/modulator framework. Instead, the circuitry of the claustrum suggests an integration of convergent cortical inputs, gated by GABAergic circuits. J. Comp. Neurol. 525:1403-1420, 2017. © 2016 Wiley Periodicals, Inc.

Untangling the Web between Eye and Brain.

How is the picture of the visual scene that the eye encodes represented by neural circuits in the brain? In this issue of Cell, Morgan et al. address this question by forming an ultrastructural "connectome" of the mouse's visual thalamus that depicts individual retinal afferents and every contact these form with target relay cells.

Ultrastructure of geniculocortical synaptic connections in the tree shrew striate cortex.

To determine whether thalamocortical synaptic circuits differ across cortical areas, we examined the ultrastructure of geniculocortical terminals in the tree shrew striate cortex to compare directly the characteristics of these terminals with those of pulvinocortical terminals (examined previously in the temporal cortex of the same species; Chomsung et al. [] Cereb Cortex 20:997-1011). Tree shrews are considered to represent a prototype of early prosimian primates but are unique in that sublaminae of striate cortex layer IV respond preferentially to light onset (IVa) or offset (IVb). We examined geniculocortical inputs to these two sublayers labeled by tracer or virus injections or an antibody against the type 2 vesicular glutamate antibody (vGLUT2). We found that layer IV geniculocortical terminals, as well as their postsynaptic targets, were significantly larger than pulvinocortical terminals and their postsynaptic targets. In addition, we found that 9-10% of geniculocortical terminals in each sublamina contacted GABAergic interneurons, whereas pulvinocortical terminals were not found to contact any interneurons. Moreover, we found that the majority of geniculocortical terminals in both IVa and IVb contained dendritic protrusions, whereas pulvinocortical terminals do not contain these structures. Finally, we found that synaptopodin, a protein uniquely associated with the spine apparatus, and telencephalin (TLCN, or intercellular adhesion molecule type 5), a protein associated with maturation of dendritic spines, are largely excluded from geniculocortical recipient layers of the striate cortex. Together our results suggest major differences in the synaptic organization of thalamocortical pathways in striate and extrastriate areas.

Spatiotemporal Profile of Voltage-Sensitive Dye Responses in the Visual Cortex of Tree Shrews Evoked by Electric Microstimulation of the Dorsal Lateral Geniculate and Pulvinar Nuclei.

The primary visual cortex (V1) receives its main thalamic drive from the dorsal lateral geniculate nucleus (dLGN) through synaptic contacts terminating primarily in cortical layer IV. In contrast, the projections from the pulvinar nucleus to the cortex are less clearly defined. The pulvinar projects predominantly to layer I in V1, and layer IV in extrastriate areas. These projection patterns suggest that the pulvinar nucleus most strongly influences (drives) activity in cortical areas beyond V1. Should this hypothesis be true, one would expect the spatiotemporal responses evoked by pulvinar activation to be different in V1 and extrastriate areas, reflecting the different connectivity patterns. We investigated this issue by analyzing the spatiotemporal dynamics of cortical visual areas' activity following thalamic electrical microstimulation in tree shrews, using optical imaging and voltage-sensitive dyes. As expected, electrical stimulation of the dLGN induced fast and local responses in V1, as well as in extrastriate and contralateral cortical areas. In contrast, electrical stimulation of the pulvinar induced fast and local responses in extrastriate areas, followed by weak and diffuse activation in V1 and contralateral cortical areas. This study highlights spatiotemporal cortical activation characteristics induced by stimulation of first (dLGN) and high-order (pulvinar) thalamic nuclei. SIGNIFICANCE STATEMENT: The pulvinar nucleus represents the main extrageniculate thalamic visual structure in higher-order mammals, but its exact role remains enigmatic. The pulvinar receive prominent inputs from virtually all visual cortical areas. Cortico-thalamo-cortical pathways through the pulvinar nuclei may then provide a complementary route for corticocortical information flow. One step toward the understanding of the role of transthalamic corticocortical pathways is to determine the nature of the signals transmitted between the cortex and the thalamus. By performing, for the first time, high spatiotemporal mesoscopic imaging on tree shrews (the primate's closest relative) through the combination of voltage-sensitive dye recordings and brain stimulation, we revealed clear evidence of distinct thalamocortical functional connectivity pattern originating from the geniculate nucleus and the pulvinar nuclei.

Retinal and Tectal "Driver-Like" Inputs Converge in the Shell of the Mouse Dorsal Lateral Geniculate Nucleus.

The dorsal lateral geniculate nucleus (dLGN) is a model system for understanding thalamic organization and the classification of inputs as "drivers" or "modulators." Retinogeniculate terminals provide the primary excitatory drive for the relay of information to visual cortex (V1), while nonretinal inputs act in concert to modulate the gain of retinogeniculate signal transmission. How do inputs from the superior colliculus, a visuomotor structure, fit into this schema? Using a variety of anatomical, optogenetic, and in vitro physiological techniques in mice, we show that dLGN inputs from the superior colliculus (tectogeniculate) possess many of the ultrastructural and synaptic properties that define drivers. Tectogeniculate and retinogeniculate terminals converge to innervate one class of dLGN neurons within the dorsolateral shell, the primary terminal domain of direction-selective retinal ganglion cells. These dLGN neurons project to layer I of V1 to form synaptic contacts with dendrites of deeper-layer neurons. We suggest that tectogeniculate inputs act as "backseat drivers," which may alert shell neurons to movement commands generated by the superior colliculus. Significance statement: The conventional view of the dorsal lateral geniculate nucleus (dLGN) is that of a simple relay of visual information between the retina and cortex. Here we show that the dLGN receives strong excitatory input from both the retina and the superior colliculus. Thus, the dLGN is part of a specialized visual channel that provides cortex with convergent information about stimulus motion and eye movement and positioning.

Developmental remodeling of relay cells in the dorsal lateral geniculate nucleus in the absence of retinal input.

BACKGROUND: The dorsal lateral geniculate nucleus (dLGN) of the mouse has been an important experimental model for understanding thalamic circuit development. The developmental remodeling of retinal projections has been the primary focus, however much less is known about the maturation of their synaptic targets, the relay cells of the dLGN. Here we examined the growth and maturation of relay cells during the first few weeks of life and addressed whether early retinal innervation affects their development. To accomplish this we utilized the math5 null (math5 (-/-) ) mouse, a mutant lacking retinal ganglion cells and central projections. RESULTS: The absence of retinogeniculate axon innervation led to an overall shrinkage of dLGN and disrupted the pattern of dendritic growth among developing relay cells. 3-D reconstructions of biocytin filled neurons from math5 (-/-) mice showed that in the absence of retinal input relay cells undergo a period of exuberant dendritic growth and branching, followed by branch elimination and an overall attenuation in dendritic field size. However, math5 (-/-) relay cells retained a sufficient degree of complexity and class specificity, as well as their basic membrane properties and spike firing characteristics. CONCLUSIONS: Retinal innervation plays an important trophic role in dLGN development. Additional support perhaps arising from non-retinal innervation and signaling is likely to contribute to the stabilization of their dendritic form and function.

Thalamic Circuit Diversity: Modulation of the Driver/Modulator Framework.

The idea that dorsal thalamic inputs can be divided into "drivers", which provide the primary excitatory drive for the relay of information to cortex, and "modulators", which alter the gain of signal transmission, has provided a valuable organizing principle for the study of thalamic function. This view further promoted the identification of "first order" and "higher order" thalamic nuclei, based on the origin of their driving inputs. Since the introduction of this influential terminology, a number of studies have revealed the existence of a wide variety of thalamic organizational schemes. For example, some thalamic nuclei are not innervated by typical driver inputs, but instead receive input from terminals which exhibit features distinct from those of either classic drivers or modulators. In addition, many thalamic nuclei contain unique combinations of convergent first order, higher order, and/or other "driver-like" inputs that do not conform with the driver/modulator framework. The assortment of synaptic arrangements identified in the thalamus are reviewed and discussed from the perspective that this organizational diversity can dramatically increase the computational capabilities of the thalamus, reflecting its essential roles in sensory, motor, and sensory-motor circuits.

Descending projections from auditory cortex to excitatory and inhibitory cells in the nucleus of the brachium of the inferior colliculus.

Descending projections from the auditory cortex (AC) terminate in subcortical auditory centers from the medial geniculate nucleus (MG) to the cochlear nucleus, allowing the AC to modulate the processing of acoustic information at many levels of the auditory system. The nucleus of the brachium of the inferior colliculus (NBIC) is a large midbrain auditory nucleus that is a target of these descending cortical projections. The NBIC is a source of several auditory projections, including an ascending projection to the MG. This ascending projection appears to originate from both excitatory and inhibitory NBIC cells, but whether the cortical projections contact either of these cell groups is unknown. In this study, we first combined retrograde tracing and immunochemistry for glutamic acid decarboxylase (GAD, a marker of GABAergic cells) to identify GABAergic and non-GABAergic NBIC projections to the MG. Our first result is that GAD-immunopositive cells constitute ~17% of the NBIC to MG projection. We then used anterograde labeling and electron microscopy to examine the AC projection to the NBIC. Our second result is that cortical boutons in the NBIC form synapses with round vesicles and asymmetric synapses, consistent with excitatory effects. Finally, we combined fluorescent anterograde labeling of corticofugal axons with immunochemistry and retrograde labeling of NBIC cells that project to the MG. These final results suggest first that AC axons contact both GAD-negative and GAD-positive NBIC cells and, second, that some of cortically-contacted cells project to the MG. Overall, the results imply that corticofugal projections can modulate both excitatory and inhibitory ascending projections from the NBIC to the auditory thalamus.