Detailed mapping of the complex fiber structure and white matter pathways of the chimpanzee brain.

Long-standing questions about human brain evolution may only be resolved through comparisons with close living evolutionary relatives, such as chimpanzees. This applies in particular to structural white matter (WM) connectivity, which continuously expanded throughout evolution. However, due to legal restrictions on chimpanzee research, neuroscience research currently relies largely on data with limited detail or on comparisons with evolutionarily distant monkeys. Here, we present a detailed magnetic resonance imaging resource to study structural WM connectivity in the chimpanzee. This open-access resource contains (1) WM reconstructions of a postmortem chimpanzee brain, using the highest-quality diffusion magnetic resonance imaging data yet acquired from great apes; (2) an optimized and validated method for high-quality fiber orientation reconstructions; and (3) major fiber tract segmentations for cross-species morphological comparisons. This dataset enabled us to identify phylogenetically relevant details of the chimpanzee connectome, and we anticipate that it will substantially contribute to understanding human brain evolution.

Selective Interruption of Auditory Interhemispheric Cross Talk Impairs Discrimination Learning of Frequency-Modulated Tone Direction But Not Gap Detection and Discrimination.

Functional hemispheric lateralization is a basic principle of brain organization. In the auditory domain, the right auditory cortex (AC) determines the pitch direction of continuous auditory stimuli whereas the left AC discriminates gaps in these stimuli. The involved functional interactions between the two sides, mediated by commissural connections, are poorly understood. Here, we selectively disrupted the interhemispheric cross talk from the left to the right primary AC and vice versa using chromophore-targeted laser-induced apoptosis of the respective projection neurons, which make up 6-17% of all AC neurons in Layers III, V, and VI. Following photolysis, male gerbils were trained in a first experimental set to discriminate between rising and falling frequency-modulated (FM) tone sweeps. The acquisition of the task was significantly delayed in lesioned animals of either lesion direction. However, the final discrimination performance and hit rate was lowest for animals with left-side lesioned commissural neurons, demonstrating that also information from the left AC is relevant for FM direction learning. Photolysis after successful learning did not affect the retrieval of the learned task, indicating that the disruption during learning was not because of a general functional impairment. In a second experimental set, the gerbil's ability to detect and discriminate small silent gaps of varying length within FM sweeps was tested. This ability was also preserved after interhemispheric disruption. Taken together, interhemispheric communication between the left and right AC is important for the acquisition of FM tone direction learning but not for its retrieval and for gap detection and gap duration discrimination.SIGNIFICANCE STATEMENT Hemispheric lateralization of neuronal functions such as speech and music processing in humans are common throughout the brain; however, the involved interhemispheric interactions are ill-defined. Here, we show that the selective photolytic disruption of auditory cortical commissural connections in rodents impairs the acquisition but not retrieval of a frequency-modulated tone direction discrimination task. The final discrimination performance and hit rate was lowest for animals with lesioned left-to-right-side projections; thus, although right auditory cortex is dominant, left auditory cortex is also relevant for learning this task. The detection and discrimination of small gaps within the tone sweeps remain intact, suggesting a pathway for the processing of these temporal structures, which could be independent from the lesioned interhemispheric cross talk.

Ultrastructure of giant thalamic terminals in the auditory cortex.

The auditory system comprises some very large axonal terminals like the endbulb and calyx of Held and "giant" corticothalamic synapses. Previously, we described a hitherto unknown population of giant thalamocortical boutons arising from the medial division of the medial geniculate body (MGm) in the Mongolian gerbil, which terminate over a wide cortical range but in a columnar manner particularly in the extragranular layers of the auditory cortex. As a first step towards an understanding of their potential functional role, we here describe their ultrastructure combining anterograde tract-tracing with biocytin and electron microscopy. Quantitative ultrastructural analyses revealed that biocytin-labelled MGm boutons reach much larger sizes than other, non-labelled boutons. Also, mitochondria occupy more space within labelled boutons whereas synapses are of similar size. Labelled boutons are very heterogeneous in size but homogeneous with respect to their ultrastructural characteristics, with asymmetric synapses containing clear, round vesicles and targeting dendritic spines. Functionally, the ultrastructure of the MGm terminals indicates that they form excitatory contacts, which may transmit their information in a rapid, powerful and high-fidelity manner onto strategically advantageous compartments of their cortical target cells.

ß-adrenergic modulation of discrimination learning and memory in the auditory cortex.

Despite vast literature on catecholaminergic neuromodulation of auditory cortex functioning in general, knowledge about its role for long-term memory formation is scarce. Our previous pharmacological studies on cortex-dependent frequency-modulated tone-sweep discrimination learning of Mongolian gerbils showed that auditory-cortical D1/5 -dopamine receptor activity facilitates memory consolidation and anterograde memory formation. Considering overlapping functions of D1/5 -dopamine receptors and ß-adrenoceptors, we hypothesised a role of ß-adrenergic signalling in the auditory cortex for sweep discrimination learning and memory. Supporting this hypothesis, the ß1/2 -adrenoceptor antagonist propranolol bilaterally applied to the gerbil auditory cortex after task acquisition prevented the discrimination increment that was normally monitored 1 day later. The increment in the total number of hurdle crossings performed in response to the sweeps per se was normal. Propranolol infusion after the seventh training session suppressed the previously established sweep discrimination. The suppressive effect required antagonist injection in a narrow post-session time window. When applied to the auditory cortex 1 day before initial conditioning, ß1 -adrenoceptor-antagonising and ß1 -adrenoceptor-stimulating agents retarded and facilitated, respectively, sweep discrimination learning, whereas ß2 -selective drugs were ineffective. In contrast, single-sweep detection learning was normal after propranolol infusion. By immunohistochemistry, ß1 - and ß2 -adrenoceptors were identified on the neuropil and somata of pyramidal and non-pyramidal neurons of the gerbil auditory cortex. The present findings suggest that ß-adrenergic signalling in the auditory cortex has task-related importance for discrimination learning of complex sounds: as previously shown for D1/5 -dopamine receptor signalling, ß-adrenoceptor activity supports long-term memory consolidation and reconsolidation; additionally, tonic input through ß1 -adrenoceptors may control mechanisms permissive for memory acquisition.

A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia.

Neuronal correlates of auditory streaming in the auditory cortex of behaving monkeys.

This study tested the hypothesis that spiking activity in the primary auditory cortex of monkeys is related to auditory stream formation. Evidence for this hypothesis was previously obtained in animals that were passively exposed to stimuli and in which differences in the streaming percept were confounded with differences between the stimuli. In this study, monkeys performed an operant task on sequences that were composed of light flashes and tones. The tones alternated between a high and a low frequency and could be perceived either as one auditory stream or two auditory streams. The flashes promoted either a one-stream percept or a two-stream percept. Comparison of different types of sequences revealed that the neuronal responses to the alternating tones were more similar when the flashes promoted auditory stream integration, and were more dissimilar when the flashes promoted auditory stream segregation. Thus our findings show that the spiking activity in the monkey primary auditory cortex is related to auditory stream formation.

Mapping fine-scale anatomy of gray matter, white matter, and trigeminal-root region applying spherical deconvolution to high-resolution 7-T diffusion MRI.

OBJECTIVES: We assessed the use of high-resolution ultra-high-field diffusion magnetic resonance imaging (dMRI) to determine neuronal fiber orientation density functions (fODFs) throughout the human brain, including gray matter (GM), white matter (WM), and small intertwined structures in the cerebellopontine region. MATERIALS AND METHODS: We acquired 7-T whole-brain dMRI data of 23 volunteers with 1.4-mm isotropic resolution; fODFs were estimated using constrained spherical deconvolution. RESULTS: High-resolution fODFs enabled a detailed view of the intravoxel distributions of fiber populations in the whole brain. In the brainstem region, the fODF of the extra- and intrapontine parts of the trigeminus could be resolved. Intrapontine trigeminal fiber populations were crossed in a network-like fashion by fiber populations of the surrounding cerebellopontine tracts. In cortical GM, additional evidence was found that in parts of primary somatosensory cortex, fODFs seem to be oriented less perpendicular to the cortical surface than in GM of motor, premotor, and secondary somatosensory cortices. CONCLUSION: With 7-T MRI being introduced into clinical routine, high-resolution dMRI and derived measures such as fODFs can serve to characterize fine-scale anatomic structures as a prerequisite to detecting pathologies in GM and small or intertwined WM tracts.

The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth.

Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of ß1-integrin to stimulate the PKC- and CaMKII-dependent activation of ß1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated ß1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

Visualization of acute focal lesions in rats with experimental autoimmune encephalomyelitis by magnetic nanoparticles, comparing different MRI sequences including phase imaging.

PURPOSE: To compare the sensitivity and specificity of phase imaging (PI) with other magnetic resonance imaging (MRI) methods in lesion detection in rats with experimental autoimmune encephalomyelitis (EAE), as an animal model for multiple sclerosis (MS). MATERIALS AND METHODS: EAE was induced in rats (n = 14) by subcutaneous (s.c.) injection of myelin basic protein (MBP) and complete Freund's adjuvant (CFA). Control animals (n = 4) were given an s.c. injection of phosphate-buffered saline mixed with CFA. The development of local inflammatory processes, demyelinations, and blood-brain barrier (BBB) disruptions were monitored over 7 weeks in a 4.7T animal scanner by T1-, T2-, T2*-weighted images, magnetization transfer, and PI in the presence or absence of very small superparamagnetic iron oxide particles (VSOP) and confirmed by immunostaining using CD31, CD68, MBP, and albumin antibodies and Gallyas silver staining. RESULTS: EAE rats developed time-dependent local inflammations and BBB disruptions but no clear demyelinizations. In histological stainings these processes were trackable as accumulations of phagocytic monocytes and extravasal albumin. In MRI without application of VSOP inflammatory processes were not detectable. MRI in the presence of VSOP revealed inflammatory processes by the appearance of hypointense spots (hs). The specificity of PI to detect hs was similar to T1- and T2*-weighted images The calculated sensitivity was less than in corresponding T2*-weighted images. CONCLUSION: The diagnostic use of PI without VSOP as contrast agent to detect lesions is not recommended at field strength of 4.7T or lower.

Different synchronization rules in primary and nonprimary auditory cortex of monkeys.

Synchronized neuronal firing in cortex has been implicated in feature binding, attentional selection, and other cognitive processes. This study addressed the question whether different cortical fields are distinct by rules according to which neurons engage in synchronous firing. To this end, we simultaneously recorded the multiunit firing at several sites within the primary and the caudomedial auditory cortical field of anesthetized macaque monkeys, determined their responses to pure tones, and calculated the cross-correlation function of the spontaneous firing of pairs of units. In the primary field, the likelihood of synchronous firing of pairs of units increased with the similarity of their frequency tuning and their response latencies. In the caudomedial field, by contrast, the likelihood of synchronization was highest when pairs of units had an octave and other harmonic relationships and when units had different response latencies. The differences in synchrony of the two fields were not paralleled by differences in distributions of best frequency, bandwidth of tuning curves, and response latency. Our findings suggest that neuronal synchrony in different cortical fields may underlie the establishment of specific relationships between the sound features that are represented by the firing of the neurons and which follow the Gestalt laws of similarity in the primary field and good continuation in the caudomedial field.

Task-demands and audio-visual stimulus configurations modulate neural activity in the human thalamus.

Recent electrophysiological studies have reported short latency modulations in cortical regions for multisensory stimuli, thereby suggesting a subcortical, possibly thalamic origin of these modulations. Concurrently, there is an ongoing debate, whether multisensory interplay reflects automatic, bottom-up driven processes or relies on top-down influences. Here, we dissociated the effects of task set and stimulus configurations on BOLD-signals in the human thalamus with event-related functional magnetic resonance imaging (fMRI). We orthogonally manipulated temporal and spatial congruency of audio-visual stimulus configurations, while subjects judged either their temporal or spatial congruency. Voxel-based fMRI results revealed increased fMRI-signals for the temporal versus spatial task in posterior and central thalamus, respectively. A more sensitive region of interest (ROI)-analysis confirmed that the posterior thalamic nuclei showed a preference for the temporal task and central thalamic nuclei for the spatial task. Moreover, the ROI-analysis also revealed enhanced fMRI-signals for spatially incongruent stimuli in the central thalamus. Together, our results demonstrate that both audio-visual stimulus configurations and task-related processing of spatial or temporal stimulus features selectively modulate thalamic processing and thus are in a position to influence cortical processing at an early stage.

Altered neuronal activity patterns in the visual cortex of the adult rat after partial optic nerve crush--a single-cell resolution metabolic mapping study.

Thallium autometallography (TIAMG) is a novel method for high-resolution mapping of neuronal activity. With this method, we found that a general depression of neuronal activity occurs in response to optic nerve crush (ONC) within the first 2 weeks postinjury in the contralateral dorsal lateral geniculate nucleus (dLGN) as well as in the contralateral primary visual cortex (V1). Interestingly, the neuronal activity recovered thereafter in both brain regions and reached a plateau in the tenth week postinjury in layers IV and V of V1, monocular area (V1m). Several clusters of highly active neurons in V1m were found 6 weeks after ONC in layers IV and V on the side contralateral to the lesion. We reasoned that these clusters appeared due to a reorganization of the corticocolliucular projections. Employing a combination of biotinylated dextran amine retrograde tract tracing from the superior colliculus (SC) with TIAMG in the same animal, we indeed found that the clusters of neurons with high Tl(+) uptake in V1m are spatially in register with those neuronal subpopulations that project to the SC. These data suggest that extensive reorganization plasticity exists in the adult rat visual cortex following ONC.

Rabies virus-based labeling of layer 6 corticothalamic neurons for two-photon imaging in vivo.

Neocortical layer 6 (L6) is less understood than other more superficial layers, largely owing to limitations of performing high-resolution investigations in vivo. Here, we show that labeling with the Challenge Virus Standard (CVS) rabies virus strain enables high-quality imaging of L6 neurons by conventional two-photon microscopes. CVS virus injection into the medial geniculate body can selectively label L6 neurons in the auditory cortex. Only three days after injection, dendrites and cell bodies of L6 neurons could be imaged across all cortical layers. Ca2+ imaging in awake mice showed that sound stimulation evokes neuronal responses from cell bodies with minimal contamination from neuropil signals. In addition, dendritic Ca2+ imaging revealed significant responses from spines and trunks across all layers. These results demonstrate a reliable method capable of rapid, high-quality labeling of L6 neurons that can be readily extended to other brain regions.

Sound-induced enhancement of low-intensity vision: multisensory influences on human sensory-specific cortices and thalamic bodies relate to perceptual enhancement of visual detection sensitivity.

Combining information across modalities can affect sensory performance. We studied how co-occurring sounds modulate behavioral visual detection sensitivity (d'), and neural responses, for visual stimuli of higher or lower intensity. Co-occurrence of a sound enhanced human detection sensitivity for lower- but not higher-intensity visual targets. Functional magnetic resonance imaging (fMRI) linked this to boosts in activity-levels for sensory-specific visual and auditory cortex, plus multisensory superior temporal sulcus (STS), specifically for a lower-intensity visual event when paired with a sound. Thalamic structures in visual and auditory pathways, the lateral and medial geniculate bodies, respectively (LGB, MGB), showed a similar pattern. Subject-by-subject psychophysical benefits correlated with corresponding fMRI signals in visual, auditory, and multisensory regions. We also analyzed differential "coupling" patterns of LGB and MGB with other regions in the different experimental conditions. Effective-connectivity analyses showed enhanced coupling of sensory-specific thalamic bodies with the affected cortical sites during enhanced detection of lower-intensity visual events paired with sounds. Coupling strength between visual and auditory thalamus with cortical regions, including STS, covaried parametrically with the psychophysical benefit for this specific multisensory context. Our results indicate that multisensory enhancement of detection sensitivity for low-contrast visual stimuli by co-occurring sounds reflects a brain network involving not only established multisensory STS and sensory-specific cortex but also visual and auditory thalamus.

Laser-Induced Apoptosis of Corticothalamic Neurons in Layer VI of Auditory Cortex Impact on Cortical Frequency Processing.

Corticofugal projections outnumber subcortical input projections by far. However, the specific role for signal processing of corticofugal feedback is still less well understood in comparisonto the feedforward projection. Here, we lesioned corticothalamic (CT) neurons in layers V and/or VI of the auditory cortex of Mongolian gerbils by laser-induced photolysis to investigate their contribution to cortical activation patterns. We have used laminar current-source density (CSD) recordings of tone-evoked responses and could show that, particularly, lesion of CT neurons in layer VI affected cortical frequency processing. Specifically, we found a decreased gain of best-frequency input in thalamocortical (TC)-recipient input layers that correlated with the relative lesion of layer VI neurons, but not layer V neurons. Using cortical silencing with the GABA a -agonist muscimol and layer-specific intracortical microstimulation (ICMS), we found that direct activation of infragranular layers recruited a local recurrent cortico-thalamo-cortical loop of synaptic input. This recurrent feedback was also only interrupted when lesioning layer VI neurons, but not cells in layer V. Our study thereby shows distinct roles of these two types of CT neurons suggesting a particular impact of CT feedback from layer VI to affect the local feedforward frequency processing in auditory cortex.

The extracellular matrix regulates cortical layer dynamics and cross-columnar frequency integration in the auditory cortex.

In the adult vertebrate brain, enzymatic removal of the extracellular matrix (ECM) is increasingly recognized to promote learning, memory recall, and restorative plasticity. The impact of the ECM on translaminar dynamics during cortical circuit processing is still not understood. Here, we removed the ECM in the primary auditory cortex (ACx) of adult Mongolian gerbils using local injections of hyaluronidase (HYase). Using laminar current-source density (CSD) analysis, we found layer-specific changes of the spatiotemporal synaptic patterns with increased cross-columnar integration and simultaneous weakening of early local sensory input processing within infragranular layers Vb. These changes had an oscillatory fingerprint within beta-band (25-36 Hz) selectively within infragranular layers Vb. To understand the laminar interaction dynamics after ECM digestion, we used time-domain conditional Granger causality (GC) measures to identify the increased drive of supragranular layers towards deeper infragranular layers. These results showed that ECM degradation altered translaminar cortical network dynamics with a stronger supragranular lead of the columnar response profile.

Dopaminergic modulation of auditory cortex-dependent memory consolidation through mTOR.

Previous studies in the auditory cortex of Mongolian gerbils on discrimination learning of the direction of frequency-modulated tones (FMs) revealed that long-term memory formation involves activation of the dopaminergic system, activity of the protein kinase mammalian target of rapamycin (mTOR), and protein synthesis. This led to the hypothesis that the dopaminergic system might modulate memory formation via regulation of mTOR, which is implicated in translational control. Here, we report that the D1/D5 dopamine receptor agonist SKF-38393 substantially improved gerbils' FM discrimination learning when administered systemically or locally into the auditory cortex shortly before, shortly after, or 1 day before conditioning. Although acquisition performance during initial training was normal, the discrimination of FMs was enhanced during retraining performed hours or days after agonist injection compared with vehicle-injected controls. The D1/D5 receptor antagonist SCH-23390, the mTOR inhibitor rapamycin, and the protein synthesis blocker anisomycin suppressed this effect. By immunohistochemistry, D1 dopamine receptors were identified in the gerbil auditory cortex predominantly in the infragranular layers. Together, these findings suggest that in the gerbil auditory cortex dopaminergic inputs regulate mTOR-mediated, protein synthesis-dependent mechanisms, thus controlling for hours or days the consolidation of memory required for the discrimination of complex auditory stimuli.

Early Sensory Loss Alters the Dendritic Branching and Spine Density of Supragranular Pyramidal Neurons in Rodent Primary Sensory Cortices.

Multisensory integration in primary auditory (A1), visual (V1), and somatosensory cortex (S1) is substantially mediated by their direct interconnections and by thalamic inputs across the sensory modalities. We have previously shown in rodents (Mongolian gerbils) that during postnatal development, the anatomical and functional strengths of these crossmodal and also of sensory matched connections are determined by early auditory, somatosensory, and visual experience. Because supragranular layer III pyramidal neurons are major targets of corticocortical and thalamocortical connections, we investigated in this follow-up study how the loss of early sensory experience changes their dendritic morphology. Gerbils were sensory deprived early in development by either bilateral sciatic nerve transection at postnatal day (P) 5, ototoxic inner hair cell damage at P10, or eye enucleation at P10. Sholl and branch order analyses of Golgi-stained layer III pyramidal neurons at P28, which demarcates the end of the sensory critical period in this species, revealed that visual and somatosensory deprivation leads to a general increase of apical and basal dendritic branching in A1, V1, and S1. In contrast, dendritic branching, particularly of apical dendrites, decreased in all three areas following auditory deprivation. Generally, the number of spines, and consequently spine density, along the apical and basal dendrites decreased in both sensory deprived and non-deprived cortical areas. Therefore, we conclude that the loss of early sensory experience induces a refinement of corticocortical crossmodal and other cortical and thalamic connections by pruning of dendritic spines at the end of the critical period. Based on present and previous own results and on findings from the literature, we propose a scenario for multisensory development following early sensory loss.

Non-sensory cortical and subcortical connections of the primary auditory cortex in Mongolian gerbils: bottom-up and top-down processing of neuronal information via field AI.

In the present study, we will provide further anatomical evidence that the primary auditory cortex (field AI) is not only involved in sensory processing of its own modality, but also in complex bottom-up and top-down processing of multimodal information. We have recently shown that AI in the Mongolian gerbil (Meriones unguiculatus) has substantial connections with non-auditory sensory and multisensory brain structures [Budinger, E., Heil, P., Hess, A., Scheich, H., 2006. Multisensory processing via early cortical stages: Connections of the primary auditory cortical field with other sensory systems. Neuroscience 143, 1065-1083]. Here we will report about the direct connections of AI with non-sensory cortical areas and subcortical structures. We approached this issue by means of the axonal transport of the sensitive bidirectional neuronal tracers fluorescein-labelled (FD) and tetramethylrhodamine-labelled dextran (TMRD), which were simultaneously injected into different frequency regions of the gerbil's AI. Of the total number of retrogradely labelled cell bodies found in non-sensory brain areas, which identify cells of origin of direct projections to AI, approximately 24% were in cortical areas and 76% in subcortical structures. Of the cell bodies in the cortical areas, about 4.4% were located in the orbital, 11.1% in the infralimbic medial prefrontal (areas DPC, IL), 18.2% in the cingulate (3.2% in CG1, 2.9% in CG2, 12.1% in CG3), 9.5% in the frontal association (area Fr2), 12.0% in the insular (areas AI, DI), 10.8% in the retrosplenial, and 34.0% in the perirhinal cortex. The cortical regions with retrogradely labelled cells, as well as the entorhinal cortex, also contained anterogradely labelled axons and their terminations, which means that they are also target areas of direct projections from AI. The laminar pattern of corticocortical connections indicates that AI receives primarily cortical feedback-type inputs and projects in a feedforward manner to its target areas. The high number of double-labelled somata, the non-topographic distribution of single FD- and TMRD-labelled somata, and the overlapping spatial distribution of FD- and TMRD-labelled axonal elements suggest rather non-tonotopic connections between AI and the multimodal cortices. Of the labelled cell bodies in the subcortical structures, about 38.8% were located in the ipsilateral basal forebrain (10.6% in the lateral amygdala LA, 11.5% in the globus pallidus GP, 3.7% in the ventral pallidum VPa, 13.0% in the nucleus basalis NB), 13.1% in the ipsi- and contralateral diencephalon (6.4% in the posterior paraventricular thalamic nuclei, 6.7% in the hypothalamic area), and 48.1% in the midbrain (20.0% in the ipsilateral substantia nigra, 9.8% in the ipsi- and contralateral ventral tegmental area, 5.0% in the ipsi- and contralateral locus coeruleus, 13.3% the ipsi- and contralateral dorsal raphe nuclei). Thus, the majority of subcortical inputs to AI was related to different neurotransmitter systems. Anterograde labelling was only found in some ipsilateral basal forebrain structures, namely, the LA, basolateral amygdala, GP, VPa, and NB. As for the cortex, the proportion and spatial distribution of single FD-, TMRD-, and double-labelled neuronal elements suggests rather non-tonotopic connections between AI and the neuromodulatory subcortical structures.