Abstract Multisensory integration is a fundamental feature of cortical processing, yet the functional pathways that deliver visual signals to the auditory cortex remain poorly understood. While anatomical studies reveal multiple candidate projection routes, demonstrating their causal contribution requires targeted manipulation of neural activity. Here, we used cortical cooling to reversibly inactivate the posteromedial lateral suprasylvian cortex (PMLS) and the adjacent area 21 to determine the functional role of higher-order visual areas in generating visual responses within the auditory cortex of the ferret. Units responsive to sound, light, or combined audiovisual stimuli were found across all sampled auditory fields and cortical depths, with visual responses most prominent within the infragranular layers and the non-tonotopic secondary auditory cortex of the Anterior Ectosylvian Gyrus (AEG). Cortical cooling induced robust, bi-directional, and stimulus-specific modulations of firing rates in AC. Approximately 50% of visually responsive units exhibited a significant decrease or complete elimination of visual activity during cooling, confirming a functional role for visual input from PLMS/area 21 to AC. Surprisingly, cooling also revealed circuit-level complexities: a subset (∼5%) of units showed enhanced or newly emergent visual responses during inactivation, suggesting that PMLS/area 21 normally exerts a gating influence over alternative visual pathways. Furthermore, contrary to feedforward anatomical predictions, neurons in the AEG—the region most heavily innervated by the cooled visual areas —were less frequently impacted by cooling than those in PEG. Together, these findings demonstrate that higher visual areas causally shape cross-modal processing in the auditory cortex through a complex mixture of direct excitation and network-level modulation.

The auditory system relies on head-centered localisation cues to estimate sound source position. Yet listeners and single neurons can encode sound in world-centred spatial reference frames. However, most studies of sound localisation constrain the subject’s orientation within a fixed speaker ring, making it difficult to distinguish which reference frame underlies spatial coding. We utilised a sound localisation task that required ferrets localise sound location, relative to the head, across rotations in the starting platform within the testing arena. Two ferrets were trained to discriminate front from back sounds. Once trained, the orientation of both the central platform and the target speakers were rotated between testing sessions, retaining the task contingency in head-centered coordinates, but breaking the alignment of head and world centered reference frames. Probe trials were presented from the other speakers so that spatial receptive fields could be constructed and compared across central platform rotations. Neural activity was recorded from auditory cortex using chronically implanted 32-channel arrays. Across 1,777 recorded units, 596 were stimulus driven. Spatially tuned neurons were predominantly head-centred, with 208 and 232 units classified as head-centred in the two animals, compared with 48 and 49 world-centred units and 33 and 26 un-tuned units, respectively. Temporal analysis showed that most units retained the same reference-frame classification from onset to offset, but the magnitude of their head-versus-world preference diminished over time, with offset responses shifting towards more ambiguous and, in some cases, world-centred coding. Together, these results demonstrate that auditory cortex maintains parallel head centered and allocentric representations that are head dominated at onset but incorporate increased world-centred information at offset, supporting perceptual constancy during dynamic listening.

A key question in auditory neuroscience is to what extent are brain regions functionally specialized for processing specific sound features, such as location and identity. In auditory cortex, correlations between neural activity and sounds support both the specialization of distinct cortical subfields, and encoding of multiple sound features within individual cortical areas. However, few studies have tested the contribution of auditory cortex to hearing in multiple contexts. Here we determined the role of ferret primary auditory cortex in both spatial and nonspatial hearing by reversibly inactivating the middle ectosylvian gyrus during behavior using cooling (n = 2 females) or optogenetics (n = 1 female). Optogenetic experiments used the mDLx promoter to express Channelrhodopsin-2 in GABAergic interneurons, and we confirmed both viral expression (n = 2 females) and light-driven suppression of spiking activity in auditory cortex, recorded using Neuropixels under anesthesia (n = 465 units from 2 additional untrained female ferrets). Cortical inactivation via cooling or optogenetics impaired vowel discrimination in colocated noise. Ferrets implanted with cooling loops were tested in additional conditions that revealed no deficit when identifying vowels in clean conditions, or when the temporally coincident vowel and noise were spatially separated by 180 degrees. These animals did, however, show impaired sound localization when inactivating the same auditory cortical region implicated in vowel discrimination in noise. Our results demonstrate that, as a brain region showing mixed selectivity for spatial and nonspatial features of sound, primary auditory cortex contributes to multiple forms of hearing. SIGNIFICANCE STATEMENT Neurons in primary auditory cortex are often sensitive to the location and identity of sounds. Here we inactivated auditory cortex during spatial and nonspatial listening tasks using cooling, or optogenetics. Auditory cortical inactivation impaired multiple behaviors, demonstrating a role in both the analysis of sound location and identity and confirming a functional contribution of mixed selectivity observed in neural activity. Parallel optogenetic experiments in two additional untrained ferrets linked behavior to physiology by demonstrating that expression of Channelrhodopsin-2 permitted rapid light-driven suppression of auditory cortical activity recorded under anesthesia.

Theta oscillations are a hallmark of hippocampal activity across mammals and play a critical role in many hippocampal models of memory and spatial navigation. To reconcile the cross-species differences observed in the presence and properties of theta, we recorded hippocampal local field potentials in rats and ferrets during auditory and visual localisation tasks designed to vary locomotion and sensory attention. Here, we show that theta oscillations occur during locomotion in both ferrets and rats, however during periods of immobility, theta oscillations persist in the ferret, contrasting starkly with the switch to large irregular activity (LIA) in the rat. Theta during immobility in the ferret is identified as analogous to Type 2 theta that has been observed in rodents due to its sensitivity to atropine, and is modulated by behavioural state with the strongest theta observed during reward epochs. These results demonstrate that even under similar behavioural conditions, differences exist between species in the relationship between theta and behavioural state.

The location of sounds can be described in multiple coordinate systems that are defined relative to ourselves, or the world around us. Evidence from neural recordings in animals point toward the existence of both head-centered and world-centered representations of sound location in the brain; however, it is unclear whether such neural representations have perceptual correlates in the sound localization abilities of nonhuman listeners. Here, we establish novel behavioral tests to determine the coordinate systems in which ferrets can localize sounds. We found that ferrets could learn to discriminate between sound locations that were fixed in either world-centered or head-centered space, across wide variations in sound location in the alternative coordinate system. Using probe sounds to assess broader generalization of spatial hearing, we demonstrated that in both head and world-centered tasks, animals used continuous maps of auditory space to guide behavior. Single trial responses of individual animals were sufficiently informative that we could then model sound localization using speaker position in specific coordinate systems and accurately predict ferrets' actions in held-out data. Our results demonstrate that ferrets, an animal model in which neurons are known to be tuned to sound location in egocentric and allocentric reference frames, can also localize sounds in multiple head and world-centered spaces. SIGNIFICANCE STATEMENT Humans can describe the location of sounds either relative to themselves, or in the world, independent of their momentary position. These different spaces are also represented in the activity of neurons in animals, but it is not clear whether nonhuman listeners also perceive both head and world-centered sound location. Here, we designed behavioral tasks in which ferrets discriminated between sounds using their position in the world, or relative to the head. Subjects learnt to solve both problems and generalized sound location in each space when presented with infrequent probe sounds. These findings reveal a perceptual correlate of neural sensitivity previously observed in the ferret brain and establish that, like humans, ferrets can access an auditory map of their local environment.

Binaural cue processing requires central auditory function as damage to the auditory cortex and other cortical regions impairs sound localization. Sound localization cues are initially extracted by brainstem nuclei, but how the cerebral cortex supports spatial sound perception remains unclear. This chapter reviews the evidence that spatial encoding within and beyond the auditory cortex supports sound localization, including the integration of information across sound frequencies and localization cues. In particular, this chapter discusses the role of brain regions across the cerebral cortex that may be specialized for extracting and transforming the spatial aspects of sounds and extends from sensory to parietal and prefrontal cortices. The chapter considers how the encoding of spatial information changes with attention and how spatial processing fits within the broader context of auditory scene analysis by cortical networks. The importance of neural plasticity in binaural processing is outlined, including a discussion of how changes in the mapping of localization cues to spatial position allow listeners to adapt to changes in auditory input throughout life and after hearing loss. The chapter ends by summarizing some of the open questions about the central processing of binaural cues and how they may be answered.

Recent studies of the neurobiology of the dorsal frontal cortex (FC) of the ferret have illuminated its key role in the attention network, top-down cognitive control of sensory processing, and goal directed behavior. To elucidate the neuroanatomical regions of the dorsal FC, and delineate the boundary between premotor cortex (PMC) and dorsal prefrontal cortex (dPFC), we placed retrograde tracers in adult ferret dorsal FC anterior to primary motor cortex and analyzed thalamo-cortical connectivity. Cyto- and myeloarchitectural differences across dorsal FC and the distinctive projection patterns from thalamic nuclei, especially from the subnuclei of the medial dorsal (MD) nucleus and the ventral thalamic nuclear group, make it possible to clearly differentiate three separate dorsal FC fields anterior to primary motor cortex: polar dPFC (dPFCpol), dPFC, and PMC. Based on the thalamic connectivity, there is a striking similarity of the ferret’s dorsal FC fields with other species. This possible homology opens up new questions for future comparative neuroanatomical and functional studies.

Ferrets (Mustela putorius furo) are a valuable animal model used in biomedical research. Like many animals, ferrets undergo significant variation in body weight seasonally, affected by photoperiod, and these variations complicate the use weight as an indicator of health status. To overcome this requires a better understanding of these seasonal weight changes. We provide a normative weight data set for the female ferret accounting for seasonal changes, and also investigate the effect of fluid regulation on weight change. Female ferrets (n = 39) underwent behavioural testing from May 2017 to August 2019 and were weighed daily, while housed in an animal care facility with controlled light exposure. In the winter (October to March), animals experienced 10 hours of light and 14 hours of dark, while in summer (March to October), this contingency was reversed. Individual animals varied in their body weight from approximately 700 to 1200 g. However, weights fluctuated with light cycle, with animals losing weight in summer, and gaining weight in winter such that they fluctuated between approximately 80% and 120% of their long-term average. Ferrets were weighed as part of their health assessment while experiencing water regulation for behavioural training. Water regulation superimposed additional weight changes on these seasonal fluctuations, with weight loss during the 5-day water regulation period being greater in summer than winter. Analysing the data with a Generalised Linear Model confirmed that the percentage decrease in weight per week was relatively constant throughout the summer months, while the percentage increase in body weight per week in winter decreased through the season. Finally, we noted that the timing of oestrus was reliably triggered by the increase in day length in spring. These data establish a normative benchmark for seasonal weight variation in female ferrets that can be incorporated into the health assessment of an animal's condition.

The ability to localize sounds is central to healthy hearing. We can perceive sound location in multiple coordinate systems including those defined by the observer (e.g. “the phone is on my right”) or by the environment (e.g. “the phone is in the office”). Although we can describe sound locations in multiple spaces, the coordinate frames in which non-human animals can perceive sounds remains unclear. Here, we designed a task that required subjects (ferrets) to report the location of sounds in the world across changes in head pose. We developed simulations of the task using world-centered (allocentric) or head-centered (egocentric) models of spatial processing, and compared model predictions to animal behavior. We found that observed behavior most closely matched performance of allocentric models, indicating that subjects solved the task using a world-centered strategy. Our findings indicate that ferrets, like humans, can perceive allocentric sound space and thus abstract sound location beyond momentary head-centered acoustic cues.

The ability to recognize sounds in noise is a key part of hearing, and the mechanisms by which the brain identifies sounds in noise are of considerable interest to scientists, clinicians and engineers. Yet we know little about the necessity of regions such as auditory cortex for hearing in noise, or how cortical processing of sounds is adversely affected by noise. Here we used reversible cortical inactivation and extracellular electrophysiology in ferrets performing a vowel discrimination task to identify and understand the causal contribution of auditory cortex to hearing in noise. Cortical inactivation by cooling impaired task performance in noisy but not clean conditions, while responses of auditory cortical neurons were less informative about vowel identity in noise. Simulations mimicking cortical inactivation indicated that effects of inactivation were related to the loss of information about sounds represented across neural populations. The addition of noise to target sounds drove spiking activity in auditory cortex and recruitment of additional neural populations that were linked to degraded behavioral performance. To suppress noise-related activity, we used continuous exposure to background noise to adapt the auditory system and recover behavioral performance in both ferrets and humans. Inactivation by cooling revealed that the benefits of continuous exposure were not cortically dependent. Together our results highlight the importance of auditory cortex in sound discrimination in noise and the underlying mechanisms through which noise-related activity and adaptation shape hearing.

Auditory cortex is required for sound localisation, but how neural firing in auditory cortex underlies our perception of sound sources in space remains unclear. Specifically, whether neurons in auditory cortex represent spatial cues or an integrated representation of auditory space across cues is not known. Here, we measured the spatial receptive fields of neurons in primary auditory cortex (A1) while ferrets performed a relative localisation task. Manipulating the availability of binaural and spectral localisation cues had little impact on ferrets' performance, or on neural spatial tuning. A subpopulation of neurons encoded spatial position consistently across localisation cue type. Furthermore, neural firing pattern decoders outperformed two-channel model decoders using population activity. Together, these observations suggest that A1 encodes the location of sound sources, as opposed to spatial cue values.

How and where in the brain audio-visual signals are bound to create multimodal objects remains unknown. One hypothesis is that temporal coherence between dynamic multisensory signals provides a mechanism for binding stimulus features across sensory modalities. Here, we report that when the luminance of a visual stimulus is temporally coherent with the amplitude fluctuations of one sound in a mixture, the representation of that sound is enhanced in auditory cortex. Critically, this enhancement extends to include both binding and non-binding features of the sound. We demonstrate that visual information conveyed from visual cortex via the phase of the local field potential is combined with auditory information within auditory cortex. These data provide evidence that early cross-sensory binding provides a bottom-up mechanism for the formation of cross-sensory objects and that one role for multisensory binding in auditory cortex is to support auditory scene analysis.

Perceptual constancy requires neural representations that are selective for object identity, but also tolerant across identity-preserving transformations. How such representations arise in the brain and support perception remains unclear. Here, we study tolerant representation of sound identity in the auditory system by recording neural activity in auditory cortex of ferrets during perceptual constancy. Ferrets generalize vowel identity across variations in fundamental frequency, sound level and location, while neurons represent sound identity robustly across acoustic variations. Stimulus features are encoded with distinct time-courses in all conditions, however encoding of sound identity is delayed when animals fail to generalize and during passive listening. Neurons also encode information about task-irrelevant sound features, as well as animals' choices and accuracy, while population decoding out-performs animals' behavior. Our results show that during perceptual constancy, sound identity is represented robustly in auditory cortex across widely varying conditions, and behavioral generalization requires conserved timing of identity information.

Auditory cortex is required for sound localisation, but how neural firing in auditory cortex underlies our perception of sources in space remains unknown. We measured spatial receptive fields in animals actively attending to spatial location while they performed a relative localisation task using stimuli that varied in the spatial cues that they provided. Manipulating the availability of binaural and spectral localisation cues had mild effects on the ferret9s performance and little impact on the spatial tuning of neurons in primary auditory cortex (A1). Consistent with a representation of space, a subpopulation of neurons encoded spatial position across localisation cue types. Spatial receptive fields measured in the presence of a competing sound source were sharper than those measured in a single-source configuration. Together these observations suggest that A1 encodes the location of auditory objects as opposed to spatial cue values. We compared our data to predictions generated from two theories about how space is represented in auditory cortex: The two-channel model, where location is encoded by the relative activity in each hemisphere, and the labelled-line model where location is represented by the activity pattern of individual cells. The representation of sound location in A1 was mainly contralateral but peak firing rates were distributed across the hemifield consistent with a labelled line model in each hemisphere representing contralateral space. Comparing reconstructions of sound location from neural activity, we found that a labelled line architecture far outperformed two channel systems. Reconstruction ability increased with increasing channel number, saturating at around 20 channels.

A key function of the brain is to provide a stable representation of an object's location in the world. In hearing, sound azimuth and elevation are encoded by neurons throughout the auditory system, and auditory cortex is necessary for sound localization. However, the coordinate frame in which neurons represent sound space remains undefined: classical spatial receptive fields in head-fixed subjects can be explained either by sensitivity to sound source location relative to the head (egocentric) or relative to the world (allocentric encoding). This coordinate frame ambiguity can be resolved by studying freely moving subjects; here we recorded spatial receptive fields in the auditory cortex of freely moving ferrets. We found that most spatially tuned neurons represented sound source location relative to the head across changes in head position and direction. In addition, we also recorded a small number of neurons in which sound location was represented in a world-centered coordinate frame. We used measurements of spatial tuning across changes in head position and direction to explore the influence of sound source distance and speed of head movement on auditory cortical activity and spatial tuning. Modulation depth of spatial tuning increased with distance for egocentric but not allocentric units, whereas, for both populations, modulation was stronger at faster movement speeds. Our findings suggest that early auditory cortex primarily represents sound source location relative to ourselves but that a minority of cells can represent sound location in the world independent of our own position.

The objective of this study was to demonstrate the efficacy of acute inactivation of brain areas by cooling in the behaving ferret and to demonstrate that cooling auditory cortex produced a localisation deficit that was specific to auditory stimuli. The effect of cooling on neural activity was measured in anesthetized ferret cortex. The behavioural effect of cooling was determined in a benchmark sound localisation task in which inactivation of primary auditory cortex (A1) is known to impair performance. Cooling strongly suppressed the spontaneous and stimulus-evoked firing rates of cortical neurons when the cooling loop was held at temperatures below 10°C, and this suppression was reversed when the cortical temperature recovered. Cooling of ferret auditory cortex during behavioural testing impaired sound localisation performance, with unilateral cooling producing selective deficits in the hemifield contralateral to cooling, and bilateral cooling producing deficits on both sides of space. The deficit in sound localisation induced by inactivation of A1 was not caused by motivational or locomotor changes since inactivation of A1 did not affect localisation of visual stimuli in the same context.

Perceptual constancy describes the ability to represent objects in the world across variation in sensory input such as recognizing a person from different angles or a spoken word across talkers. This ability requires neural representations that are sensitive to some aspects of a stimulus (such as the spectral envelope of a sound) while tolerant to other variations in stimuli (such periodicity). In hearing, such representations have been observed in auditory cortex but never in combination with behavioural testing, which is essential in order to link neural codes to perceptual constancy. By testing ferrets in a vowel discrimination task which they perform across multiple stimulus dimensions and recording neuronal activity in auditory cortex we directly correlate neural tolerance with perceptual constancy. Subjects reported vowel identity across variations in fundamental frequency, sound location, and sound level, but failed to consistently generalize across voicing from voiced to whispered sounds. We decoded the responses of simultaneously recorded units in auditory cortex to identity units informative about vowel identity across each of these task-orthogonal variations in acoustic input. Significant proportions of units were vowel informative across each of these conditions, although fewer units were informative about vowel identity across voicing. For about half of vowel informative units, information about vowel identity was conserved across multiple orthogonal variables. The time of best decoding was also used to identify the relative timing and temporal multiplexing of sound features. Our results show that neural tolerance can be observed within single units in auditory cortex in animals demonstrating perceptual constancy.

Perceptual constancy describes the ability to represent objects in the world across variation in sensory input such as recognizing a person from different angles or a spoken word across talkers. This ability requires neural representations that are sensitive to some aspects of a stimulus (such as the spectral envelope of a sound) while tolerant to other variations in stimuli (such periodicity). In hearing, such representations have been observed in auditory cortex but never in combination with behavioural testing, which is essential in order to link neural codes to perceptual constancy. By testing ferrets in a vowel discrimination task which they perform across multiple stimulus dimensions and recording neuronal activity in auditory cortex we directly correlate neural tolerance with perceptual constancy. Subjects reported vowel identity across variations in fundamental frequency, sound location, and sound level, but failed to consistently generalize across voicing from voiced to whispered sounds. We decoded the responses of simultaneously recorded units in auditory cortex to identity units informative about vowel identity across each of these task-orthogonal variations in acoustic input. Significant proportions of units were vowel informative across each of these conditions, although fewer units were informative about vowel identity across voicing. For about half of vowel informative units, information about vowel identity was conserved across multiple orthogonal variables. The time of best decoding was also used to identify the relative timing and temporal multiplexing of sound features. Our results show that neural tolerance can be observed within single units in auditory cortex in animals demonstrating perceptual constancy.

Multisensory integration is observed in many subcortical and cortical locations including primary and non-primary sensory cortex, and higher cortical areas including frontal and parietal cortex. During unisensory perceptual tasks many of these same brain areas show neural signatures associated with decision-making. It is unclear whether multisensory representations in sensory cortex directly inform decision-making in a multisensory task, or if cross-modal signals are only combined after the accumulation of unisensory evidence at a final decision-making stage in higher cortical areas. Manipulations of neuronal activity are required to establish causal roles for given brain regions in multisensory perceptual decision-making, and so far indicate that distributed networks underlie multisensory decision-making. Understanding multisensory integration requires synthesis of small-scale pathway specific and large-scale network level manipulations.

Timbre distinguishes sounds of equal loudness, pitch, and duration; however, little is known about the neural mechanisms underlying timbre perception. Such understanding requires animal models such as the ferret in which neuronal and behavioral observation can be combined. The current study asked what spectral cues ferrets use to discriminate between synthetic vowels. Ferrets were trained to discriminate vowels differing in the position of the first (F1) and second formants (F2), inter-formant distance, and spectral centroid. In experiment 1, ferrets responded to probe trials containing novel vowels in which the spectral cues of trained vowels were mismatched. Regression models fitted to behavioral responses determined that F2 and spectral centroid were stronger predictors of ferrets' behavior than either F1 or inter-formant distance. Experiment 2 examined responses to single formant vowels and found that individual spectral peaks failed to account for multi-formant vowel perception. Experiment 3 measured responses to unvoiced vowels and showed that ferrets could generalize vowel identity across voicing conditions. Experiment 4 employed the same design as experiment 1 but with human participants. Their responses were also predicted by F2 and spectral centroid. Together these findings further support the ferret as a model for studying the neural processes underlying timbre perception.