The role of cerebellum in learned vocal communication in adult songbirds.

Injury, tumors, ischemia, and lesions in the cerebellum show the involvement of this region in human speech. The association of the cerebellum with learned birdsong has only been identified recently. Cerebellar dysfunction in young songbirds causes learning disabilities, but its role in adult songbirds has not been established. The aim of this study was to investigate the role of the deep cerebellar nuclei (DCN) in adult birdsong. We created bilateral excitotoxic lesions in the DCN of adult male zebra finches (Taeniopygia guttata) and recorded their songs for up to 4 months. Using magnetic resonance imaging (MRI) and immunohistochemistry, we validated the lesion efficacy. We found that the song duration significantly increased from 14 weeks post-op; the increase in duration was caused by a greater number of introductory notes as well as a greater number of syllables sung after the introductory notes. On the other hand, the motif duration decreased from 8 weeks after DCN lesions were induced, which was due to faster singing of syllables, not changes in inter-syllable interval length. DCN lesions also caused a decrease in the fundamental frequency of syllables. In summary, we showed that DCN lesions influence the temporal and acoustic features of birdsong. These results suggest that the cerebellum influences singing in adult songbirds.

Mulberry Posterior Inferior Nasal Turbinate Is Associated with a Lower Pharyngeal pH Environment.

OBJECTIVES: Mulberry-like changes of the posterior inferior nasal turbinate (MPINT) can lead to nasal obstruction. Extraesophageal reflux (EER) characterized by lower pH causes mucosal inflammation and therefore can contribute to sinonasal pathologies. No prior studies have objectively examined the possible association between acidic pH and MPINT formation. Therefore, this study is aimed to investigate the 24-h pharyngeal pH value in patients with MPINT. STUDY DESIGN: Prospective case-control multi-center study. METHODS: Fifty-five patients with chronic EER symptoms were included in the study. They filled in questionnaires aimed at reflux and sinonasal symptoms (RSI®, SNOT-22) and underwent video endoscopy evaluating the laryngeal findings (RFS®) and the presence or absence of the MPINT. And, 24-h oropharyngeal pH monitoring was used to detect the acidic pH environment in the pharynx. RESULTS: Out of the 55 analyzed patients, 38 had the MPINT (group 1), and in 17 patients, the MPINT was absent (group 2). Based on the pathological RYAN Score, in 29 (52.7%) patients, severe acidic pH drops were detected. In group 1, the acidic pH drops were diagnosed significantly more often (68.4%) compared with those in group 2 (p = 0.001). Moreover, in group 1, a significantly higher median total percentage of time spent below pH 5.5 (p = 0.005), as well as a higher median number of events lasting more than 5 min (p = 0.006), and higher median total number of events with pH drops (p = 0.017) were observed. CONCLUSION: In this study, the MPINT was significantly more often present in patients with acidic pH events detected by 24-h oropharyngeal pH monitoring. Acidic pH in the pharynx might lead to MPINT formation. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:62-68, 2024.

Association Between Inferior Turbinate Hypertrophy and Extraesophageal Reflux.

Importance: To the authors' knowledge, no prior studies have examined the association between inferior turbinate hypertrophy (ITH) and extraesophageal reflux (EER). If EER were a cause or cofactor of ITH, antireflux treatment can be considered prior to surgical intervention. Objective: To evaluate EER presence and severity in patients with different degrees of ITH. Design, Setting, and Participants: Prospective multicentric cohort study conducted at 3 referral centers treating patients with EER and certified for 24-hour monitoring of oropharyngeal pH. The monitoring was performed between October 2020 and October 2021. A total of 94 adult patients with EER symptoms were recruited, 90 of whom were analyzed. Interventions: Nasal endoscopy was performed to determine the degree of ITH, according to the Camacho classification. Presence and severity of EER were examined using 24-hour monitoring of oropharyngeal pH. Main Outcomes and Measures: Primary outcomes were presence of EER according to RYAN Score, total percentage of time below pH 5.5, and total numbers of EER events below pH 5.5. Results: Of the 90 analyzed patients (median [IQR] age, 46 [33-58] years; 36 [40%] male patients), 41 had a maximum of second-degree ITH (group 1), and 49 patients had at least third-degree ITH (group 2), according to the Camacho classification. On the basis of the RYAN Score, EER was diagnosed more often in group 2 (69.4%) than in group 1 (34.1%; difference, 35.3% [95% CI, 13.5%-56.9%]). Moreover, compared with group 1, group 2 exhibited higher median total percentage of time below pH 5.5 (median [IQR], group 1: 2.1% [0.0%-9.4%], group 2: 11.2% [1.5%-15.8%]; difference, 9.1% [95% CI, 4.1%-11.8%]) and higher median total number of EER events (median [IQR], group 1: 6 [1-14] events, group 2: 14 [4-26] events; difference, 8 [95% CI, 2-15] events). Patients with proven EER demonstrated no difference in the degree of ITH between the right and left nasal cavity (Cohen g, -0.17 [95% CI, -0.50 to 0.30]), or between the anterior and posterior parts of the nasal cavity (Cohen g, -0.21 [95% CI, -0.50 to 0.17]). Conclusions and Relevance: In this cohort study, patients with a higher degree of ITH had more severe EER. A possible association between severe ITH and EER was demonstrated.

Striatal Injury Induces Overall Brain Alteration at the Pallial, Thalamic, and Cerebellar Levels.

The striatal region Area X plays an important role during song learning, sequencing, and variability in songbirds. A previous study revealed that neurotoxic damage within Area X results in micro and macrostructural changes across the entire brain, including the downstream dorsal thalamus and both the upstream pallial nucleus HVC (proper name) and the deep cerebellar nuclei (DCN). Here, we specify these changes on cellular and gene expression levels. We found decreased cell density in the thalamic and cerebellar areas and HVC, but it was not related to neuronal loss. On the contrary, perineuronal nets (PNNs) in HVC increased for up to 2 months post-lesion, suggesting their protecting role. The synaptic plasticity marker Forkhead box protein P2 (FoxP2) showed a bi-phasic increase at 8 days and 3 months post-lesion, indicating a massive synaptic rebuilding. The later increase in HVC was associated with the increased number of new neurons. These data suggest that the damage in the striatal vocal nucleus induces cellular and gene expression alterations in both the efferent and afferent destinations. These changes may be long-lasting and involve plasticity and neural protection mechanisms in the areas directly connected to the injury site and also to distant areas, such as the cerebellum.

Effectivity of Two Cell Proliferation Markers in Brain of a Songbird Zebra Finch.

There are two most heavily used markers of cell proliferation, thymidine analogues 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) that are incorporated into the DNA during its synthesis. In neurosciences, they are often used consecutively in the same animal to detect neuronal populations arising at multiple time points, their migration and incorporation. The effectivity of these markers, however, is not well established. Here, we studied the effectivity of equimolar doses of BrdU and EdU to label new cells and looked for the dose that will label the highest number of proliferating cells in the neurogenic ventricular zone (VZ) of adult songbirds. We found that, in male zebra finches (Taeniopygia guttata), the equimolar doses of BrdU and EdU did not label the same number of cells, with BrdU being more effective than EdU. Similarly, in liver, BrdU was more effective. The saturation of the detected brain cells occurred at 50 mg/kg BrdU and above 41 mg/kg EdU. Higher dose of 225 mg/kg BrdU or the equimolar dose of EdU did not result in any further significant increases. These results show that both markers are reliable for the detection of proliferating cells in birds, but the numbers obtained with BrdU and EdU should not be compared.

In vivo assessment of the neural substrate linked with vocal imitation accuracy.

Human speech and bird song are acoustically complex communication signals that are learned by imitation during a sensitive period early in life. Although the brain areas indispensable for speech and song learning are known, the neural circuits important for enhanced or reduced vocal performance remain unclear. By combining in vivo structural Magnetic Resonance Imaging with song analyses in juvenile male zebra finches during song learning and beyond, we reveal that song imitation accuracy correlates with the structural architecture of four distinct brain areas, none of which pertain to the song control system. Furthermore, the structural properties of a secondary auditory area in the left hemisphere, are capable to predict future song copying accuracy, already at the earliest stages of learning, before initiating vocal practicing. These findings appoint novel brain regions important for song learning outcome and inform that ultimate performance in part depends on factors experienced before vocal practicing.

Is neurogenesis in two songbird species related to their song sequence variability?

Neurogenesis takes part in the adult songbird brain and new neurons are integrated into the forebrain including defined areas involved in the control of song learning and production. It has been suggested that the new neurons in the song system might enable vocal variability. Here, we examined the basal levels of neurogenesis in two songbird species, zebra finch ( Taeniopygia guttata) and Bengalese finch ( Lonchura striata var. domestica), which do not learn new song elements as adults but differ in the level of song sequence variability. We found that Bengalese finches had less linear and stereotyped song sequence and a higher number of newborn cells in the neurogenic subventricular zone (SVZ) as well as the number of newly born neurons incorporated into the vocal nucleus HVC (used as a proper name) in comparison to zebra finches. Importantly, this vocal sequence variability in Bengalese finches correlated with the number of new neurons in the vocal nucleus HVC and more plastic song was associated with higher neuronal incorporation. In summary, our data support the hypothesis that newly generated neurons facilitate behavioural variability.

Volumetric development of the zebra finch brain throughout the first 200 days of post-hatch life traced by in vivo MRI.

The first months of life are characterized by massive neuroplastic processes that parallel the acquisition of skills and abilities vital for proper functioning in later life. Likewise, juvenile songbirds learn the song sung by their tutor during the first months after hatching. To date, most studies targeting brain development in songbirds exclusively focus on the song control and auditory pathways. To gain a comprehensive insight into structural developmental plasticity of the entire zebra finch brain throughout the different subphases of song learning, we designed a longitudinal study in a group of male (16) and female (19) zebra finches. We collected T2-weighted 3-dimensional anatomical scans at six developmental milestones throughout the process of song learning, i.e. 20, 30, 40, 65, 90 and 120 days post hatching (dph), and one additional time point well after song crystallization, i.e. 200 dph. We observed that the total brain volume initially increases, peaks around 30-40 dph and decreases towards the end of the study. Further, we performed brain-wide voxel-based volumetric analyses to create spatio-temporal maps indicating when specific brain areas increase or decrease in volume, relative to the subphases of song learning. These maps informed (1) that most areas implicated in song control change early, i.e. between 20 and 65 dph, and are embedded in large clusters that cover major subdivisions of the zebra finch brain, (2) that volume changes between consecutive subphases of vocal learning appear highly similar in males and females, and (3) that only more rostrally situated brain regions change in volume towards later ages. Lastly, besides detecting sex differences in local tissue volume that align with previous studies, we uncovered two additional brain loci that are larger in male compared to female zebra finches. These volume differences co-localize with areas related to the song control and auditory pathways and can therefore be associated to the behavioral difference as only male zebra finches sing. In sum, our data point to clear heterochronous patterns of brain development similar to brain development in mammalian species and this work can serve as a reference for future neurodevelopmental imaging studies in zebra finches.

Neuroplasticity in the cerebello-thalamo-basal ganglia pathway: A longitudinal in vivo MRI study in male songbirds.

Similar to human speech, bird song is controlled by several pathways including a cortico-basal ganglia-thalamo-cortical (C-BG-T-C) loop. Neurotoxic disengagement of the basal ganglia component, i.e. Area X, induces long-term changes in song performance, while most of the lesioned area regenerates within the first months. Importantly however, the timing and spatial extent of structural neuroplastic events potentially affecting other constituents of the C-BG-T-C loop is not clear. We designed a longitudinal MRI study where changes in brain structure were evaluated relative to the time after neurotoxic lesioning or to vocal performance. By acquiring both Diffusion Tensor Imaging and 3-dimensional anatomical scans, we were able to track alterations in respectively intrinsic tissue properties and local volume. Voxel-based statistical analyses revealed structural remodeling remote to the lesion, i.e. in the thalamus and, surprisingly, the cerebellum, both peaking within the first two months after lesioning Area X. Voxel-wise correlations between song performance and MRI parameters uncovered intriguing brain-behavior relationships in several brain areas pertaining to the C-BG-T-C loop supervising vocal motor control. Our results clearly point to structural neuroplasticity in the cerebellum induced by basal ganglia (striatal) damage and might point to the existence of a human-like cerebello-thalamic-basal ganglia pathway capable of modifying vocal motor output.

Imaging of striatal injury in a songbird brain.

Neurological insults affect both, brain structure and behavior. The injury-induced brain plasticity and associated changes in behavior are difficult to study using classical histological methods. The magnetic resonance imaging (MRI), however, enables repeated inspection of the brain in the same individual. Here we took advantage of the songbird model with discrete brain circuitry controlling song learning and production and assessed if a conventional MRI is suitable to detect even relatively small brain changes. Our aim was to monitor injury and the following regeneration in the striatal vocal nucleus Area X that controls vocal learning in juveniles and affects song in adult songbird zebra finch (Taeniopygia guttata). The regeneration process was detected using T2-weighted images and validated by immunohistochemical (IHC) staining up to 6 months after the injury. Despite the small volume of the zebra finch brain, a satisfactory signal-to-noise ratio was achieved with reasonably short measurement times. No significant difference was found between the measurements of the lesion size obtained by MRI and IHC staining. Our data show that the non-invasive MRI technique can reliably measure and quantify the regeneration process even in a relatively small part of the brain and that the avian striatum progressively regenerates after its neurotoxic injury.

Dopamine D3 receptors modulate the rate of neuronal recovery, cell recruitment in Area X, and song tempo after neurotoxic damage in songbirds.

Songbirds, like humans, learn vocalizations and their striatum recruits new neurons in adulthood. Injury in striatal vocal nucleus Area X, involved in song learning and production in songbirds, is followed by massive regeneration. The newborn neurons arise from the subventricular zone (SVZ) rich in dopamine D3 receptors (D3Rs). The aim of this study was to investigate whether the D3Rs affect the rate of neuronal recovery in Area X. Male zebra finches (Taeniopygia guttata) received bilateral neurotoxic lesion of Area X and were implanted with osmotic minipumps containing D3R agonist 7-OH-DPAT, antagonist U99194, or saline. Treatment with 7-OH-DPAT but not U99194 led to significant reduction of lesion size and increased numbers of migrating neuroblasts and newborn cells in the Area X. These cells were detected in the lesion border as well as the lesion center. Lesion also led to increased mRNA expression of the D3Rs in the neurogenic SVZ and in the nucleus robustus arcopallialis (RA) involved in song production. Moreover, lesion alone prolonged the song duration and this may be facilitated by D3Rs in RA. Parallel lesion and stimulation of D3Rs prolonged it even more, while blocking of D3Rs abolished the lesion-induced effect. These data suggest that D3R stimulation after striatal injury accelerates the striatal recovery and can cause behavioral alterations.

Basal ganglia function, stuttering, sequencing, and repair in adult songbirds.

A pallial-basal-ganglia-thalamic-pallial loop in songbirds is involved in vocal motor learning. Damage to its basal ganglia part, Area X, in adult zebra finches has been noted to have no strong effects on song and its function is unclear. Here we report that neurotoxic damage to adult Area X induced changes in singing tempo and global syllable sequencing in all animals, and considerably increased syllable repetition in birds whose song motifs ended with minor repetitions before lesioning. This stuttering-like behavior started at one month, and improved over six months. Unexpectedly, the lesioned region showed considerable recovery, including immigration of newly generated or repaired neurons that became active during singing. The timing of the recovery and stuttering suggest that immature recovering activity of the circuit might be associated with stuttering. These findings indicate that even after juvenile learning is complete, the adult striatum plays a role in higher level organization of learned vocalizations.