Human GNPTAB stuttering mutations engineered into mice cause vocalization deficits and astrocyte pathology in the corpus callosum.

Stuttering is a common neurodevelopmental disorder that has been associated with mutations in genes involved in intracellular trafficking. However, the cellular mechanisms leading to stuttering remain unknown. Engineering a mutation in N-acetylglucosamine-1-phosphate transferase subunits α and ß (GNPTAB) found in humans who stutter into the mouse Gnptab gene resulted in deficits in the flow of ultrasonic vocalizations similar to speech deficits of humans who stutter. Here we show that other human stuttering mutations introduced into this mouse gene, Gnptab Ser321Gly and Ala455Ser, produce the same vocalization deficit in 8-day-old pup isolation calls and do not affect other nonvocal behaviors. Immunohistochemistry showed a marked decrease in staining of astrocytes, particularly in the corpus callosum of the Gnptab Ser321Gly homozygote mice compared to wild-type littermates, while the staining of cerebellar Purkinje cells, oligodendrocytes, microglial cells, and dopaminergic neurons was not significantly different. Diffusion tensor imaging also detected deficits in the corpus callosum of the Gnptab Ser321Gly mice. Using a range of cell type-specific Cre-drivers and a Gnptab conditional knockout line, we found that only astrocyte-specific Gnptab-deficient mice displayed a similar vocalization deficit. These data suggest that vocalization defects in mice carrying human stuttering mutations in Gnptab derive from abnormalities in astrocytes, particularly in the corpus callosum, and provide support for hypotheses that focus on deficits in interhemispheric communication in stuttering.

Advance cueing produces enhanced action-boundary patterns of spike activity in the sensorimotor striatum.

One of the most characteristic features of habitual behaviors is that they can be evoked by a single cue. In the experiments reported here, we tested for the effects of such advance cueing on the firing patterns of striatal neurons in the sensorimotor striatum. Rats ran in a T-maze with instruction cues about the location of reward given at the start of the runs. This advance cueing about reward produced a highly augmented task-bracketing pattern of activity at the beginning and end of procedural task performance relative to the patterns found previously with midtask cueing. Remarkably, the largest increase in activity early during the T-maze runs was not associated with the instruction cues themselves, the earliest predictors of reward; instead, the highest peak of early activity was associated with the beginning of the motor period of the task. We suggest that the advance cueing, reducing midrun demands for decision making but adding a working-memory load, facilitated chunking of the maze runs as executable scripts anchored to sensorimotor aspects of the task and unencumbered by midtask decision-making demands. Our findings suggest that the acquisition of stronger task-bracketing patterns of striatal activity in the sensorimotor striatum could reflect this enhancement of behavioral chunking. Deficits in such representations of learned sequential behaviors could contribute to motor and cognitive problems in a range of neurological disorders affecting the basal ganglia, including Parkinson's disease.

Activity of striatal neurons reflects dynamic encoding and recoding of procedural memories.

Learning to perform a behavioural procedure as a well-ingrained habit requires extensive repetition of the behavioural sequence, and learning not to perform such behaviours is notoriously difficult. Yet regaining a habit can occur quickly, with even one or a few exposures to cues previously triggering the behaviour. To identify neural mechanisms that might underlie such learning dynamics, we made long-term recordings from multiple neurons in the sensorimotor striatum, a basal ganglia structure implicated in habit formation, in rats successively trained on a reward-based procedural task, given extinction training and then given reacquisition training. The spike activity of striatal output neurons, nodal points in cortico-basal ganglia circuits, changed markedly across multiple dimensions during each of these phases of learning. First, new patterns of task-related ensemble firing successively formed, reversed and then re-emerged. Second, task-irrelevant firing was suppressed, then rebounded, and then was suppressed again. These changing spike activity patterns were highly correlated with changes in behavioural performance. We propose that these changes in task representation in cortico-basal ganglia circuits represent neural equivalents of the explore-exploit behaviour characteristic of habit learning.

Group and Individual Variability in Mouse Pup Isolation Calls Recorded on the Same Day Show Stability.

Mice produce ultrasonic vocalizations (USVs) in a variety of social situations, and USVs have been leveraged to study many neurological diseases including verbal dyspraxia, depression, autism and stuttering. Pups produce isolation calls, a common USV, spontaneously when they are isolated from their mother during the first 2 weeks of life. Several genetic manipulations affect (and often reduce) pup isolation calls in mice. To facilitate the use of this assay as a means of testing whether significant functional differences in genotypes exist instead of contextual differences, we test the variability inherent in many commons measures of mouse vocalizations. Here we use biological consistency as a way of determining which are reproducible in mouse pup vocalizations. We present a comprehensive analysis of the normal variability of these vocalizations in groups of mice, individual mice and different strains of mice. To control for maturation effects, we recorded pup isolation calls in the same group of C57BL/6J 5 days old mice twice, with 1 h of rest in between recordings. In almost all cases, the group averages between the first and second recordings were the same. We also found that there were high correlations in some parameters in individual mice across recording while others were not well correlated. These findings could be replicated for the majority of features in a separate group of C57BL/6J mice and a group of 129/SvEvBrd-C57BL/6J mice. The averages of these mouse USV features are highly consistent and represent a robust assay to test the effects of genetic and other interventions in the experimental setting.

Knockout of Lysosomal Enzyme-Targeting Gene Causes Abnormalities in Mouse Pup Isolation Calls.

Humans lacking a working copy of the GNPTAB gene suffer from the metabolic disease Mucolipidosis type II (MLII). MLII symptoms include mental retardation, skeletal deformities and cartilage defects as well as a speech delay with most subjects unable to utter single words (Otomo et al., 2009; Cathey et al., 2010; Leroy et al., 2012). Here we asked whether mice lacking a copy of Gnptab gene exhibited vocal abnormities. We recorded ultrasonic vocalizations from 5 to 8 day old mice separated from their mother and littermates. Although Gnptab-/- pups emitted a similar number of calls, several features of the calls were different from their wild type littermates. Gnptab-/- mice showed a decrease in the length of calls, an increase in the intra-bout pause duration, significantly fewer pitch jumps with smaller mean size, and an increase in the number of isolated calls. In addition, Gnptab-/- mice vocalizations had less power, particularly in the higher frequencies. Gnptab+/- mouse vocalizations did not appear to be affected. We then attempted to classify these recordings using these features to determine the genotype of the animal. We were able to correctly identify 87% of the recordings as either Gnptab-/- or Gnptab+/+ pup, significantly better than chance, demonstrating that genotype is a strong predictor of vocalization phenotype. These data show that deletion of genes in the lysosomal enzyme targeting pathway affect mouse pup isolation calls.

A Mutation Associated with Stuttering Alters Mouse Pup Ultrasonic Vocalizations.

A promising approach to understanding the mechanistic basis of speech is to study disorders that affect speech without compromising other cognitive or motor functions. Stuttering, also known as stammering, has been linked to mutations in the lysosomal enzyme-targeting pathway, but how this remarkably specific speech deficit arises from mutations in a family of general "cellular housekeeping" genes is unknown. To address this question, we asked whether a missense mutation associated with human stuttering causes vocal or other abnormalities in mice. We compared vocalizations from mice engineered to carry a mutation in the Gnptab (N-acetylglucosamine-1-phosphotransferase subunits alpha/beta) gene with wild-type littermates. We found significant differences in the vocalizations of pups with the human Gnptab stuttering mutation compared to littermate controls. Specifically, we found that mice with the mutation emitted fewer vocalizations per unit time and had longer pauses between vocalizations and that the entropy of the temporal sequence was significantly reduced. Furthermore, Gnptab missense mice were similar to wild-type mice on an extensive battery of non-vocal behaviors. We then used the same language-agnostic metrics for auditory signal analysis of human speech. We analyzed speech from people who stutter with mutations in this pathway and compared it to control speech and found abnormalities similar to those found in the mouse vocalizations. These data show that mutations in the lysosomal enzyme-targeting pathway produce highly specific effects in mouse pup vocalizations and establish the mouse as an attractive model for studying this disorder.