Human mutations in the dystroglycan complex (DGC) result in not only muscular dystrophy but also cognitive impairments. However, the molecular architecture critical for the synaptic organization of the DGC in neurons remains elusive. Here, we report Inhibitory Synaptic protein 1 (InSyn1) is a critical component of the DGC whose loss alters the composition of the GABAergic synapses, excitatory/inhibitory balance in vitro and in vivo, and cognitive behavior. Association of InSyn1 with DGC subunits is required for InSyn1 synaptic localization. InSyn1 null neurons also show a significant reduction in DGC and GABA receptor distribution as well as abnormal neuronal network activity. Moreover, InSyn1 null mice exhibit elevated neuronal firing patterns in the hippocampus and deficits in fear conditioning memory. Our results support the dysregulation of the DGC at inhibitory synapses and altered neuronal network activity and specific cognitive tasks via loss of a novel component, InSyn1.

Virtuosic motor performance requires the ability to evaluate and modify individual gestures within a complex motor sequence. Where and how the evaluative and premotor circuits operate within the brain to enable such temporally precise learning are poorly understood. Songbirds can learn to modify individual syllables within their complex vocal sequences, providing a system for elucidating the underlying evaluative and premotor circuits. We combined behavioral and optogenetic methods to identify two afferents to the ventral tegmental area (VTA) that serve evaluative roles in syllable-specific learning and to establish that downstream cortico-basal ganglia circuits serve a learning role that is only premotor. Further, song performance-contingent
optogenetic stimulation of either VTA afferent was sufficient to drive syllable-specific learning, and these learning effects were of opposite valence. Finally, functional, anatomical, and molecular
studies support the idea that these evaluative afferents bi-directionally modulate VTA dopamine
neurons to enable temporally precise vocal learning.

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study
of all cells, including the diversity of cell types in the brain. However, current approaches are often low-throughput and resource-intensive. Here we describe a CRISPR/Cas9-based Homology independent Universal Genome Engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs AAV vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications, that can integrate into any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural circuit-specific protein modification, subcellular rerouting and sequestration, as well as truncation-based structure-function analysis. Thus, the “plug and play” nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.

The complex skills underlying verbal and musical expression can be learned without external
punishment or reward, indicating their learning is internally guided. The neural mechanisms that
mediate internally guided learning are poorly understood, but a circuit comprising dopamine
releasing neurons in the midbrain ventral tegmental area (VTA) and their targets in the basal
ganglia (BG) are important to externally reinforced learning. Juvenile zebra finches copy a tutor
song in a process that is internally guided and, in adulthood, can learn to modify the fundamental
frequency (pitch) of a target syllable in response to external reinforcement with white noise. Here
we combined intersectional genetic ablation of VTA neurons, reversible blockade of dopamine receptors in the BG, and singing-triggered optogenetic stimulation of VTA terminals to establish
that a common VTA – BG circuit enables internally-guided song copying and externally reinforced syllable pitch learning.

Learning to vocalize depends on the ability to adaptively modify the temporal and spectral features of vocal elements. Neurons that convey motor-related signals to the auditory system are theorized
to facilitate vocal learning, but the identity and function of such neurons remain unknown. Here
we identify a previously unknown neuron type in the songbird brain that transmits vocal motor
signals to the auditory cortex. Genetically ablating these neurons in juveniles disrupted their
ability to imitate features of an adult tutor’s song. Ablating these neurons in adults had little effect
on previously learned songs, but interfered with their ability to adaptively modify the duration of
vocal elements and largely prevented the degradation of song’s temporal features normally caused by deafening. These findings identify a motor to auditory circuit essential to vocal imitation and to the adaptive modification of vocal timing.