A histological and diceCT-derived 3D reconstruction of the avian visual thalamofugal pathway.

Amniotes feature two principal visual processing systems: the tectofugal and thalamofugal pathways. In most mammals, the thalamofugal pathway predominates, routing retinal afferents through the dorsolateral geniculate complex to the visual cortex. In most birds, the thalamofugal pathway often plays the lesser role with retinal afferents projecting to the principal optic thalami, a complex of several nuclei that resides in the dorsal thalamus. This thalamic complex sends projections to a forebrain structure called the Wulst, the terminus of the thalamofugal visual system. The thalamofugal pathway in birds serves many functions such as pattern discrimination, spatial memory, and navigation/migration. A comprehensive analysis of avian species has unveiled diverse subdivisions within the thalamic and forebrain structures, contingent on species, age, and techniques utilized. In this study, we documented the thalamofugal system in three dimensions by integrating histological and contrast-enhanced computed tomography imaging of the avian brain. Sections of two-week-old chick brains were cut in either coronal, sagittal, or horizontal planes and stained with Nissl and either Gallyas silver or Luxol Fast Blue. The thalamic principal optic complex and pallial Wulst were subdivided on the basis of cell and fiber density. Additionally, we utilized the technique of diffusible iodine-based contrast-enhanced computed tomography (diceCT) on a 5-week-old chick brain, and right eyeball. By merging diceCT data, stained histological sections, and information from the existing literature, a comprehensive three-dimensional model of the avian thalamofugal pathway was constructed. The use of a 3D model provides a clearer understanding of the structural and spatial organization of the thalamofugal system. The ability to integrate histochemical sections with diceCT 3D modeling is critical to better understanding the anatomical and physiologic organization of complex pathways such as the thalamofugal visual system.

Functional neurogenomic responses to acoustic threats, including a heterospecific referential alarm call and its referent, in the auditory forebrain of red-winged blackbirds.

In animal communication, functionally referential alarm calls elicit the same behavioral responses as their referents, despite their typically distinct bioacoustic traits. Yet the auditory forebrain in at least one songbird species, the black-capped chickadee Poecile atricapillus, responds similarly to threat calls and their referent predatory owl calls, as assessed by immediate early gene responses in the secondary auditory forebrain nuclei. Whether and where in the brain such perceptual and cognitive equivalence is processed remains to be understood in most other avian systems. Here, we studied the functional neurogenomic (non-) equivalence of acoustic threat stimuli perception by the red-winged blackbird Agelaius phoeniceus in response to the actual calls of the obligate brood parasitic brown-headed cowbird Molothrus ater and the referential anti-parasitic alarm calls of the yellow warbler Setophaga petechia, upon which the blackbird is known to eavesdrop. Using RNA-sequencing from neural tissue in the auditory lobule (primary and secondary auditory nuclei combined), in contrast to previous findings, we found significant differences in the gene expression profiles of both an immediate early gene, ZENK (egr-1), and other song-system relevant gene-products in blackbirds responding to cowbird vs. warbler calls. In turn, direct cues of threats (including conspecific intruder calls and nest-predator calls) elicited higher ZENK and other differential gene expression patterns compared to harmless heterospecific calls. These patterns are consistent with a perceptual non-equivalence in the auditory forebrain of adult male red-winged blackbirds in response to referential calls and the calls of their referents.

Src-NADH dehydrogenase subunit 2 complex and recognition memory of imprinting in domestic chicks.

Src is a non-receptor tyrosine kinase participating in a range of neuronal processes, including synaptic plasticity. We have recently shown that the amounts of total Src and its two phosphorylated forms, at tyrosine-416 (activated) and tyrosine-527 (inhibited), undergoes time-dependent, region-specific learning-related changes in the domestic chick forebrain after visual imprinting. These changes occur in the intermediate medial mesopallium (IMM), a site of memory formation for visual imprinting, but not the posterior pole of the nidopallium (PPN), a control brain region not involved in imprinting. Src interacts with mitochondrial genome-coded NADH dehydrogenase subunit 2 (NADH2), a component of mitochondrial respiratory complex I. This interaction occurs at brain excitatory synapses bearing NMDA glutamate receptors. The involvement of Src-NADH2 complexes in learning and memory is not yet explored. We show for the first time that, independently of changes in total Src or total NADH2, NADH2 bound to Src immunoprecipitated from the P2 plasma membrane-mitochondrial fraction: (i) is increased in a learning-related manner in the left IMM 1 h after the end of training; (ii), is decreased in the right IMM in a learning-related way 24 h after training. These changes occurred in the IMM but not the PPN. They are attributable to learning occurring during training rather than a predisposition to learn. Learning-related changes in Src-bound NADH2 are thus time- and region-dependent.

A Purkinje cell to parabrachial nucleus pathway enables broad cerebellar influence over the forebrain.

In addition to its motor functions, the cerebellum is involved in emotional regulation, anxiety and affect. We found that suppressing the firing of cerebellar Purkinje cells (PCs) rapidly excites forebrain areas that contribute to such functions (including the amygdala, basal forebrain and septum), but that the classic cerebellar outputs, the deep cerebellar nuclei, do not directly project there. We show that PCs directly inhibit parabrachial nuclei (PBN) neurons that project to numerous forebrain regions. Suppressing the PC-PBN pathway influences many regions in the forebrain and is aversive. Molecular profiling shows that PCs directly inhibit numerous types of PBN neurons that control diverse behaviors that are not involved in motor control. Therefore, the PC-PBN pathway allows the cerebellum to directly regulate activity in the forebrain, and may be an important substrate for cerebellar disorders arising from damage to the posterior vermis.

Neural circuit-wide analysis of changes to gene expression during deafening-induced birdsong destabilization.

Sensory feedback is required for the stable execution of learned motor skills, and its loss can severely disrupt motor performance. The neural mechanisms that mediate sensorimotor stability have been extensively studied at systems and physiological levels, yet relatively little is known about how disruptions to sensory input alter the molecular properties of associated motor systems. Songbird courtship song, a model for skilled behavior, is a learned and highly structured vocalization that is destabilized following deafening. Here, we sought to determine how the loss of auditory feedback modifies gene expression and its coordination across the birdsong sensorimotor circuit. To facilitate this system-wide analysis of transcriptional responses, we developed a gene expression profiling approach that enables the construction of hundreds of spatially-defined RNA-sequencing libraries. Using this method, we found that deafening preferentially alters gene expression across birdsong neural circuitry relative to surrounding areas, particularly in premotor and striatal regions. Genes with altered expression are associated with synaptic transmission, neuronal spines, and neuromodulation and show a bias toward expression in glutamatergic neurons and Pvalb/Sst-class GABAergic interneurons. We also found that connected song regions exhibit correlations in gene expression that were reduced in deafened birds relative to hearing birds, suggesting that song destabilization alters the inter-region coordination of transcriptional states. Finally, lesioning LMAN, a forebrain afferent of RA required for deafening-induced song plasticity, had the largest effect on groups of genes that were also most affected by deafening. Combined, this integrated transcriptomics analysis demonstrates that the loss of peripheral sensory input drives a distributed gene expression response throughout associated sensorimotor neural circuitry and identifies specific candidate molecular and cellular mechanisms that support the stability and plasticity of learned motor skills.

Structure-function subsystem model and computational lesions of the central nervous system's rostral sector (forebrain and midbrain).

The craniote central nervous system has been divided into rostral, intermediate, and caudal sectors, with the rostral sector containing the vertebrate forebrain and midbrain. Here, network science tools were used to create and analyze a rat hierarchical structure-function subsystem model of intrarostral sector neural connectivity between gray matter regions. The hierarchy has 109 bottom-level subsystems and three upper-level subsystems corresponding to voluntary behavior control, cognition, and affect; instinctive survival behaviors and homeostasis; and oculomotor control. As in previous work, subsystems identified based on their coclassification as network communities are revealed as functionally related. We carried out focal perturbations of neural structural connectivity comprehensively by computationally lesioning each region of the network, and the resulting effects on the network's modular (subsystem) organization were systematically mapped and measured. The pattern of changes was found to be correlated with three structural attributes of the lesioned region: region centrality (degree, strength, and betweenness), region position in the hierarchy, and subsystem distribution of region neural outputs and inputs. As expected, greater region centrality results, on average, in stronger lesion impact and more distributed lesion effects. In addition, our analysis suggests that strongly functionally related regions, belonging to the same bottom-level subsystem, exhibit similar effects after lesioning. These similarities account for coherent patterns of disturbances that align with subsystem boundaries and propagate through the network. These systematic lesion effects and their similarity across functionally related regions are of potential interest for theoretical, experimental, and clinical studies.

Forebrain Network Associated With Cardiovascular Control in Exercising Humans.

This article describes the forebrain neurocircuitry associated with rapid heart rate response at the exercise onset with attention to ascending somatosensory information from the Type I and II afferents from the contracting muscle and potential influence of sensory information related to blood pressure and changes in heart rate.

Responses to Song Playback Differ in Sleeping versus Anesthetized Songbirds.

Vocal learning in songbirds is mediated by a highly localized system of interconnected forebrain regions, including recurrent loops that traverse the cortex, basal ganglia, and thalamus. This brain-behavior system provides a powerful model for elucidating mechanisms of vocal learning, with implications for learning speech in human infants, as well as for advancing our understanding of skill learning in general. A long history of experiments in this area has tested neural responses to playback of different song stimuli in anesthetized birds at different stages of vocal development. These studies have demonstrated selectivity for different song types that provide neural signatures of learning. In contrast to the ease of obtaining responses to song playback in anesthetized birds, song-evoked responses in awake birds are greatly reduced or absent, indicating that behavioral state is an important determinant of neural responsivity. Song-evoked responses can be elicited during sleep as well as anesthesia, and the selectivity of responses to song playback in adult birds is highly similar between anesthetized and sleeping states, encouraging the idea that anesthesia and sleep are similar. In contrast to that idea, we report evidence that cortical responses to song playback in juvenile zebra finches (Taeniopygia guttata) differ greatly between sleep and urethane anesthesia. This finding indicates that behavioral states differ in sleep versus anesthesia and raises questions about relationships between developmental changes in sleep activity, selectivity for different song types, and the neural substrate for vocal learning.

Vocal changes in a zebra finch model of Parkinson's disease characterized by alpha-synuclein overexpression in the song-dedicated anterior forebrain pathway.

Deterioration in the quality of a person's voice and speech is an early marker of Parkinson's disease (PD). In humans, the neural circuit that supports vocal motor control consists of a cortico-basal ganglia-thalamo-cortico loop. The basal ganglia regions, striatum and globus pallidus, in this loop play a role in modulating the acoustic features of vocal behavior such as loudness, pitch, and articulatory rate. In PD, this area is implicated in pathogenesis. In animal models of PD, the accumulation of toxic aggregates containing the neuronal protein alpha-synuclein (αsyn) in the midbrain and striatum result in limb and vocal motor impairments. It has been challenging to study vocal impairments given the lack of well-defined cortico-basal ganglia circuitry for vocalization in rodent models. Furthermore, whether deterioration of voice quality early in PD is a direct result of αsyn-induced neuropathology is not yet known. Here, we take advantage of the well-characterized vocal circuits of the adult male zebra finch songbird to experimentally target a song-dedicated pathway, the anterior forebrain pathway, using an adeno-associated virus expressing the human wild-type αsyn gene, SNCA. We found that overexpression of αsyn in this pathway coincides with higher levels of insoluble, monomeric αsyn compared to control finches. Impairments in song production were also detected along with shorter and poorer quality syllables, which are the most basic unit of song. These vocal changes are similar to the vocal abnormalities observed in individuals with PD.

Mapping the vocal circuitry of Alston's singing mouse with pseudorabies virus.

Vocalizations are often elaborate, rhythmically structured behaviors. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alston's singing mouse (Scotinomys teguina) each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening, respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguus for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally colabel neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong colabeling in compartments of the midbrain including the ventrolateral periaqueductal gray and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive colabeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. The progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis.

During mammalian brain development, how different astrocytes are specified from progenitor cells is not well understood. In particular, whether astrocyte progenitor cells (APCs) start as a relatively homogenous population or whether there is early heterogeneity remains unclear. Here, we have dissected subpopulations of embryonic mouse forebrain progenitors using single-cell transcriptome analyses. Our sequencing data revealed two molecularly distinct APC subgroups at the start of gliogenesis from both dorsal and ventral forebrains. The two APC subgroups were marked, respectively, by specific expression of Sparc and Sparcl1, which are known to function in mature astrocytes with opposing activities for regulating synapse formation. Expression analyses showed that SPARC and SPARCL1 mark APC subgroups that display distinct temporal and spatial patterns, correlating with major waves of astrogliogenesis during development. Our results uncover an early molecular divergence of APCs in the mammalian brain and provide a useful transcriptome resource for the study of glial cell specification.

The Direction of response selectivity between conspecific and heterospecific auditory stimuli varies with response metric.

Species recognition is an essential behavioral outcome of social discrimination, flocking, mobbing, mating, and/or parental care. In songbirds, auditory species recognition cues are processed through specialized forebrain circuits dedicated to acoustic discrimination. Here we addressed the direction of behavioral and neural metrics of zebra finches' (Taeniopygia guttata) responses to acoustic cues of unfamiliar conspecifics vs. heterospecifics. Behaviorally, vocal response rates were greater for conspecific male zebra finch songs over heterospecific Pin-tailed Whydah (Vidua macroura) songs, which paralleled greater multiunit spike rates in the auditory forebrain in response to the same type of conspecific over heterospecific auditory stimuli. In contrast, forebrain activation levels were reversed to species-specific song playbacks during two functional magnetic resonance imaging experiments: we detected consistently greater responses to whydah songs over finch songs and did so independently of whether subjects had been co-housed or not with heterospecifics. These results imply that the directionality of behavioral and neural response selectivity metrics are not always consistent and appear to be experience-independent in this set of stimulus-and-subject experimental paradigms.

Prediction errors disrupt hippocampal representations and update episodic memories.

The brain supports adaptive behavior by generating predictions, learning from errors, and updating memories to incorporate new information. Prediction error, or surprise, triggers learning when reality contradicts expectations. Prior studies have shown that the hippocampus signals prediction errors, but the hypothesized link to memory updating has not been demonstrated. In a human functional MRI study, we elicited mnemonic prediction errors by interrupting familiar narrative videos immediately before the expected endings. We found that prediction errors reversed the relationship between univariate hippocampal activation and memory: greater hippocampal activation predicted memory preservation after expected endings, but memory updating after surprising endings. In contrast to previous studies, we show that univariate activation was insufficient for understanding hippocampal prediction error signals. We explain this surprising finding by tracking both the evolution of hippocampal activation patterns and the connectivity between the hippocampus and neuromodulatory regions. We found that hippocampal activation patterns stabilized as each narrative episode unfolded, suggesting sustained episodic representations. Prediction errors disrupted these sustained representations and the degree of disruption predicted memory updating. The relationship between hippocampal activation and subsequent memory depended on concurrent basal forebrain activation, supporting the idea that cholinergic modulation regulates attention and memory. We conclude that prediction errors create conditions that favor memory updating, prompting the hippocampus to abandon ongoing predictions and make memories malleable.

Cholinergic Deep Brain Stimulation for Memory and Cognitive Disorders.

Memory and cognitive impairment as sequelae of neurodegeneration in Alzheimer's disease and age-related dementia are major health issues with increasing social and economic burden. Deep brain stimulation (DBS) has emerged as a potential treatment to slow or halt progression of the disease state. The selection of stimulation target is critical, and structures that have been targeted for memory and cognitive enhancement include the Papez circuit, structures projecting to the frontal lobe such as the ventral internal capsule, and the cholinergic forebrain. Recent human clinical and animal model results imply that DBS of the nucleus basalis of Meynert can induce a therapeutic modulation of neuronal activity. Benefits include enhanced activity across the cortical mantle, and potential for amelioration of neuropathological mechanisms associated with Alzheimer's disease. The choice of stimulation parameters is also critical. High-frequency, continuous stimulation is used for movement disorders as a way of inhibiting their output; however, no overexcitation has been hypothesized in Alzheimer's disease and lower stimulation frequency or intermittent patterns of stimulation (periods of stimulation interleaved with periods of no stimulation) are likely to be more effective for stimulation of the cholinergic forebrain. Efficacy and long-term tolerance in human patients remain open questions, though the cumulative experience gained by DBS for movement disorders provides assurance for the safety of the procedure.

Theta-gamma coupling emerges from spatially heterogeneous cholinergic neuromodulation.

Theta and gamma rhythms and their cross-frequency coupling play critical roles in perception, attention, learning, and memory. Available data suggest that forebrain acetylcholine (ACh) signaling promotes theta-gamma coupling, although the mechanism has not been identified. Recent evidence suggests that cholinergic signaling is both temporally and spatially constrained, in contrast to the traditional notion of slow, spatially homogeneous, and diffuse neuromodulation. Here, we find that spatially constrained cholinergic stimulation can generate theta-modulated gamma rhythms. Using biophysically-based excitatory-inhibitory (E-I) neural network models, we simulate the effects of ACh on neural excitability by varying the conductance of a muscarinic receptor-regulated K+ current. In E-I networks with local excitatory connectivity and global inhibitory connectivity, we demonstrate that theta-gamma-coupled firing patterns emerge in ACh modulated network regions. Stable gamma-modulated firing arises within regions with high ACh signaling, while theta or mixed theta-gamma activity occurs at the peripheries of these regions. High gamma activity also alternates between different high-ACh regions, at theta frequency. Our results are the first to indicate a causal role for spatially heterogenous ACh signaling in the emergence of localized theta-gamma rhythmicity. Our findings also provide novel insights into mechanisms by which ACh signaling supports the brain region-specific attentional processing of sensory information.

Metabolic adaptations to hypoxia in the neonatal mouse forebrain can occur independently of the transporters SLC7A5 and SLC3A2.

Neonatal encephalopathy due to hypoxia-ischemia is associated with adverse neurodevelopmental effects. The involvement of branched chain amino acids (BCAAs) in this is largely unexplored. Transport of BCAAs at the plasma membrane is facilitated by SLC7A5/SLC3A2, which increase with hypoxia. We hypothesized that hypoxia would alter BCAA transport and metabolism in the neonatal brain. We investigated this using an organotypic forebrain slice culture model with, the SLC7A5/SLC3A2 inhibitor, 2-Amino-2-norbornanecarboxylic acid (BCH) under normoxic or hypoxic conditions. We subsequently analysed the metabolome and candidate gene expression. Hypoxia was associated with increased expression of SLC7A5 and SLC3A2 and an increased tissue abundance of BCAAs. Incubation of slices with 13C-leucine confirmed that this was due to increased cellular uptake. BCH had little effect on metabolite abundance under normoxic or hypoxic conditions. This suggests hypoxia drives increased cellular uptake of BCAAs in the neonatal mouse forebrain, and membrane mediated transport through SLC7A5 and SLC3A2 is not essential for this process. This indicates mechanisms exist to generate the compounds required to maintain essential metabolism in the absence of external nutrient supply. Moreover, excess BCAAs have been associated with developmental delay, providing an unexplored mechanism of hypoxia mediated pathogenesis in the developing forebrain.

Long-Range GABAergic Inhibition Modulates Spatiotemporal Dynamics of the Output Neurons in the Olfactory Bulb.

Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in male and female mice acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased on activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits.SIGNIFICANCE STATEMENT Disruption of GABAergic inhibition from the basal forebrain (BF) to the olfactory bulb (OB) impairs the discrimination of similar odors, yet how this centrifugal inhibition influences neuronal circuits in the OB remains unclear. Here, we show that the BF GABAergic neurons exclusively target local inhibitory neurons in the OB, having a functional disinhibitory effect on the output neurons, the mitral cells. Phasic inhibition by BF GABAergic neurons reduces spike precision of mitral cells and lowers the intensity of oscillatory activity in the OB, while directly modulating the extent of dendrodendritic inhibition. These circuit-level effects of this centrifugal inhibition can influence the temporal and spatial dynamics of odor coding in the OB.

Basal forebrain mediates prosocial behavior via disinhibition of midbrain dopamine neurons.

Sociability is fundamental for our daily life and is compromised in major neuropsychiatric disorders. However, the neuronal circuit mechanisms underlying prosocial behavior are still elusive. Here we identify a causal role of the basal forebrain (BF) in the control of prosocial behavior via inhibitory projections that disinhibit the midbrain ventral tegmental area (VTA) dopamine (DA) neurons. Specifically, BF somatostatin-positive (SST) inhibitory neurons were robustly activated during social interaction. Optogenetic inhibition of these neurons in BF or their axon terminals in the VTA largely abolished social preference. Electrophysiological examinations further revealed that SST neurons predominantly targeted VTA GABA neurons rather than DA neurons. Consistently, optical inhibition of SST neuron axon terminals in the VTA decreased DA release in the nucleus accumbens during social interaction, confirming a disinhibitory action. These data reveal a previously unappreciated function of the BF in prosocial behavior through a disinhibitory circuitry connected to the brain's reward system.

Functional parcellation of mouse visual cortex using statistical techniques reveals response-dependent clustering of cortical processing areas.

The visual cortex of the mouse brain can be divided into ten or more areas that each contain complete or partial retinotopic maps of the contralateral visual field. It is generally assumed that these areas represent discrete processing regions. In contrast to the conventional input-output characterizations of neuronal responses to standard visual stimuli, here we asked whether six of the core visual areas have responses that are functionally distinct from each other for a given visual stimulus set, by applying machine learning techniques to distinguish the areas based on their activity patterns. Visual areas defined by retinotopic mapping were examined using supervised classifiers applied to responses elicited by a range of stimuli. Using two distinct datasets obtained using wide-field and two-photon imaging, we show that the area labels predicted by the classifiers were highly consistent with the labels obtained using retinotopy. Furthermore, the classifiers were able to model the boundaries of visual areas using resting state cortical responses obtained without any overt stimulus, in both datasets. With the wide-field dataset, clustering neuronal responses using a constrained semi-supervised classifier showed graceful degradation of accuracy. The results suggest that responses from visual cortical areas can be classified effectively using data-driven models. These responses likely reflect unique circuits within each area that give rise to activity with stronger intra-areal than inter-areal correlations, and their responses to controlled visual stimuli across trials drive higher areal classification accuracy than resting state responses.

Imaging of spine synapses using super-resolution microscopy.

Neuronal circuits in the neocortex and hippocampus are essential for higher brain functions such as motor learning and spatial memory. In the mammalian forebrain, most excitatory synapses of pyramidal neurons are formed on spines, which are tiny protrusions extending from the dendritic shaft. The spine contains specialized molecular machinery that regulates synaptic transmission and plasticity. Spine size correlates with the efficacy of synaptic transmission, and spine morphology affects signal transduction at the post-synaptic compartment. Plasticity-related changes in the structural and molecular organization of spine synapses are thought to underlie the cellular basis of learning and memory. Recent advances in super-resolution microscopy have revealed the molecular mechanisms of the nanoscale synaptic structures regulating synaptic transmission and plasticity in living neurons, which are difficult to investigate using electron microscopy alone. In this review, we summarize recent advances in super-resolution imaging of spine synapses and discuss the implications of nanoscale structures in the regulation of synaptic function, learning, and memory.