Variation of connectivity across exemplar sensory and associative thalamocortical loops in the mouse.

The thalamus engages in sensation, action, and cognition, but the structure underlying these functions is poorly understood. Thalamic innervation of associative cortex targets several interneuron types, modulating dynamics and influencing plasticity. Is this structure-function relationship distinct from that of sensory thalamocortical systems? Here, we systematically compared function and structure across a sensory and an associative thalamocortical loop in the mouse. Enhancing excitability of mediodorsal thalamus, an associative structure, resulted in prefrontal activity dominated by inhibition. Equivalent enhancement of medial geniculate excitability robustly drove auditory cortical excitation. Structurally, geniculate axons innervated excitatory cortical targets in a preferential manner and with larger synaptic terminals, providing a putative explanation for functional divergence. The two thalamic circuits also had distinct input patterns, with mediodorsal thalamus receiving innervation from a diverse set of cortical areas. Altogether, our findings contribute to the emerging view of functional diversity across thalamic microcircuits and its structural basis.

Retinal Inputs to the Thalamus Are Selectively Gated by Arousal.

The brain can flexibly filter out sensory information in a manner that depends on behavioral state. In the visual thalamus and cortex, arousal and locomotion are associated with changes in the magnitude of responses to visual stimuli. Here, we asked whether such modulation of visual responses might already occur at an earlier stage in this visual pathway. We measured neural activity of retinal axons using wide-field and two-photon calcium imaging in awake mouse thalamus across arousal states associated with different pupil sizes. Surprisingly, visual responses to drifting gratings in retinal axonal boutons were robustly modulated by arousal level in a manner that varied across stimulus dimensions and across functionally distinct subsets of boutons. At low and intermediate spatial frequencies, the majority of boutons were suppressed by arousal. In contrast, at high spatial frequencies, boutons tuned to regions of visual space ahead of the mouse showed enhancement of responses. Arousal-related modulation also varied with a bouton's preference for luminance changes and direction or axis of motion, with greater response suppression in boutons tuned to luminance decrements versus increments, and in boutons preferring motion along directions or axes of optic flow. Together, our results suggest that differential modulation of distinct visual information channels by arousal state occurs at very early stages of visual processing, before the information is transmitted to neurons in visual thalamus. Such early filtering may provide an efficient means of optimizing central visual processing and perception across behavioral contexts.

Structural dynamics and stability of corticocortical and thalamocortical axon terminals during motor learning.

Synaptic plasticity is the cellular basis of learning and memory. When animals learn a novel motor skill, synaptic modifications are induced in the primary motor cortex (M1), and new postsynaptic dendritic spines relevant to motor memory are formed in the early stage of learning. However, it is poorly understood how presynaptic axonal boutons are formed, eliminated, and maintained during motor learning, and whether long-range corticocortical and thalamocortical axonal boutons show distinct structural changes during learning. In this study, we conducted two-photon imaging of presynaptic boutons of long-range axons in layer 1 (L1) of the mouse M1 during the 7-day learning of an accelerating rotarod task. The training-period-averaged rate of formation of boutons on axons projecting from the secondary motor cortical area increased, while the average rate of elimination of those from the motor thalamus (thalamic boutons) decreased. In particular, the elimination rate of thalamic boutons during days 4-7 was lower than that in untrained mice, and the fraction of pre-existing thalamic boutons that survived until day 7 was higher than that in untrained mice. Our results suggest that the late stabilization of thalamic boutons in M1 contributes to motor skill learning.

VGluT1 Deficiency Impairs Visual Attention and Reduces the Dynamic Range of Short-Term Plasticity at Corticothalamic Synapses.

The most common excitatory neurotransmitter in the central nervous system, glutamate, is loaded into synaptic vesicles by vesicular glutamate transporters (VGluTs). The primary isoforms, VGluT1 and 2, are expressed in complementary patterns throughout the brain and correlate with short-term synaptic plasticity. VGluT1 deficiency is observed in certain neurological disorders, and hemizygous (VGluT1+/-) mice display increased anxiety and depression, altered sensorimotor gating, and impairments in learning and memory. The synaptic mechanisms underlying these behavioral deficits are unknown. Here, we show that VGluT1+/- mice had decreased visual processing speeds during a sustained visual-spatial attention task. Furthermore, in vitro recordings of corticothalamic (CT) synapses revealed dramatic reductions in short-term facilitation, increased initial release probability, and earlier synaptic depression in VGluT1+/- mice. Our electron microscopy results show that VGluT1 concentration is reduced at CT synapses of hemizygous mice, but other features (such as vesicle number and active zone size) are unchanged. We conclude that VGluT1-haploinsuficiency decreases the dynamic range of gain modulation provided by CT feedback to the thalamus, and this deficiency contributes to the observed attentional processing deficit. We further hypothesize that VGluT1 concentration regulates release probability by applying a "brake" to an unidentified presynaptic protein that typically acts as a positive regulator of release.

Spatiotemporal model of tripartite synapse with perinodal astrocytic process.

Information transfer may not be limited only to synapses. Therefore, the processes and dynamics of biological neuron-astrocyte coupling and intercellular interaction within this domain are worth investigating. Existing models of tripartite synapse consider an astrocyte as a point process. Here, we extended the tripartite synapse model by considering the astrocytic processes (synaptic and perinodal) as compartments. The scattered extrinsic signals in the extracellular space and the presence of calcium stores in different astrocytic sites create local transient [Ca2+]. We investigated the Ca2+ dynamics and found that the increase in astrocytic intracellular [Ca2+] enhances the probability of neurotransmitter release. However, the period in which the extrasynaptic glutamate lingers in the extracellular space may cause excitotoxicity. We propose further biological investigation on intercellular communication, considering that unconventional sources (nonsynaptic) of glutamate may improve information processing in neuron-astrocyte networks.

Relation of koniocellular layers of dorsal lateral geniculate to inferior pulvinar nuclei in common marmosets.

Traditionally, the dorsal lateral geniculate nucleus (LGN) and the inferior pulvinar (IPul) nucleus are considered as anatomically and functionally distinct thalamic nuclei. However, in several primate species it has also been established that the koniocellular (K) layers of LGN and parts of the IPul have a shared pattern of immunoreactivity for the calcium-binding protein calbindin. These calbindin-rich cells constitute a thalamic matrix system which is implicated in thalamocortical synchronisation. Further, the K layers and IPul are both involved in visual processing and have similar connections with retina and superior colliculus. Here, we confirmed the continuity between calbindin-rich cells in LGN K layers and the central lateral division of IPul (IPulCL) in marmoset monkeys. By employing a high-throughput neuronal tracing method, we found that both the K layers and IPulCL form comparable patterns of connections with striate and extrastriate cortices; these connections are largely different to those of the parvocellular and magnocellular laminae of LGN. Retrograde tracer-labelled cells and anterograde tracer-labelled axon terminals merged seamlessly from IPulCL into LGN K layers. These results support continuity between LGN K layers and IPulCL, providing an anatomical basis for functional congruity of this region of the dorsal thalamic matrix and calling into question the traditional segregation between LGN and the inferior pulvinar nucleus.

Major Feedforward Thalamic Input Into Layer 4C of Primary Visual Cortex in Primate.

One of the underlying principles of how mammalian circuits are constructed is the relative influence of feedforward to recurrent synaptic drive. It has been dogma in sensory systems that the thalamic feedforward input is relatively weak and that there is a large amplification of the input signal by recurrent feedback. Here we show that in trichromatic primates there is a major feedforward input to layer 4C of primary visual cortex. Using a combination of 3D-electron-microscopy and 3D-confocal imaging of thalamic boutons we found that the average feedforward contribution was about 20% of the total excitatory input in the parvocellular (P) pathway, about 3 times the currently accepted values for primates. In the magnocellular (M) pathway it was around 15%, nearly twice the currently accepted values. New methods showed the total synaptic and cell densities were as much as 150% of currently accepted values. The new estimates of contributions of feedforward synaptic inputs into visual cortex call for a major revision of the design of the canonical cortical circuit.

A Sensorimotor Pathway via Higher-Order Thalamus.

We now know that sensory processing in cortex occurs not only via direct communication between primary to secondary areas, but also via their parallel cortico-thalamo-cortical (i.e., trans-thalamic) pathways. Both corticocortical and trans-thalamic pathways mainly signal through glutamatergic class 1 (driver) synapses, which have robust and efficient synaptic dynamics suited for the transfer of information such as receptive field properties, suggesting the importance of class 1 synapses in feedforward, hierarchical processing. However, such a parallel arrangement has only been identified in sensory cortical areas: visual, somatosensory, and auditory. To test the generality of trans-thalamic pathways, we sought to establish its presence beyond purely sensory cortices to determine whether there is a trans-thalamic pathway parallel to the established primary somatosensory (S1) to primary motor (M1) pathway. We used trans-synaptic viral tracing, optogenetics in slice preparations, and bouton size analysis in the mouse (both sexes) to document that a circuit exists from layer 5 of S1 through the posterior medial nucleus of the thalamus to M1 with glutamatergic class 1 properties. This represents a hitherto unknown, robust sensorimotor linkage and suggests that the arrangement of parallel direct and trans-thalamic corticocortical circuits may be present as a general feature of cortical functioning.SIGNIFICANCE STATEMENT During sensory processing, feedforward pathways carry information such as receptive field properties via glutamatergic class 1 synapses, which have robust and efficient synaptic dynamics. As expected, class 1 synapses subserve the feedforward projection from primary to secondary sensory cortex, but also a route through specific higher-order thalamic nuclei, creating a parallel feedforward trans-thalamic pathway. We now extend the concept of cortical areas being connected via parallel, direct, and trans-thalamic circuits from purely sensory cortices to a sensorimotor cortical circuit (i.e., primary sensory cortex to primary motor cortex). This suggests a generalized arrangement for corticocortical communication.

Secretory vesicle trafficking in awake and anaesthetized mice: differential speeds in axons versus synapses.

KEY POINTS: Despite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain. DCVs are transported at similar average speeds in the anaesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher. Microtubule plus (+)-end extension imaging visualized microtubular growth at 0.12 µm/s and revealed that DCVs were transported faster in the anterograde direction. DCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release. Our work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveals novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality. ABSTRACT: Neuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamocortical projections of anaesthetized and awake mice. We found an average speed of 1 µm/s, maximal speeds of up to 5 µm/s and a pausing fraction of ∼11%. Directionality of transport differed between anaesthetized and awake mice. In vivo microtubule +-end extension imaging using MACF18-GFP revealed microtubular growth at 0.12 µm/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (∼2.1 µm/s) than retrograde transport (∼1.4 µm/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.

A Fine-Scale Functional Logic to Convergence from Retina to Thalamus.

Numerous well-defined classes of retinal ganglion cells innervate the thalamus to guide image-forming vision, yet the rules governing their convergence and divergence remain unknown. Using two-photon calcium imaging in awake mouse thalamus, we observed a functional arrangement of retinal ganglion cell axonal boutons in which coarse-scale retinotopic ordering gives way to fine-scale organization based on shared preferences for other visual features. Specifically, at the ∼6 µm scale, clusters of boutons from different axons often showed similar preferences for either one or multiple features, including axis and direction of motion, spatial frequency, and changes in luminance. Conversely, individual axons could "de-multiplex" information channels by participating in multiple, functionally distinct bouton clusters. Finally, ultrastructural analyses demonstrated that retinal axonal boutons in a local cluster often target the same dendritic domain. These data suggest that functionally specific convergence and divergence of retinal axons may impart diverse, robust, and often novel feature selectivity to visual thalamus.

In vivo wide-field calcium imaging of mouse thalamocortical synapses with an 8 K ultra-high-definition camera.

In vivo wide-field imaging of neural activity with a high spatio-temporal resolution is a challenge in modern neuroscience. Although two-photon imaging is very powerful, high-speed imaging of the activity of individual synapses is mostly limited to a field of approximately 200 µm on a side. Wide-field one-photon epifluorescence imaging can reveal neuronal activity over a field of ≥1 mm2 at a high speed, but is not able to resolve a single synapse. Here, to achieve a high spatio-temporal resolution, we combine an 8 K ultra-high-definition camera with spinning-disk one-photon confocal microscopy. This combination allowed us to image a 1 mm2 field with a pixel resolution of 0.21 µm at 60 fps. When we imaged motor cortical layer 1 in a behaving head-restrained mouse, calcium transients were detected in presynaptic boutons of thalamocortical axons sparsely labeled with GCaMP6s, although their density was lower than when two-photon imaging was used. The effects of out-of-focus fluorescence changes on calcium transients in individual boutons appeared minimal. Axonal boutons with highly correlated activity were detected over the 1 mm2 field, and were probably distributed on multiple axonal arbors originating from the same thalamic neuron. This new microscopy with an 8 K ultra-high-definition camera should serve to clarify the activity and plasticity of widely distributed cortical synapses.

Single excitatory axons form clustered synapses onto CA1 pyramidal cell dendrites.

CA1 pyramidal neurons are a major output of the hippocampus and encode features of experience that constitute episodic memories. Feature-selective firing of these neurons results from the dendritic integration of inputs from multiple brain regions. While it is known that synchronous activation of spatially clustered inputs can contribute to firing through the generation of dendritic spikes, there is no established mechanism for spatiotemporal synaptic clustering. Here we show that single presynaptic axons form multiple, spatially clustered inputs onto the distal, but not proximal, dendrites of CA1 pyramidal neurons. These compound connections exhibit ultrastructural features indicative of strong synapses and occur much more commonly in entorhinal than in thalamic afferents. Computational simulations revealed that compound connections depolarize dendrites in a biophysically efficient manner, owing to their inherent spatiotemporal clustering. Our results suggest that distinct afferent projections use different connectivity motifs that differentially contribute to dendritic integration.

Parvalbumin-producing striatal interneurons receive excitatory inputs onto proximal dendrites from the motor thalamus in male mice.

In rodents, the dorsolateral striatum regulates voluntary movement by integrating excitatory inputs from the motor-related cerebral cortex and thalamus to produce contingent inhibitory output to other basal ganglia nuclei. Striatal parvalbumin (PV)-producing interneurons receiving this excitatory input then inhibit medium spiny neurons (MSNs) and modify their outputs. To understand basal ganglia function in motor control, it is important to reveal the precise synaptic organization of motor-related cortical and thalamic inputs to striatal PV interneurons. To examine which domains of the PV neurons receive these excitatory inputs, we used male bacterial artificial chromosome transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein in PV neurons. An anterograde tracing study with the adeno-associated virus vector combined with immunodetection of pre- and postsynaptic markers visualized the distribution of the excitatory appositions on PV dendrites. Statistical analysis revealed that the density of thalamostriatal appositions along the dendrites was significantly higher on the proximal than distal dendrites. In contrast, there was no positional preference in the density of appositions from axons of the dorsofrontal cortex. Population observations of thalamostriatal and corticostriatal appositions by immunohistochemistry for pathway-specific vesicular glutamate transporters confirmed that thalamic inputs preferentially, and cortical ones less preferentially, made apposition on proximal dendrites of PV neurons. This axodendritic organization suggests that PV neurons produce fast and reliable inhibition of MSNs in response to thalamic inputs and process excitatory inputs from motor cortices locally and plastically, possibly together with other GABAergic and dopaminergic dendritic inputs, to modulate MSN inhibition.

Distinct kinetics of inhibitory currents in thalamocortical neurons that arise from dendritic or axonal origin.

Thalamocortical neurons in the dorsal lateral geniculate nucleus (dLGN) transfer visual information from retina to primary visual cortex. This information is modulated by inhibitory input arising from local interneurons and thalamic reticular nucleus (TRN) neurons, leading to alterations of receptive field properties of thalamocortical neurons. Local GABAergic interneurons provide two distinct synaptic outputs: axonal (F1 terminals) and dendritic (F2 terminals) onto dLGN thalamocortical neurons. By contrast, TRN neurons provide only axonal output (F1 terminals) onto dLGN thalamocortical neurons. It is unclear if GABAA receptor-mediated currents originating from F1 and F2 terminals have different characteristics. In the present study, we examined multiple characteristics (rise time, slope, halfwidth and decay τ) of GABAA receptor-mediated miniature inhibitory postsynaptic synaptic currents (mIPSCs) originating from F1 and F2 terminals. The mIPSCs arising from F2 terminals showed slower kinetics relative to those from F1 terminals. Such differential kinetics of GABAAR-mediated responses could be an important role in temporal coding of visual signals.

Optogenetic Visualization of Presynaptic Tonic Inhibition of Cerebellar Parallel Fibers.

UNLABELLED: Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABAA receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABAA receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABAA receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits. SIGNIFICANCE STATEMENT: This paper demonstrates that an unconventional form of signaling, known as tonic inhibition, is found in presynaptic terminals and affects conventional synaptic communication. Our results establish the basic characteristics and mechanisms of presynaptic tonic inhibition and show that it occurs in vivo as well as in isolated brain tissue.

Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs.

Understanding the functions of a brain region requires knowing the neural representations of its myriad inputs, local neurons and outputs. Primary visual cortex (V1) has long been thought to compute visual orientation from untuned thalamic inputs, but very few thalamic inputs have been measured in any mammal. We determined the response properties of ∼ 28,000 thalamic boutons and ∼ 4,000 cortical neurons in layers 1-5 of awake mouse V1. Using adaptive optics that allows accurate measurement of bouton activity deep in cortex, we found that around half of the boutons in the main thalamorecipient L4 carried orientation-tuned information and that their orientation and direction biases were also dominant in the L4 neuron population, suggesting that these neurons may inherit their selectivity from tuned thalamic inputs. Cortical neurons in all layers exhibited sharper tuning than thalamic boutons and a greater diversity of preferred orientations. Our results provide data-rich constraints for refining mechanistic models of cortical computation.

Modifications of excitatory and inhibitory transmission in rat hippocampal pyramidal neurons by acute lithium treatment.

The acute effects of high-dose Li(+) treatment on glutamatergic and GABAergic transmissions were studied in the "synaptic bouton" preparation of isolated rat hippocampal pyramidal neurons by using focal electrical stimulation. Both action potential-dependent glutamatergic excitatory and GABAergic inhibitory postsynaptic currents (eEPSC and eIPSC, respectively) were dose-dependently inhibited in the external media containing 30-150 mM Li(+), but the sensitivity for Li(+) was greater tendency for eEPSCs than for eIPSCs. When the effects of Li(+) on glutamate or GABAA receptor-mediated whole-cell responses (IGlu and IGABA) elicited by an exogenous application of glutamate or GABA were examined in the postsynaptic soma membrane of CA3 neurons, Li(+) slightly inhibited both IGlu and IGABA at the 150 mM Li(+) concentration. Present results suggest that acute treatment with high concentrations of Li(+) acts preferentially on presynaptic terminals, and that the Li(+)-induced inhibition may be greater for excitatory than for inhibitory transmission.

Orexins/hypocretins modulate the activity of NPY-positive and -negative neurons in the rat intergeniculate leaflet via OX1 and OX2 receptors.

Orexins/hypocretins (OXA and OXB) are two hypothalamic peptides involved in the regulation of many physiological processes including the sleep-wake cycle, food intake and arousal. The orexinergic system of the lateral hypothalamus is considered a non-specific peptidergic system, and its nerve fibers innervate numerous brain areas. Among many targets of orexinergic neurons is the intergeniculate leaflet (IGL) of the thalamus - a small but important structure of the mammalian biological clock. In rats, the IGL consists of GABAergic cells which also synthesize different neuropeptides. One group of neurons produces neuropeptide Y (NPY) and sends its axons to the master biological clock known as the suprachiasmatic nuclei. Another neuronal group produces enkephalin and is known to connect contralateral IGLs. This study evaluated the effects of orexins on identified IGL neurons revealing that 58% of the recorded neurons were sensitive to OXA (200nM) and OXB (200nM) administration. Both NPY-positive and -negative neurons were depolarized by these neuropeptides. Experiments using selective orexin receptor antagonists (SB-334867, 10µM and TCS-OX2-29, 10µM) suggested that both orexin receptors participate in the recorded OXA effects. In addition, IGL neurons were either directly depolarized by OXA or their activity was altered by changes in presynaptic inputs. We observed an increase of GABA release onto the investigated IGL neuron after OXA application, consistent with a presynaptic localization of the orexin receptors. An increase in miniature excitatory postsynaptic current frequency was not observed within the IGL. Our findings reinforce the connection between circadian clock physiology and the orexinergic system.

Artemisia santolinifolia enhances glutamatergic neurotransmission in the nucleus of the solitary tract.

Artemisia extracts have been used as remedies for a variety of maladies related to metabolic and gastrointestinal control. Because the vagal afferent-nucleus of the solitary tract (NST) synapse regulates the same homeostatic functions affected by Artemisia, it is possible that these extracts may have activity at the synaptic level in the NST. Therefore, we evaluated how extracts of three common medicinal Artemisia species, Artemisia santolinifolia (SANT), Artemisia scoparia (SCO), and Artemisia dracunculus L (PMI-5011), modulate the excitability of the glutamatergic vagal afferent-NST synapse. Our in vitro live cell calcium imaging data from prelabeled vagal afferent terminals show that SANT extract is a positive modulator of vagal afferent calcium levels, as the extract significantly increased the calcium signal relative to the time control. Neither SCO nor PMI-5011 extract altered the vagal calcium signals compared to the time control. Furthermore, whole cell voltage-clamp recordings from NST neurons corroborated the vagal terminal calcium data in that SANT extract also significantly increased miniature excitatory postsynaptic current (mEPSC) frequency in NST neurons. These data suggest that SANT extract could be a pharmacologically significant mediator of glutamatergic neurotransmission within the CNS.

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction.

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, we investigated the formation of new boutons in NMJs lacking synapsin [Syn(-)], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. We found that budding of new boutons at Syn(-) NMJs was significantly diminished, and that new boutons in Syn(-) preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), we assayed whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway. We pretreated preparations with forskolin to raise cAMP levels and found this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn(-) preparations. To examine the trafficking of synapsin during synaptic growth, we generated transgenic animals expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway.