GluN2C/D-containing NMDA receptors enhance temporal summation and increase sound-evoked and spontaneous firing in the inferior colliculus.

Along the ascending auditory pathway, there is a broad shift from temporal coding, which is common in the lower auditory brainstem, to rate coding, which predominates in auditory cortex. This temporal-to-rate transition is particularly prominent in the inferior colliculus (IC), the midbrain hub of the auditory system, but the mechanisms that govern how individual IC neurons integrate information across time remain largely unknown. Here, we report the widespread expression of Glun2c and Glun2d mRNA in IC neurons. GluN2C/D-containing NMDA receptors are relatively insensitive to voltage-dependent Mg2+ blockade, and thus can conduct current at resting membrane potential. Using in situ hybridization and pharmacology, we show that vasoactive intestinal peptide neurons in the IC express GluN2D-containing NMDA receptors that are activatable by commissural inputs from the contralateral IC. In addition, GluN2C/D-containing receptors have much slower kinetics than other NMDA receptors, and we found that GluN2D-containing receptors facilitate temporal summation of synaptic inputs in vasoactive intestinal peptide neurons. In a model neuron, we show that a GluN2C/D-like conductance interacts with the passive membrane properties of the neuron to alter temporal and rate coding of stimulus trains. Consistent with this, we show in vivo that blocking GluN2C/D-containing receptors decreases both the spontaneous firing rate and the overall firing rate elicited by amplitude-modulated sounds in many IC neurons. These results suggest that GluN2C/D-containing NMDA receptors influence rate coding for auditory stimuli in the IC by facilitating the temporal integration of synaptic inputs. KEY POINTS: NMDA receptors are critical components of most glutamatergic circuits in the brain, and the diversity of NMDA receptor subtypes yields receptors with a variety of functions. We found that many neurons in the auditory midbrain express GluN2C and/or GluN2D NMDA receptor subunits, which are less sensitive to Mg2+ blockade than the more commonly expressed GluN2A/B subunits. We show that GluN2C/D-containing receptors conducted current at resting membrane potential and enhanced temporal summation of synaptic inputs. In a model, we show that GluN2C/D-containing receptors provide additive gain for input-output functions driven by trains of synaptic inputs. In line with this, we found that blocking GluN2C/D-containing NMDA receptors in vivo decreased both spontaneous firing rates and firing evoked by amplitude-modulated sounds.

A dendritic mechanism for balancing synaptic flexibility and stability.

Biological and artificial neural networks learn by modifying synaptic weights, but it is unclear how these systems retain previous knowledge and also acquire new information. Here, we show that cortical pyramidal neurons can solve this plasticity-versus-stability dilemma by differentially regulating synaptic plasticity at distinct dendritic compartments. Oblique dendrites of adult mouse layer 5 cortical pyramidal neurons selectively receive monosynaptic thalamic input, integrate linearly, and lack burst-timing synaptic potentiation. In contrast, basal dendrites, which do not receive thalamic input, exhibit conventional NMDA receptor (NMDAR)-mediated supralinear integration and synaptic potentiation. Congruently, spiny synapses on oblique branches show decreased structural plasticity in vivo. The selective decline in NMDAR activity and expression at synapses on oblique dendrites is controlled by a critical period of visual experience. Our results demonstrate a biological mechanism for how single neurons can safeguard a set of inputs from ongoing plasticity by altering synaptic properties at distinct dendritic domains.

Demonstration that sublinear dendrites enable linearly non-separable computations.

Theory predicts that nonlinear summation of synaptic potentials within dendrites allows neurons to perform linearly non-separable computations (LNSCs). Using Boolean analysis approaches, we predicted that both supralinear and sublinear synaptic summation could allow single neurons to implement a type of LNSC, the feature binding problem (FBP), which does not require inhibition contrary to the exclusive-or function (XOR). Notably, sublinear dendritic operations enable LNSCs when scattered synaptic activation generates increased somatic spike output. However, experimental demonstrations of scatter-sensitive neuronal computations have not yet been described. Using glutamate uncaging onto cerebellar molecular layer interneurons, we show that scattered synaptic-like activation of dendrites evoked larger compound EPSPs than clustered synaptic activation, generating a higher output spiking probability. Moreover, we also demonstrate that single interneurons can indeed implement the FBP. Using a biophysical model to explore the conditions in which a neuron might be expected to implement the FBP, we establish that sublinear summation is necessary but not sufficient. Other parameters such as the relative sublinearity, the EPSP size, depolarization amplitude relative to action potential threshold, and voltage fluctuations all influence whether the FBP can be performed. Since sublinear synaptic summation is a property of passive dendrites, we expect that many different neuron types can implement LNSCs.

Survival and Functional Integration of Human Embryonic Stem Cell-Derived Retinal Organoids After Shipping and Transplantation into Retinal Degeneration Rats.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Complex Synaptic and Intrinsic Interactions Disrupt Input/Output Functions in the Hippocampus of Scn1b Knock-Out Mice.

Pathogenic variants in SCN1B have been linked to severe developmental epileptic encephalopathies including Dravet syndrome. Scn1b knock-out (KO) mice model SCN1B loss-of-function (LOF) disorders, demonstrating seizures, developmental delays, and early death. SCN1B encodes the protein ß1, an ion channel auxiliary subunit that also has roles in cell adhesion, neurite outgrowth, and gene expression. The goal of this project is to better understand of how loss of Scn1b alters information processing in the brain, resulting in seizures and associated cognitive dysfunction. Using slice electrophysiology in the CA1 region of the hippocampus from male and female Scn1b KO mice and wild-type (WT) littermates, we found that processing of physiologically relevant patterned Schaffer collateral (SC) stimulation produces larger, prolonged depolarizations and increased spiking in KO neurons compared with WTs. KO neurons exhibit enhanced intrinsic excitability, firing more action potentials with current injection. Interestingly, SC stimulation produces smaller, more facilitating excitatory and IPSCs in KO pyramidal neurons, but larger postsynaptic potentials (PSPs) with the same stimulation. We also found reduced intrinsic firing of parvalbumin (PV)-expressing interneurons and disrupted recruitment of both parvalbumin-expressing and somatostatin (SST)-expressing interneurons in response to patterned synaptic stimulation. Neuronal information processing relies on the interplay between synaptic properties, intrinsic properties that amplify or suppress incoming synaptic signals, and firing properties that produce cellular output. We found changes at each of these levels in Scn1b KO pyramidal neurons, resulting in fundamentally altered cellular information processing in the hippocampus that likely contributes to the complex phenotypes of SCN1B-linked epileptic encephalopathies.SIGNIFICANCE STATEMENT Genetic developmental epileptic encephalopathies have limited treatment options, in part because of our lack of understanding of how genetic changes result in dysfunction at the cellular and circuit levels. SCN1B is a gene linked to Dravet syndrome and other developmental epileptic encephalopathies, and Scn1b knock-out (KO) mice phenocopy the human disease, allowing us to study underlying neurophysiological changes. Here, we found changes at all levels of neuronal information processing in brains lacking Scn1b, including intrinsic excitability, synaptic properties, and synaptic integration, resulting in greatly enhanced input/output functions of the hippocampus. Our study shows that loss of Scn1b results in a complex array of cellular and network changes that fundamentally alters information processing in the hippocampus.

High-resolution volumetric imaging constrains compartmental models to explore synaptic integration and temporal processing by cochlear nucleus globular bushy cells.

Globular bushy cells (GBCs) of the cochlear nucleus play central roles in the temporal processing of sound. Despite investigation over many decades, fundamental questions remain about their dendrite structure, afferent innervation, and integration of synaptic inputs. Here, we use volume electron microscopy (EM) of the mouse cochlear nucleus to construct synaptic maps that precisely specify convergence ratios and synaptic weights for auditory nerve innervation and accurate surface areas of all postsynaptic compartments. Detailed biophysically based compartmental models can help develop hypotheses regarding how GBCs integrate inputs to yield their recorded responses to sound. We established a pipeline to export a precise reconstruction of auditory nerve axons and their endbulb terminals together with high-resolution dendrite, soma, and axon reconstructions into biophysically detailed compartmental models that could be activated by a standard cochlear transduction model. With these constraints, the models predict auditory nerve input profiles whereby all endbulbs onto a GBC are subthreshold (coincidence detection mode), or one or two inputs are suprathreshold (mixed mode). The models also predict the relative importance of dendrite geometry, soma size, and axon initial segment length in setting action potential threshold and generating heterogeneity in sound-evoked responses, and thereby propose mechanisms by which GBCs may homeostatically adjust their excitability. Volume EM also reveals new dendritic structures and dendrites that lack innervation. This framework defines a pathway from subcellular morphology to synaptic connectivity, and facilitates investigation into the roles of specific cellular features in sound encoding. We also clarify the need for new experimental measurements to provide missing cellular parameters, and predict responses to sound for further in vivo studies, thereby serving as a template for investigation of other neuron classes.

Altered integration of excitatory inputs onto the basal dendrites of layer 5 pyramidal neurons in a mouse model of Fragile X syndrome.

Layer 5 (L5) pyramidal neurons receive predictive and sensory inputs in a compartmentalized manner at their apical and basal dendrites, respectively. To uncover how integration of sensory inputs is affected in autism spectrum disorders (ASD), we used two-photon glutamate uncaging to activate spines in the basal dendrites of L5 pyramidal neurons from a mouse model of Fragile X syndrome (FXS), the most common genetic cause of ASD. While subthreshold excitatory inputs integrate linearly in wild-type animals, surprisingly those with FXS summate sublinearly, contradicting what would be expected of sensory hypersensitivity classically associated with ASD. We next investigated the mechanism underlying this sublinearity by performing knockdown of the regulatory ß4 subunit of BK channels, which rescued the synaptic integration, a result that was corroborated with numerical simulations. Taken together, these findings suggest that there is a differential impairment in the integration of feedforward sensory and feedback predictive inputs in L5 pyramidal neurons in FXS and potentially other forms of ASD, as a result of specifically localized subcellular channelopathies. These results challenge the traditional view that FXS and other ASD are characterized by sensory hypersensitivity, proposing instead a hyposensitivity of sensory inputs and hypersensitivity of predictive inputs onto cortical neurons.

Bi-directional Control of Synaptic Input Summation and Spike Generation by GABAergic Inputs at the Axon Initial Segment.

Differing from other subtypes of inhibitory interneuron, chandelier or axo-axonic cells form depolarizing GABAergic synapses exclusively onto the axon initial segment (AIS) of targeted pyramidal cells (PCs). However, the debate whether these AIS-GABAergic inputs produce excitation or inhibition in neuronal processing is not resolved. Using realistic NEURON modeling and electrophysiological recording of cortical layer-5 PCs, we quantitatively demonstrate that the onset-timing of AIS-GABAergic input, relative to dendritic excitatory glutamatergic inputs, determines its bi-directional regulation of the efficacy of synaptic integration and spike generation in a PC. More specifically, AIS-GABAergic inputs promote the boosting effect of voltage-activated Na+ channels on summed synaptic excitation when they precede glutamatergic inputs by >15 ms, while for nearly concurrent excitatory inputs, they primarily produce a shunting inhibition at the AIS. Thus, our findings offer an integrative mechanism by which AIS-targeting interneurons exert sophisticated regulation of the input-output function in targeted PCs.

Patch-clamp analysis of nicotinic synapses whose strength straddles the firing threshold of rat sympathetic neurons.

Neurons in paravertebral sympathetic ganglia are innervated by converging nicotinic synapses of varying strength. Based upon intracellular recordings of excitatory postsynaptic potentials (EPSPs) with sharp microelectrodes these synapses were classified in the past as either primary (strong) or secondary (weak) by their ability to trigger postsynaptic action potentials. Here we present an analysis of 22 synapses whose strength straddled threshold, thereby distinguishing them from the original classification scheme for primary and secondary synapses. Recordings at 36°C were made from intact superior cervical ganglia isolated from 13 male and 3 female Sprague-Dawley rats and from 4 male spontaneously hypertensive (SHR) rats. Ganglia were pretreated with collagenase to permit patch recording. By dissecting a 1 cm length of the presynaptic cervical sympathetic nerve as part of the preparation and through use of graded presynaptic stimulation it was possible to fractionate synaptic inputs by their distinct latencies and magnitudes, and by the presynaptic stimulus threshold for each component. Comparison of cell-attached extracellular recordings with intracellular recordings of synaptic potentials and synaptic currents indicated that straddling EPSPs are not an artifact of shunting damage caused by intracellular recording. The results also showed that in cells where a single presynaptic shock elicits multiple action potentials, the response is driven by multiple synapses and not by repetitive postsynaptic firing. The conductance of straddling synapses also provides a direct estimate of the threshold synaptic conductance (9.8 nS ± 7.6 nS, n = 22, mean ± SD). The results are discussed in terms of their implications for ganglionic integration and an existing model of synaptic amplification.

Mixed synapses reconcile violations of the size principle in zebrafish spinal cord.

Mixed electrical-chemical synapses potentially complicate electrophysiological interpretations of neuronal excitability and connectivity. Here, we disentangle the impact of mixed synapses within the spinal locomotor circuitry of larval zebrafish. We demonstrate that soma size is not linked to input resistance for interneurons, contrary to the biophysical predictions of the 'size principle' for motor neurons. Next, we show that time constants are faster, excitatory currents stronger, and mixed potentials larger in lower resistance neurons, linking mixed synapse density to resting excitability. Using a computational model, we verify the impact of weighted electrical synapses on membrane properties, synaptic integration and the low-pass filtering and distribution of coupling potentials. We conclude differences in mixed synapse density can contribute to excitability underestimations and connectivity overestimations. The contribution of mixed synaptic inputs to resting excitability helps explain 'violations' of the size principle, where neuron size, resistance and recruitment order are unrelated.

Cell-type-specific integration of feedforward and feedback synaptic inputs in the posterior parietal cortex.

The integration of feedforward (sensory) and feedback (top-down) neuronal signals is a principal function of the neocortex. Yet, we have limited insight into how these information streams are combined by individual neurons. Using a two-color optogenetic strategy, we found that layer 5 pyramidal neurons in the posterior parietal cortex receive monosynaptic dual innervation, combining sensory inputs with top-down signals. Subclasses of layer 5 pyramidal neurons integrated these synapses with distinct temporal dynamics. Specifically, regular spiking cells exhibited supralinear enhancement of delayed-but not coincident-inputs, while intrinsic burst-firing neurons selectively boosted coincident synaptic events. These subthreshold integration characteristics translated to a nonlinear summation of action potential firing. Complementing electrophysiology with computational modeling, we found that distinct integration profiles arose from a cell-type-specific interaction of ionic mechanisms and feedforward inhibition. These data provide insight into the cellular properties that guide the nonlinear interaction of distinct long-range afferents in the neocortex.

Dendritic Morphology Affects the Velocity and Amplitude of Back-propagating Action Potentials.

The back-propagating action potential (bpAP) is crucial for neuronal signal integration and synaptic plasticity in dendritic trees. Its properties (velocity and amplitude) can be affected by dendritic morphology. Due to limited spatial resolution, it has been difficult to explore the specific propagation process of bpAPs along dendrites and examine the influence of dendritic morphology, such as the dendrite diameter and branching pattern, using patch-clamp recording. By taking advantage of Optopatch, an all-optical electrophysiological method, we made detailed recordings of the real-time propagation of bpAPs in dendritic trees. We found that the velocity of bpAPs was not uniform in a single dendrite, and the bpAP velocity differed among distinct dendrites of the same neuron. The velocity of a bpAP was positively correlated with the diameter of the dendrite on which it propagated. In addition, when bpAPs passed through a dendritic branch point, their velocity decreased significantly. Similar to velocity, the amplitude of bpAPs was also positively correlated with dendritic diameter, and the attenuation patterns of bpAPs differed among different dendrites. Simulation results from neuron models with different dendritic morphology corresponded well with the experimental results. These findings indicate that the dendritic diameter and branching pattern significantly influence the properties of bpAPs. The diversity among the bpAPs recorded in different neurons was mainly due to differences in dendritic morphology. These results may inspire the construction of neuronal models to predict the propagation of bpAPs in dendrites with enormous variation in morphology, to further illuminate the role of bpAPs in neuronal communication.

Parvalbumin interneuron dendrites enhance gamma oscillations.

Dendrites are essential determinants of the input-output relationship of single neurons, but their role in network computations is not well understood. Here, we use a combination of dendritic patch-clamp recordings and in silico modeling to determine how dendrites of parvalbumin (PV)-expressing basket cells contribute to network oscillations in the gamma frequency band. Simultaneous soma-dendrite recordings from PV basket cells in the dentate gyrus reveal that the slope, or gain, of the dendritic input-output relationship is exceptionally low, thereby reducing the cell's sensitivity to changes in its input. By simulating gamma oscillations in detailed network models, we demonstrate that the low gain is key to increase spike synchrony in PV basket cell assemblies when cells are driven by spatially and temporally heterogeneous synaptic inputs. These results highlight the role of inhibitory neuron dendrites in synchronized network oscillations.

Synaptic Integration in CA1 Pyramidal Neurons Is Intact despite Deficits in GABAergic Transmission in the Scn1a Haploinsufficiency Mouse Model of Dravet Syndrome.

Mutations of SCN1A, which encodes the voltage-gated sodium channel Nav1.1, can cause epilepsy disorders such as Dravet syndrome (DS) that are comorbid with wide-ranging neurologic dysfunction. Many studies suggest that Nav1.1 haploinsufficiency causes forebrain GABAergic interneuron hypoexcitability, while pyramidal neuron physiology is mostly unaltered, and that this serves as a primary cell physiology phenotype linking mutation to disease. We hypothesized that deficits in inhibition would alter synaptic integration during activation of the hippocampal microcircuit, thus disrupting cellular information processing and leading to seizures and cognitive deficits. We tested this hypothesis using ex vivo whole-cell recordings from CA1 pyramidal neurons in a heterozygous Scn1a knock-out mouse model and wild-type (WT) littermates, measuring responses to single and patterned synaptic stimulation and spontaneous synaptic activity. Overall, our experiments reveal a surprising normalcy of excitatory and inhibitory synaptic temporal integration in the hippocampus of Scn1a haploinsufficient mice. While miniature IPSCs and feedforward inhibition and were decreased, we did not identify a pattern or frequency of input that caused a failure of synaptic inhibition. We further show that reduced GABA release probability and subsequent reduced short-term depression may act to overcome deficits in inhibition normalizing input/output functions in the Scn1a haploinsufficient hippocampus. These experiments show that CA1 pyramidal neuron synaptic processing is surprisingly robust, even during decreased interneuron function, and more complex circuit activity is likely required to reveal altered function in the hippocampal microcircuit.

Somatic Depolarization Enhances Hippocampal CA1 Dendritic Spike Propagation and Distal Input-Driven Synaptic Plasticity.

Synaptic inputs that target distal regions of neuronal dendrites can often generate local dendritic spikes that can amplify synaptic depolarization, induce synaptic plasticity, and enhance neuronal output. However, distal dendritic spikes are subject to significant attenuation by dendritic cable properties, and often produce only a weak subthreshold depolarization of the soma. Nonetheless, such spikes have been implicated in memory storage, sensory perception and place field formation. How can such a weak somatic response produce such powerful behavioral effects? Here, we use dual dendritic and somatic recordings in acute hippocampal slices of male mice to reveal that dendritic spike propagation, but not spike initiation, is strongly enhanced when the somatic resting potential is depolarized, likely as a result of increased inactivation of A-type K+ channels. Somatic depolarization also facilitates the induction of a form of dendritic spike driven heterosynaptic plasticity that enhances memory specificity. Thus, the effect of somatic membrane depolarization to enhance dendritic spike propagation and long-term synaptic plasticity is likely to play an important role in hippocampal-dependent spatial representations as well as learning and memory.SIGNIFICANCE STATEMENT Neurons receive synaptic input along their dendrites but produce action potential (AP) output at their soma. Signals arriving at the distal dendrites of pyramidal neurons (PNs) have little impact on the soma unless they combine to initiate a dendritic spike, which needs to propagate to the soma to trigger an AP. This study shows that small subthreshold depolarization of the soma powerfully enhances the propagation of dendritic spikes, through inactivation of dendritic A-type potassium channels. Enhanced dendritic spike propagation also markedly facilitates the induction of a form of plasticity driven by the distal synaptic inputs. Thus, small changes in somatic membrane potential, similar to those observed in vivo, act as a powerful gate of neuronal information transfer.

Input rate encoding and gain control in dendrites of neocortical pyramidal neurons.

Elucidating how neurons encode network activity is essential to understanding how the brain processes information. Neocortical pyramidal cells receive excitatory input onto spines distributed along dendritic branches. Local dendritic branch nonlinearities can boost the response to spatially clustered and synchronous input, but how this translates into the integration of patterns of ongoing activity remains unclear. To examine dendritic integration under naturalistic stimulus regimes, we use two-photon glutamate uncaging to repeatedly activate multiple dendritic spines at random intervals. In the proximal dendrites of two populations of layer 5 pyramidal neurons in the mouse motor cortex, spatially restricted synchrony is not a prerequisite for dendritic boosting. Branches encode afferent inputs with distinct rate sensitivities depending upon cell and branch type. Thus, inputs distributed along a dendritic branch can recruit supralinear boosting and the window of this nonlinearity may provide a mechanism by which dendrites can preferentially amplify slow-frequency network oscillations.

Dendritic Morphology of an Inhibitory Retinal Interneuron Enables Simultaneous Local and Global Synaptic Integration.

Amacrine cells, inhibitory interneurons of the retina, feature synaptic inputs and outputs in close proximity throughout their dendritic trees, making them notable exceptions to prototypical somato-dendritic integration with output transmitted via axonal action potentials. The extent of dendritic compartmentalization in amacrine cells with widely differing dendritic tree morphology, however, is largely unexplored. Combining compartmental modeling, dendritic Ca2+ imaging, targeted microiontophoresis and multielectrode patch-clamp recording (voltage and current clamp, capacitance measurement of exocytosis), we investigated integration in the AII amacrine cell, a narrow-field electrically coupled interneuron that participates in multiple, distinct microcircuits. Physiological experiments were performed with in vitro slices prepared from retinas of both male and female rats. We found that the morphology of the AII enables simultaneous local and global integration of inputs targeted to different dendritic regions. Local integration occurs within spatially restricted dendritic subunits and narrow time windows and is largely unaffected by the strength of electrical coupling. In contrast, global integration across the dendritic tree occurs over longer time periods and is markedly influenced by the strength of electrical coupling. These integrative properties enable AII amacrines to combine local control of synaptic plasticity with location-independent global integration. Dynamic inhibitory control of dendritic subunits is likely to be of general importance for amacrine cells, including cells with small dendritic trees, as well as for inhibitory interneurons in other regions of the CNS.SIGNIFICANCE STATEMENT Our understanding of synaptic integration is based on the prototypical morphology of a neuron with multiple dendrites and a single axon at opposing ends of a cell body. Many neurons, notably retinal amacrine cells, are exceptions to this arrangement, and display input and output synapses interspersed along their dendritic branches. In the large dendritic trees of some amacrine cells, such arrangements can give rise to multiple computational subunits. Other amacrine cells, with small dendritic trees, have been assumed to operate as single computational units. Here, we report the surprising result that despite a small dendritic tree, the AII amacrine cell simultaneously performs local integration of synaptic inputs (over smaller dendritic subregions) and global integration across the entire cell.

Synaptic Integration of Subquantal Neurotransmission by Colocalized G Protein-Coupled Receptors in Presynaptic Terminals.

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gßγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gßγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gßγ, but one (a GABAB receptor) inhibits Ca2+ entry whereas another (a 5-HT1B receptor) competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, whereas 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.

Binge Alcohol Drinking Alters Synaptic Processing of Executive and Emotional Information in Core Nucleus Accumbens Medium Spiny Neurons.

The nucleus accumbens (NAc) is a forebrain region mediating the positive-reinforcing properties of drugs of abuse, including alcohol. It receives glutamatergic projections from multiple forebrain and limbic regions such as the prefrontal cortex (PFCx) and basolateral amygdala (BLA), respectively. However, it is unknown how NAc medium spiny neurons (MSNs) integrate PFCx and BLA inputs, and how this integration is affected by alcohol exposure. Because progress has been hampered by the inability to independently stimulate different pathways, we implemented a dual wavelength optogenetic approach to selectively and independently stimulate PFCx and BLA NAc inputs within the same brain slice. This approach functionally demonstrates that PFCx and BLA inputs synapse onto the same MSNs where they reciprocally inhibit each other pre-synaptically in a strict time-dependent manner. In alcohol-naïve mice, this temporal gating of BLA-inputs by PFCx afferents is stronger than the reverse, revealing that MSNs prioritize high-order executive processes information from the PFCx. Importantly, binge alcohol drinking alters this reciprocal inhibition by unilaterally strengthening BLA inhibition of PFCx inputs. In line with this observation, we demonstrate that in vivo optogenetic stimulation of the BLA, but not PFCx, blocks binge alcohol drinking escalation in mice. Overall, our results identify NAc MSNs as a key integrator of executive and emotional information and show that this integration is dysregulated during binge alcohol drinking.