Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.

In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.

The reeler mouse mutant has long served as a primary model to study the development of cortical layers, which is governed by the extracellular glycoprotein reelin secreted by Cajal-Retzius cells. Because layers organize local and long-range circuits for sensory processing, we investigated whether intracortical connectivity is compromised by reelin deficiency in this model. We generated a transgenic reeler mutant (we used both sexes), in which layer 4-fated spiny stellate neurons are labeled with tdTomato and applied slice electrophysiology and immunohistochemistry with synaptotagmin-2 to study the circuitry between the major thalamorecipient cell types, namely excitatory spiny stellate and inhibitory fast-spiking (putative basket) cells. In the reeler mouse, spiny stellate cells are clustered into barrel equivalents. In these clusters, we found that intrinsic physiology, connectivity, and morphology of spiny stellate and fast-spiking, putative basket cells does not significantly differ between reeler and controls. Properties of unitary connections, including connection probability, were very comparable in excitatory cell pairs and spiny stellate/fast-spiking cell pairs, suggesting an intact excitation-inhibition balance at the first stage of cortical sensory information processing. Together with previous findings, this suggests that thalamorecipient circuitry in the barrel cortex develops and functions independently of proper cortical lamination and postnatal reelin signaling.

Selective distribution of proteins in presynaptic active zones (AZs) is a prerequisite for generating postsynaptic target cell type-specific differences in presynaptic vesicle release probability (Pv) and short-term plasticity, a characteristic feature of cortical pyramidal cells (PCs). In the hippocampus of rodents, somatostatin and mGluR1α expressing interneurons (mGluR1α+ INs) receive small, facilitating excitatory postsynaptic currents (EPSCs) from PCs and express Elfn1 that trans-synaptically recruits mGluR7 into the presynaptic AZ of PC axons. Here we show that Elfn1 also has a role in the selective recruitment of Munc13-2, a synaptic vesicle priming and docking protein, to PC AZs that innervate mGluR1α+ INs. In Elfn1 knock-out mice, unitary EPSCs (uEPSCs) in mGluR1α+ INs have threefold larger amplitudes with less pronounced short-term facilitation, which might be the consequence of the loss of either mGluR7 or Munc13-2 or both. Conditional genetic deletion of Munc13-2 from CA1 PCs results in the loss of Munc13-2, but not mGluR7 from the AZs, and has no effect on the amplitude of uEPSCs and leaves the characteristic short-term facilitation intact at PC to mGluR1α+ IN connection. Our results demonstrate that Munc13-1 alone is capable of imposing low Pv at PC to mGluR1α+ IN synapses and Munc13-2 has yet an unknown role in this synapse.

Astrocytes and oligodendrocytes are main players in the brain to ensure ion and neurotransmitter homeostasis, metabolic supply, and fast action potential propagation in axons. These functions are fostered by the formation of large syncytia in which mainly astrocytes and oligodendrocytes are directly coupled. Panglial networks constitute on connexin-based gap junctions in the membranes of neighboring cells that allow the passage of ions, metabolites, and currents. However, these networks are not uniform but exhibit a brain region-dependent heterogeneous connectivity influencing electrical communication and intercellular ion spread. Here, we describe different approaches to analyze gap junctional communication in acute tissue slices that can be implemented easily in most electrophysiology and imaging laboratories. These approaches include paired recordings, determination of syncytial isopotentiality, tracer coupling followed by analysis of network topography, and wide field imaging of ion sensitive dyes. These approaches are capable to reveal cellular heterogeneity causing electrical isolation of functional circuits, reduced ion-transfer between different cell types, and anisotropy of tracer coupling. With a selective or combinatory use of these methods, the results will shed light on cellular properties of glial cells and their contribution to neuronal function.

Long-term synaptic plasticity is widely believed to underlie learning and memory in the brain. Whether plasticity is primarily expressed pre- or postsynaptically has been the subject of considerable debate for many decades. More recently, it is generally agreed that the locus of plasticity depends on a number of factors, such as developmental stage, induction protocol, and synapse type. Since presynaptic expression alters not just the gain but also the short-term dynamics of a synapse, whereas postsynaptic expression only modifies the gain, the locus has fundamental implications for circuits dynamics and computations in the brain. It therefore remains crucial for our understanding of neuronal circuits to know the locus of expression of long-term plasticity. One classical method for elucidating whether plasticity is pre- or postsynaptically expressed is based on analysis of the coefficient of variation (CV), which serves as a measure of noise levels of synaptic neurotransmission. Here, we provide a practical guide to using CV analysis for the purposes of exploring the locus of expression of long-term plasticity, primarily aimed at beginners in the field. We provide relatively simple intuitive background to an otherwise theoretically complex approach as well as simple mathematical derivations for key parametric relationships. We list important pitfalls of the method, accompanied by accessible computer simulations to better illustrate the problems (downloadable from GitHub), and we provide straightforward solutions for these issues.

Synaptic transmission between neurons is the basic mechanism for information processing in cortical microcircuits. To date, paired recording from synaptically coupled neurons is the most widely used method which allows a detailed functional characterization of unitary synaptic transmission at the cellular and synaptic level in combination with a structural characterization of both pre- and postsynaptic neurons at the light and electron microscopic level. In this review, we will summarize the many applications of paired recordings to investigate synaptic function and structure. Paired recordings have been used to study the detailed electrophysiological and anatomical properties of synaptically coupled cell pairs within a synaptic microcircuit; this is critical in order to understand the connectivity rules and dynamic properties of synaptic transmission. Paired recordings can also be adopted for quantal analysis of an identified synaptic connection and to study the regulation of synaptic transmission by neuromodulators such as acetylcholine, the monoamines, neuropeptides, and adenosine etc. Taken together, paired recordings from synaptically coupled neurons will remain a very useful approach for a detailed characterization of synaptic transmission not only in the rodent brain but also that of other species including humans.

Striatal output pathways are known to play a crucial role in the control of movement. One possible component for shaping the synaptic output of striatal neuron is the glutamatergic input that originates from cortex and thalamus. Although reports focusing on quantifying glutamatergic-induced morphological changes in striatum exist, the role of glutamatergic input in regulating striatal function remains poorly understood. Using primary neurons from newborn mice of either sex in a reduced two-neuron microcircuit culture system, we examined whether glutamatergic input modulates the output of striatal neurons. We found that glutamatergic input enhanced striatal inhibition in vitro With a glutamatergic partner from either cortex or thalamus, we attributed this potentiation to an increase in the size of quantal IPSC, suggesting a strengthening of the postsynaptic response to GABAergic signaling. Additionally, a differential effect of cortical and thalamic innervation onto striatal GABAergic neurons output was revealed. We observed that cortical, but not thalamic input, enhanced the number of releasable GABAergic synaptic vesicles and morphological synapses. Importantly, these alterations were reverted by blockade of neuronal activity and glutamate receptors, as well as disruption of BDNF-TrkB signaling. Together, our data indicate, for first time, that GABAergic synapse formation in corticostriatal pairs depends on two parallel, but potentially intersecting, signaling pathways that involve glutamate receptor activation in striatal neurons, as well as BDNF signaling. Understanding how cortical and thalamic inputs refine striatal output will pave the way toward dissecting basal ganglia activity in both physiological and pathological conditions.SIGNIFICANCE STATEMENT Striatal GABAergic microcircuits are critical for motor function. However, the mechanisms controlling striatal output, particularly at the level of synaptic strength, are unclear. Using two-neuron culture system, we quantified the synaptic output of individual striatal GABAergic neurons paired with a glutamatergic partner and studied the influence of the excitatory connections that are known to be interregionally formed in vivo We found that glutamatergic input potentiated striatal inhibitory output, potentially involving an increased feedback and/or feedforward inhibition. Moreover, distinct components of glutamatergic innervation, such as firing activity or release of neurotrophic factors were shown to be required for the glutamatergic-induced phenotype. Investigation, therefore, of two-neuron in vitro microcircuits could be a powerful tool to explore synaptic mechanisms or disease pathophysiology.

Cortical computations rely on functionally diverse and highly dynamic synapses. How their structural composition affects synaptic transmission and plasticity and whether they support functional diversity remains rather unclear. Here, synaptic boutons on layer 5B (L5B) pyramidal neurons in the adult rat barrel cortex were investigated. Simultaneous patch-clamp recordings from synaptically connected L5B pyramidal neurons revealed great heterogeneity in amplitudes, coefficients of variation (CVs), and failures (F%) of EPSPs. Quantal analysis indicated multivesicular release as a likely source of this variability. Trains of EPSPs decayed with fast and slow time constants, presumably representing release from small readily releasable (RRP; 5.40 ± 1.24 synaptic vesicles) and large recycling (RP; 74 ± 21 synaptic vesicles) pools that were independent and highly variable at individual synaptic contacts (RRP range 1.2-12.8 synaptic vesicles; RP range 3.4-204 synaptic vesicles). Most presynaptic boutons (~85%) had a single, often perforated active zone (AZ) with a ~2 to 5-fold larger pre- (0.29 ± 0.19 μm2) and postsynaptic density (0.31 ± 0.21 μm2) when compared with even larger CNS synaptic boutons. They contained 200-3400 vesicles (mean ~800). At the AZ, ~4 and ~12 vesicles were located within a perimeter of 10 and 20 nm, reflecting docked and readily releasable vesicles of a putative RRP. Vesicles (~160) at 60-200 nm constituting the structural estimate of the presumed RP were ~2-fold larger than our functional estimate of the RP although both with a high variability. The remaining constituted a presumed large resting pool. Multivariate analysis revealed two clusters of L5B synaptic boutons distinguished by the size of their resting pool. Our functional and ultrastructural analyses closely link stationary properties, temporal dynamics and endurance of synaptic transmission to vesicular content and distribution within the presynaptic boutons suggesting that functional diversity of L5B synapses is enhanced by their structural heterogeneity.

The olfactory bulb contains excitatory principal cells (mitral and tufted cells) that project to cortical targets as well as inhibitory interneurons. How the local circuitry in this region facilitates odor-specific output is not known, but previous work suggests that GABAergic granule cells plays an important role, especially during fine odor discrimination. Principal cells interact with granule cells through reciprocal dendrodendritic connections that are poorly understood. While many studies examined the GABAergic output side of these reciprocal connections, little is known about how granule cells are excited. Only two previous studies reported monosynaptically coupled mitral/granule cell connections and neither attempted to determine the fundamental properties of these synapses. Using dual intracellular recordings and a custom-built loose-patch amplifier, we have recorded unitary granule cell EPSPs evoked in response to mitral cell action potentials in rat (both sexes) brain slices. We find that the unitary dendrodendritic input is relatively weak with highly variable release probability and short-term depression. In contrast with the weak dendrodendritic input, the facilitating cortical input to granule cells is more powerful and less variable. Our computational simulations suggest that dendrodendritic synaptic properties prevent individual principal cells from strongly depolarizing granule cells, which likely discharge in response to either concerted activity among a large proportion of inputs or coactivation of a smaller subset of local dendrodendritic inputs with coincidence excitation from olfactory cortex. This dual-pathway requirement likely enables the sparse mitral/granule cell interconnections to develop highly odor-specific responses that facilitate fine olfactory discrimination.SIGNIFICANCE STATEMENT The olfactory bulb plays a central role in converting broad, highly overlapping, sensory input patterns into odor-selective population responses. How this occurs is not known, but experimental and theoretical studies suggest that local inhibition often plays a central role. Very little is known about how the most common local interneuron subtype, the granule cell, is excited during odor processing beyond the unusual anatomical arraignment of the interconnections (reciprocal dendrodendritic synapses). Using paired recordings and two-photon imaging, we determined the properties of the primary input to granule cells for the first time and show that these connections bias interneurons to fire in response to spiking in large populations of principal cells rather than a small group of highly active cells.