Empagliflozin attenuates arrhythmogenesis in diabetic cardiomyopathy by normalizing intracellular Ca2+ handling in ventricular cardiomyocytes.

Diabetic cardiomyopathy has been reported to increase the risk of fatal ventricular arrhythmia. The beneficial effects of the selective sodium-glucose cotransporter-2 inhibitor have not been fully examined in the context of antiarrhythmic therapy, especially its direct cardioprotective effects despite the negligible SGLT2 expression in cardiomyocytes. We aimed to examine the antiarrhythmic effects of empagliflozin (EMPA) treatment on diabetic cardiomyocytes, with a special focus on Ca2+ handling. We conducted echocardiography and hemodynamic studies and studied electrophysiology, Ca2+ handling, and protein expression in C57BLKS/J-leprdb/db mice (db/db mice) and their nondiabetic lean heterozygous Leprdb/+ littermates (db/+ mice). Preserved systolic function with diastolic dysfunction was observed in 16-wk-old db/db mice. During arrhythmia induction, db/db mice had significantly increased premature ventricular complexes (PVCs) than controls, which was attenuated by EMPA. In protein expression analyses, calmodulin-dependent protein kinase II (CaMKII) Thr287 autophosphorylation and CaMKII-dependent RyR2 phosphorylation (S2814) were significantly increased in diabetic hearts, which were inhibited by EMPA. In addition, global O-GlcNAcylation significantly decreased with EMPA treatment. Furthermore, EMPA significantly inhibited ventricular cardiomyocyte glucose uptake. Diabetic cardiomyocytes exhibited increased spontaneous Ca2+ events and decreased sarcoplasmic reticulum (SR) Ca2+ content, along with impaired Ca2+ transient, all of which normalized with EMPA treatment. Notably, most EMPA-induced improvements in Ca2+ handling were abolished by the addition of an O-GlcNAcase (OGA) inhibitor. In conclusion, EMPA attenuated ventricular arrhythmia inducibility by normalizing the intracellular Ca2+ handling, and we speculated that this effect was, at least partly, due to the inhibition of O-GlcNAcylation via the suppression of glucose uptake into cardiomyocytes.NEW & NOTEWORTHY SGLT2is are known to improve cardiovascular outcomes regardless of the presence of diabetes and decrease traditional cardiovascular risk factors. We demonstrated, for the first time, that EMPA inhibited PVCs by normalizing Ca2+ handling in diabetic mice. Our data suggest that the effects of SGLT2is on calcium handling may occur because of suppression of O-GlcNAcylation through inhibition of glucose uptake and not because of NHE inhibition, as previously suggested.

Acetylcholine from the nucleus basalis magnocellularis facilitates the retrieval of well-established memory.

Retrieval deficit of long-term memory is a cardinal symptom of dementia and has been proposed to associate with abnormalities in the central cholinergic system. Difficulty in the retrieval of memory is experienced by healthy individuals and not limited to patients with neurological disorders that result in forgetfulness. The difficulty of retrieving memories is associated with various factors, such as how often the event was experienced or remembered, but it is unclear how the cholinergic system plays a role in the retrieval of memory formed by a daily routine (accumulated experience). To investigate this point, we trained rats moderately (for a week) or extensively (for a month) to detect a visual cue in a two-alternative forced-choice task. First, we confirmed the well-established memory in the extensively trained group was more resistant to the retrieval problem than recently acquired memory in the moderately trained group. Next, we tested the effect of a cholinesterase inhibitor, donepezil, on the retrieval of memory after a long no-task period in extensively trained rats. Pre-administration of donepezil improved performance and reduced the latency of task initiation compared to the saline-treated group. Finally, we lesioned cholinergic neurons of the nucleus basalis magnocellularis (NBM), which project to the entire neocortex, by injecting the cholinergic toxin 192 IgG-saporin. NBM-lesioned rats showed severely impaired task initiation and performance. These abilities recovered as the trials progressed, though they never reached the level observed in rats with intact NBM. These results suggest that acetylcholine released from the NBM contributes to the retrieval of well-established memory developed by a daily routine.

Perineuronal Nets in the Deep Cerebellar Nuclei Regulate GABAergic Transmission and Delay Eyeblink Conditioning.

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.

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.

Pre- and Postembedding Immunoelectron Microscopy to Analyze the Central Nervous System.

Immunocytochemistry, a method of delineating the subcellular localization of target proteins, was developed from immunohistochemistry. In principle, proteins are labeled using an antigen-antibody reaction. In order to observe under an electron microscope, the reaction product must scatter the electron beam with sufficient contrast while it is necessary to have an amplifying label that can withstand the observation. We have some detailed tips on making electron microscope samples to achieve this objective, and we would be happy to help you.

Volume electron microscopy for genetically and molecularly defined neural circuits.

The brain networks responsible for adaptive behavioral changes are based on the physical connections between neurons. Light and electron microscopy have long been used to study neural projections and the physical connections between neurons. Volume electron microscopy has recently expanded its scale of analysis due to methodological advances, resulting in complete wiring maps of neurites in a large volume of brain tissues and even entire nervous systems in a growing number of species. However, structural approaches frequently suffer from inherent limitations in which elements in images are identified solely by morphological criteria. Recently, an increasing number of tools and technologies have been developed to characterize cells and cellular components in the context of molecules and gene expression. These advancements include newly developed probes for visualization in electron microscopic images as well as correlative integration methods for the same elements across multiple microscopic modalities. Such approaches advance our understanding of interactions between specific neurons and circuits and may help to elucidate novel aspects of the basal ganglia network involving dopamine neurons. These advancements are expected to reveal mechanisms for processing adaptive changes in specific neural circuits that modulate brain functions.

Globus pallidus is not independent from striatal direct pathway neurons: an up-to-date review.

Striatal projection neurons, which are classified into two groups-direct and indirect pathway neurons, play a pivotal role in our understanding of the brain's functionality. Conventional models propose that these two pathways operate independently and have contrasting functions, akin to an "accelerator" and "brake" in a vehicle. This analogy further elucidates how the depletion of dopamine neurons in Parkinson's disease can result in bradykinesia. However, the question arises: are these direct and indirect pathways truly autonomous? Despite being distinct types of neurons, their interdependence cannot be overlooked. Single-neuron tracing studies employing membrane-targeting signals have shown that the majority of direct pathway neurons terminate not only in the output nuclei, but also in the external segment of the globus pallidus (GP in rodents), a relay nucleus of the indirect pathway. Recent studies have unveiled the existence of arkypallidal neurons, which project solely to the striatum, in addition to prototypic neurons. This raises the question of which type of GP neurons receive these striatal axon collaterals. Our morphological and electrophysiological experiments showed that the striatal direct pathway neurons may affect prototypic neurons via the action of substance P on neurokinin-1 receptors. Conversely, another research group has reported that direct pathway neurons inhibit arkypallidal neurons via GABA. Regardless of the neurotransmitter involved, it can be concluded that the GP is not entirely independent of direct pathway neurons. This review article underscores the intricate interplay between different neuronal pathways and challenges the traditional understanding of their independence.

Anterograde trans-neuronal labeling of striatal interneurons in relation to dopamine neurons in the substantia nigra pars compacta.

Recent advances in neural tracing have unveiled numerous neural circuits characterized by brain region and cell type specificity, illuminating the underpinnings of specific functions and behaviors. Dopaminergic (DA) neurons in the midbrain are highly heterogeneous in terms of gene and protein expression and axonal projections. Different cell types within the substantia nigra pars compacta (SNc) tend to project to the striatum in a cell-type-dependent manner characterized by specific topography. Given the wide and dense distribution of DA axons, coupled with a combination of synaptic and volume transmission, it remains unclear how DA release is spatially and temporally regulated, to appropriately achieve specific behaviors and functions. Our hypothesis posits that hidden rules governing synapse formation between pre-synaptic DA neuron types and striatal neuron types may modulate the effect of DA at a single-cell level. To address this conjecture, we employed adeno-associated virus serotype 1 (AAV1) to visualize the neural circuitry of DA neurons. AAV1 has emerged as a potent anatomical instrument capable of labeling and visualizing pre- and post-synaptic neurons simultaneously through anterograde trans-synaptic labeling. First, AAV1-Cre was injected into the SNc, resulting in Cre expression in both medium spiny neurons and interneurons in the striatum. Due to the potential occurrence of the retrograde transfer of AAV1, only striatal interneurons were considered for trans-synaptic or trans-neuronal labeling. Interneuron types expressing parvalbumin, choline acetyltransferase, somatostatin, or nitrogen oxide synthase exhibited Cre expression. Using a combination of AAV1-Cre and Cre-driven fluorophore expressing AAVs, striatal interneurons and the axons originating from the SNc were visualized in distinct colors. Using immunofluorescence against neurotransmitter transporters, almost all axons in the striatum visualized using this approach were confirmed to be dopaminergic. Moreover, individual DA axons established multiple appositions on the somata and proximal dendrites of interneurons. This finding suggests that irrespective of the extensive and widespread axonal arborization of DA neurons, a particular DA neuron may exert a significant influence on specific interneurons. Thus, AAV1-based labeling of the DA system can be a valuable tool to uncover the concealed rules governing these intricate relationships.

descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

Specialized cortical subnetworks differentially connect frontal cortex to parahippocampal areas.

How information is manipulated and segregated within local circuits in the frontal cortex remains mysterious, in part because of inadequate knowledge regarding the connectivity of diverse pyramidal cell subtypes. The frontal cortex participates in the formation and retrieval of declarative memories through projections to the perirhinal cortex, and in procedural learning through projections to the striatum/pontine nuclei. In rat frontal cortex, we identified two pyramidal cell subtypes selectively projecting to distinct subregions of perirhinal cortex (PRC). PRC-projecting cells in upper layer 2/3 (L2/3) of the frontal cortex projected to perirhinal area 35, while neurons in L5 innervated perirhinal area 36. L2/3 PRC-projecting cells partially overlapped with those projecting to the basolateral amygdala. L5 PRC-projecting cells partially overlapped with crossed corticostriatal cells, but were distinct from neighboring corticothalamic (CTh)/corticopontine cells. L5 PRC-projecting and CTh cells were different in their electrophysiological properties and dendritic/axonal morphologies. Within the frontal cortex, L2/3 PRC-projecting cells innervated L5 PRC-projecting and CTh cells with similar probabilities, but received feedback excitation only from PRC-projecting cells. These data suggest that specific neuron subtypes in different cortical layers are reciprocally excited via interlaminar loops. Thus, two interacting output channels send information from the frontal cortex to different hierarchical stages of the parahippocampal network, areas 35 and 36, with additional collaterals selectively targeting the amygdala or basal ganglia, respectively. Combined with the hierarchical connectivity of PRC-projecting and CTh cells, these observations demonstrate an exquisite diversification of frontal projection neurons selectively connected according to their participation in distinct memory subsystems.

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Axon topography of layer IV spiny cells to orientation map in the cat primary visual cortex (area 18).

Our aim was to reveal the relationship between layer IV horizontal connections and the functional architecture of the cat primary visual cortex because these connections play important roles in the first cortical stage of visual signals integration. We investigated bouton distribution of spiny neurons over an orientation preference map using in vivo optical imaging, unit recordings, and single neuron reconstructions. The radial extent of reconstructed axons (14 star pyramidal and 9 spiny stellate cells) was ~1.5 mm. In the vicinity of the parent somata (<400 µm), boutons occupied chiefly iso-orientations, however, more distally, 7 cells projected preferentially to non-iso-orientations. Boutons of each cell were partitioned into 1-15 distinct clusters based on the mean-shift algorithm, of which 57 clusters preferred iso-orientations and 43 clusters preferred cross-orientations, each showing sharp orientation preference "tuning." However, unlike layer III/V pyramidal cells preferring chiefly iso-orientations, layer IV cells were engaged with broad orientations because each bouton cluster from the same cell could show different orientation preference. These results indicate that the circuitry of layer IV spiny cells is organized differently from that of iso-orientation dominant layer III/V cells and probably processes visual signals in a different manner from that of the superficial and deeper layers.

Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons.

Whether neocortical γ-aminobutyric acid (GABA) cells are composed of a limited number of distinct classes of neuron, or whether they are continuously differentiated with much higher diversity, remains a contentious issue for the field. Most GABA cells of rat frontal cortex have at least 1 of 6 chemical markers (parvalbumin, calretinin, alpha-actinin-2, somatostatin, vasoactive intestinal polypeptide, and cholecystokinin), with each chemical class comprising several distinct neuronal subtypes having specific physiological and morphological characteristics. To better clarify GABAergic neuron diversity, we assessed the colocalization of these 6 chemical markers with corticotropin-releasing factor (CRF), neuropeptide Y (NPY), the substance P receptor (SPR), and nitric oxide synthase (NOS); these 4 additional chemical markers suggested to be expressed diversely or specifically among cortical GABA cells. We further correlated morphological and physiological characteristics of identified some chemical subclasses of inhibitory neurons. Our results reveal expression specificity of CRF, NPY, SPR, and NOS in morphologically and physiologically distinct interneuron classes. These observations support the existence of a limited number of functionally distinct subtypes of GABA cells in the neocortex.

Conservation of the Direct and Indirect Pathway Dichotomy in Mouse Caudal Striatum With Uneven Distribution of Dopamine Receptor D1- and D2-Expressing Neurons.

The striatum is one of the key nuclei for adequate control of voluntary behaviors and reinforcement learning. Two striatal projection neuron types, expressing either dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R) constitute two independent output routes: the direct or indirect pathways, respectively. These pathways co-work in balance to achieve coordinated behavior. Two projection neuron types are equivalently intermingled in most striatal space. However, recent studies revealed two atypical zones in the caudal striatum: the zone in which D1R-neurons are the minor population (D1R-poor zone) and that in which D2R-neurons are the minority (D2R-poor zone). It remains obscure as to whether these imbalanced zones have similar properties on axonal projections and electrophysiology compared to other striatal regions. Based on morphological experiments in mice using immunofluorescence, in situ hybridization, and neural tracing, here, we revealed that the poor zones densely projected to the globus pallidus and substantia nigra pars lateralis, with a few collaterals in substantia nigra pars reticulata and compacta. Similar to that in other striatal regions, D1R-neurons were the direct pathway neurons. We also showed that the membrane properties of projection neurons in the poor zones were largely similar to those in the conventional striatum using in vitro electrophysiological recording. In addition, the poor zones existed irrespective of the age or sex of mice. We also identified the poor zones in the common marmoset as well as other rodents. These results suggest that the poor zones in the caudal striatum follow the conventional projection patterns irrespective of the imbalanced distribution of projection neurons. The poor zones could be an innate structure and common in mammals. The unique striatal zones possessing highly restricted projections could relate to functions different from those of motor-related striatum.

Modulatory Effects of Monoamines and Perineuronal Nets on Output of Cerebellar Purkinje Cells.

Classically, the cerebellum has been thought to play a significant role in motor coordination. However, a growing body of evidence for novel neural connections between the cerebellum and various brain regions indicates that the cerebellum also contributes to other brain functions implicated in reward, language, and social behavior. Cerebellar Purkinje cells (PCs) make inhibitory GABAergic synapses with their target neurons: other PCs and Lugaro/globular cells via PC axon collaterals, and neurons in the deep cerebellar nuclei (DCN) via PC primary axons. PC-Lugaro/globular cell connections form a cerebellar cortical microcircuit, which is driven by serotonin and noradrenaline. PCs' primary outputs control not only firing but also synaptic plasticity of DCN neurons following the integration of excitatory and inhibitory inputs in the cerebellar cortex. Thus, strong PC-mediated inhibition is involved in cerebellar functions as a key regulator of cerebellar neural networks. In this review, we focus on physiological characteristics of GABAergic transmission from PCs. First, we introduce monoaminergic modulation of GABAergic transmission at synapses of PC-Lugaro/globular cell as well as PC-large glutamatergic DCN neuron, and a Lugaro/globular cell-incorporated microcircuit. Second, we review the physiological roles of perineuronal nets (PNNs), which are organized components of the extracellular matrix and enwrap the cell bodies and proximal processes, in GABA release from PCs to large glutamatergic DCN neurons and in cerebellar motor learning. Recent evidence suggests that alterations in PNN density in the DCN can regulate cerebellar functions.

Quantitative chemical composition of cortical GABAergic neurons revealed in transgenic venus-expressing rats.

Although neocortical GABAergic (gamma-aminobutyric acidergic) interneurons have been the focus of intense study, especially in the rat, a consensus view of the functional diversity and organization of inhibitory cortical neurons has not yet been achieved. To better analyze GABAergic neurons in the rat, we used a bacterial artificial chromosome (BAC) construct and established 2 lines of transgenic rats that coexpress Venus, a yellow fluorescent protein, with the vesicular GABA transporter. The brain GABA content from both transgenic lines was similar to the level found in wild-type rats. In the frontal cortex, Venus was expressed in >95% of GABAergic neurons, most of which also expressed at least one of 6 biochemical markers, including alpha-actitin-2, which preferentially labeled late-spiking neurogliaform cells. Taking advantage of the fact that Venus expression allows for targeted recording from all classes of nonpyramidal cells, irrespective of their somatic morphologies, we demonstrated that fast-spiking neurons, which were heterogeneous in somatic size as well as vertical dendritic projection, had relatively uniform horizontal dimensions, suggesting a cell type-specific columnar input territory. Our data demonstrate the benefits of VGAT-Venus rats for investigating GABAergic circuits, as well as the feasibility of using BAC technology in rats to label subsets of specific, genetically defined neurons.

Thalamostriatal projections and striosome-matrix compartments.

The neostriatum has a mosaic organization consisting of striosome and matrix compartments. It receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Recent behavioral studies in rats revealed a selectively active medial prefronto-striosomal circuit during cost-benefit decision-making. However, clarifying the input/output organization of striatal compartments has been difficult because of its complex structure. We recently demonstrated that the source of thalamostriatal projections are highly organized in striatal compartments. This finding indicated that the functional properties of striatal compartments are influenced by their cortical and thalamic afferents, presumably with different time latencies. In addition, these afferents likely support the unique dynamics of striosome and matrix compartments. In this manuscript, we review the anatomy of basal ganglia networks with regard to striosome/matrix structure. We place specific focus on thalamostriatal projections at the population and single neuron level.

Motor cortex can directly drive the globus pallidus neurons in a projection neuron type-dependent manner in the rat.

The basal ganglia are critical for the control of motor behaviors and for reinforcement learning. Here, we demonstrate in rats that primary and secondary motor areas (M1 and M2) make functional synaptic connections in the globus pallidus (GP), not usually thought of as an input site of the basal ganglia. Morphological observation revealed that the density of axonal boutons from motor cortices in the GP was 47% and 78% of that in the subthalamic nucleus (STN) from M1 and M2, respectively. Cortical excitation of GP neurons was comparable to that of STN neurons in slice preparations. FoxP2-expressing arkypallidal neurons were preferentially innervated by the motor cortex. The connection probability of cortico-pallidal innervation was higher for M2 than M1. These results suggest that cortico-pallidal innervation is an additional excitatory input to the basal ganglia, and that it can affect behaviors via the cortex-basal ganglia-thalamus motor loop.

Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents.

Fast inhibition in the cortex is gated primarily at GABAergic synapses formed by local interneurons onto postsynaptic targets. Although GABAergic inputs to the somata and axon initial segments of neocortical pyramidal neurons are associated with direct inhibition of action potential generation, the role of GABAergic inputs to distal dendritic segments, including spines, is less well characterized. Because a significant proportion of inhibitory input occurs on distal dendrites and spines, it will be important to determine whether these GABAergic synapses are formed selectively by certain classes of presynaptic cells onto specific postsynaptic elements. By electron microscopic observations of synapses formed by different subtypes of nonpyramidal cells, we found that a surprisingly large fraction (33.4 +/- 9.3%) of terminals formed symmetrical synaptic junctions onto a subset of cortical spines that were mostly coinnervated by an asymmetrical terminal. Using VGLUT1 and VGLUT2 isoform of the glutamate vesicular transporter immunohistochemistry, we found that the double-innervated spines selectively received thalamocortical afferents expressing the VGLUT2 but almost never intracortical inputs expressing the VGLUT1. When comparing the volumes of differentially innervated spines and their synaptic junction areas, we found that spines innervated by VGLUT2-positive terminal were significantly larger than spines innervated by VGLUT1-positive terminal and that these spines had larger, and more often perforated, synapses than those of spines innervated by VGLUT1-positive afferent. These results demonstrate that inhibitory inputs to pyramidal cell spines may preferentially reduce thalamocortical rather than intracortical synaptic transmission and are therefore positioned to selectively gate extracortical information.

Supramammillary Nucleus Afferents to the Dentate Gyrus Co-release Glutamate and GABA and Potentiate Granule Cell Output.

The supramammillary nucleus (SuM) of the hypothalamus projects to the dentate gyrus (DG) and the CA2 region of the hippocampus. Although the SuM-to-hippocampus circuits have been implicated in spatial and emotional memory formation, little is known about precise neural connections between the SuM and hippocampus. Here, we report that axons of SuM neurons make monosynaptic connections to granule cells (GCs) and GABAergic interneurons, but not to hilar mossy cells, in the DG and co-release glutamate and γ-aminobutyric acid (GABA) at these synapses. Although inputs from the SuM can excite some interneurons, the inputs alone fail to generate spikes in GCs. However, despite the insufficient excitatory drive and GABAergic co-transmission, SuM inputs have net excitatory effects on GCs and can potentiate GC firing when temporally associated with perforant path inputs. Our results indicate that the SuM influences DG information processing by modulating GC outputs.