Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Lifelong absence of microglia alters hippocampal glutamatergic networks but not synapse and spine density.

Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1rΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r+/+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1rΔFIRE/ΔFIRE mice. Notably, these changes in Csf1rΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms.

Reduction of cortical parvalbumin-expressing GABAergic interneurons in a rodent hyperoxia model of preterm birth brain injury with deficits in social behavior and cognition.

The inhibitory GABAergic system in the brain is involved in the etiology of various psychiatric problems, including autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and others. These disorders are influenced not only by genetic but also by environmental factors, such as preterm birth, although the underlying mechanisms are not known. In a translational hyperoxia model, exposing mice pups at P5 to 80% oxygen for 48 h to mimic a steep rise of oxygen exposure caused by preterm birth from in utero into room air, we documented a persistent reduction of cortical mature parvalbumin-expressing interneurons until adulthood. Developmental delay of cortical myelin was observed, together with decreased expression of oligodendroglial glial cell-derived neurotrophic factor (GDNF), a factor involved in interneuronal development. Electrophysiological and morphological properties of remaining interneurons were unaffected. Behavioral deficits were observed for social interaction, learning and attention. These results demonstrate that neonatal oxidative stress can lead to decreased interneuron density and to psychiatric symptoms. The obtained cortical myelin deficit and decreased oligodendroglial GDNF expression indicate that an impaired oligodendroglial-interneuronal interplay contributes to interneuronal damage.

A novel giant non-cholinergic striatal interneuron restricted to the ventrolateral striatum coexpresses Kv3.3 potassium channel, parvalbumin, and the vesicular GABA transporter.

The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.

Interneuron diversity in the rat dentate gyrus: An unbiased in vitro classification.

Information processing in cortical circuits, including the hippocampus, relies on the dynamic control of neuronal activity by GABAergic interneurons (INs). INs form a heterogenous population with defined types displaying distinct morphological, molecular, and physiological characteristics. In the major input region of the hippocampus, the dentate gyrus (DG), a number of IN types have been described which provide synaptic inhibition to distinct compartments of excitatory principal cells (PrCs) and other INs. In this study, we perform an unbiased classification of GABAergic INs in the DG by combining in vitro whole-cell patch-clamp recordings, intracellular labeling, morphological analysis, and unsupervised cluster analysis to better define IN type diversity in this region. This analysis reveals that DG INs divide into at least 13 distinct morpho-physiological types which reflect the complexity of the local IN network and serve as a basis for further network analyses.

Cell-Type Specific Inhibition Controls the High-Frequency Oscillations in the Medial Entorhinal Cortex.

The medial entorhinal cortex (mEC) plays a critical role for spatial navigation and memory. While many studies have investigated the principal neurons within the entorhinal cortex, much less is known about the inhibitory circuitries within this structure. Here, we describe for the first time in the mEC a subset of parvalbumin-positive (PV+) interneurons (INs)-stuttering cells (STUT)-with morphological, intrinsic electrophysiological, and synaptic properties distinct from fast-spiking PV+ INs. In contrast to the fast-spiking PV+ INs, the axon of the STUT INs also terminated in layer 3 and showed subthreshold membrane oscillations at gamma frequencies. Whereas the synaptic output of the STUT INs was only weakly reduced by a µ-opioid agonist, their inhibitory inputs were strongly suppressed. Given these properties, STUT are ideally suited to entrain gamma activity in the pyramidal cell population of the mEC. We propose that activation of the µ-opioid receptors decreases the GABA release from the PV+ INs onto the STUT, resulting in disinhibition of the STUT cell population and the consequent increase in network gamma power. We therefore suggest that the opioid system plays a critical role, mediated by STUT INs, in the neural signaling and oscillatory network activity within the mEC.

Reciprocal Homer1a and Homer2 Isoform Expression Is a Key Mechanism for Muscle Soleus Atrophy in Spaceflown Mice.

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-form was downregulated in SOL muscle in both models. Homer immunofluorescence intensity analysis at the NMJ of BF and HU animals showed comparable outcomes in SOL but not in the extensor digitorum longus (EDL) muscle. Reduced Homer crosslinking at the NMJ consequent to increased Homer1a and/or reduced Homer2 may contribute to muscle-type specific atrophy resulting from microgravity and HU disuse suggesting mutual mechanisms.

Connectivity and Dynamics Underlying Synaptic Control of the Subthalamic Nucleus.

Adaptive motor control critically depends on the interconnected nuclei of the basal ganglia in the CNS. A pivotal element of the basal ganglia is the subthalamic nucleus (STN), which serves as a therapeutic target for deep brain stimulation (DBS) in movement disorders, such as Parkinson's disease. The functional connectivity of the STN at the microcircuit level, however, still requires rigorous investigation. Here we combine multiple simultaneous whole-cell recordings with extracellular stimulation and post hoc neuroanatomical analysis to investigate intrinsic and afferent connectivity and synaptic properties of the STN in acute brain slices obtained from rats of both sexes. Our data reveal an absence of intrinsic connectivity and an afferent innervation with low divergence, suggesting that STN neurons operate as independent processing elements driven by upstream structures. Hence, synchrony in the STN, a hallmark of motor processing, exclusively depends on the interactions and dynamics of GABAergic and glutamatergic afferents. Importantly, these inputs are subject to differential short-term depression when stimulated at high, DBS-like frequencies, shifting the balance of excitation and inhibition toward inhibition. Thus, we present a mechanism for fast yet transient decoupling of the STN from synchronizing afferent control. Together, our study provides new insights into the microcircuit organization of the STN by identifying its neurons as parallel processing units and thus sets new constraints for future computational models of the basal ganglia. The observed differential short-term plasticity of afferent inputs further offers a basis to better understand and optimize DBS algorithms.SIGNIFICANCE STATEMENT The subthalamic nucleus (STN) is a pivotal element of the basal ganglia and serves as target for deep brain stimulation, but information on the functional connectivity of its neurons is limited. To investigate the STN microcircuitry, we combined multiple simultaneous patch-clamp recordings and neuroanatomical analysis. Our results provide new insights into the synaptic organization of the STN identifying its neurons as parallel processing units and thus set new constraints for future computational models of the basal ganglia. We further find that synaptic dynamics of afferent inputs result in a rapid yet transient decoupling of the STN when stimulated at high frequencies. These results offer a better understanding of deep brain stimulation mechanisms, promoting the development of optimized algorithms.

Initiating a new national epilepsy surgery program: Experiences gathered in Georgia.

Surgery is the most effective therapeutic approach for medically refractory epilepsies and a safe and cost-efficient treatment in terms of long-term expenses of direct, indirect, and intangible costs. Georgia is a Caucasian low- to middle-income country with a remarkable effort to deal with epileptic diseases, but without an appropriate epilepsy surgery program. To address the needs for such a service in this country, two joint German-Georgian projects were initiated in 2017 and 2019. In the framework of these projects, a productive exchange program involving German and Georgian experts was undertaken in the past two years. This program included training and mentoring for Georgian clinical colleagues, as well as joint case conferences and workshops with the aim of optimizing presurgical diagnostics and preparing for an epilepsy surgery program in Georgia. Finally, a postsurgical medium- and long-term follow-up scheme was organized as the third component of this comprehensive approach. As a result of our efforts, the first patients underwent anterior temporal lobectomy and all of them remain seizure-free up to the present day. Hence, epilepsy surgery is not only feasible, but also already available in Georgia. In this report, we aim to share our experiences in the initiation and implementation of surgical epilepsy intervention in Georgia and illustrate our recent endeavor and achievements.

Differential Dependence of GABAergic and Glutamatergic Neurons on Glia for the Establishment of Synaptic Transmission.

In the mammalian cortex, GABAergic and glutamatergic neurons represent 2 major neuronal classes, which establish inhibitory and excitatory synapses, respectively. Despite differences in their anatomy, physiology and developmental origin, both cell types require support from glial cells, particularly astrocytes, for their growth and survival. Recent experiments indicate that glutamatergic neurons also depend on astrocytes for synapse formation. However, it is not clear if the same holds true for GABAergic neurons. By studying highly pure GABAergic cell cultures, established through fluorescent activated cell sorting, we find that purified GABAergic neurons are smaller and have reduced survival, nevertheless they establish robust synaptic transmission in the absence of glia. Support from glial cells reverses morphological and survival deficits, but does little to alter synaptic transmission. In contrast, in cultures of purified glutamatergic neurons, morphological development, survival and synaptic transmission are collectively dependent on glial support. Thus, our results demonstrate a fundamental difference in the way GABAergic and glutamatergic neurons depend on glia for the establishment of synaptic transmission, a finding that has important implications for our understanding of how neuronal networks develop.

BDNF Expression in Cortical GABAergic Interneurons.

Brain-derived neurotrophic factor (BDNF) is a major neuronal growth factor that is widely expressed in the central nervous system. It is synthesized as a glycosylated precursor protein, (pro)BDNF and post-translationally converted to the mature form, (m)BDNF. BDNF is known to be produced and secreted by cortical glutamatergic principal cells (PCs); however, it remains a question whether it can also be synthesized by other neuron types, in particular, GABAergic interneurons (INs). Therefore, we utilized immunocytochemical labeling and reverse transcription quantitative PCR (RT-qPCR) to investigate the cellular distribution of proBDNF and its RNA in glutamatergic and GABAergic neurons of the mouse cortex. Immunofluorescence labeling revealed that mBDNF, as well as proBDNF, localized to both the neuronal populations in the hippocampus. The precursor proBDNF protein showed a perinuclear distribution pattern, overlapping with the rough endoplasmic reticulum (ER), the site of protein synthesis. RT-qPCR of samples obtained using laser capture microdissection (LCM) or fluorescence-activated cell sorting (FACS) of hippocampal and cortical neurons further demonstrated the abundance of BDNF transcripts in both glutamatergic and GABAergic cells. Thus, our data provide compelling evidence that BDNF can be synthesized by both principal cells and INs of the cortex.

Correction to: Morphological diversity and connectivity of hippocampal interneurons.

The original version of this article inadvertently presented a mistake regarding the termination zones of entorhinal cotex in the dentate gyrus. The termination zones were erroneously swapped in both Figure 7. and the associated text.

Morphological diversity and connectivity of hippocampal interneurons.

The mammalian forebrain is constructed from ensembles of neurons that form local microcircuits giving rise to the exquisite cognitive tasks the mammalian brain can perform. Hippocampal neuronal circuits comprise populations of relatively homogenous excitatory neurons, principal cells and exceedingly heterogeneous inhibitory neurons, the interneurons. Interneurons release GABA from their axon terminals and are capable of controlling excitability in every cellular compartment of principal cells and interneurons alike; thus, they provide a brake on excess activity, control the timing of neuronal discharge and provide modulation of synaptic transmission. The dendritic and axonal morphology of interneurons, as well as their afferent and efferent connections within hippocampal circuits, is central to their ability to differentially control excitability, in a cell-type- and compartment-specific manner. This review aims to provide an up-to-date compendium of described hippocampal interneuron subtypes, with respect to their morphology, connectivity, neurochemistry and physiology, a full understanding of which will in time help to explain the rich diversity of neuronal function.

Layer-Specific Organization of Local Excitatory and Inhibitory Synaptic Connectivity in the Rat Presubiculum.

The presubiculum is part of the parahippocampal spatial navigation system and contains head direction and grid cells upstream of the medial entorhinal cortex. This position within the parahippocampal cortex renders the presubiculum uniquely suited for analyzing the circuit requirements underlying the emergence of spatially tuned neuronal activity. To identify the local circuit properties, we analyzed the topology of synaptic connections between pyramidal cells and interneurons in all layers of the presubiculum by testing 4250 potential synaptic connections using multiple whole-cell recordings of up to 8 cells simultaneously. Network topology showed layer-specific organization of microcircuits consistent with the prevailing distinction of superficial and deep layers. While connections among pyramidal cells were almost absent in superficial layers, deep layers exhibited an excitatory connectivity of 3.9%. In contrast, synaptic connectivity for inhibition was higher in superficial layers though markedly lower than in other cortical areas. Finally, synaptic amplitudes of both excitatory and inhibitory connections showed log-normal distributions suggesting a nonrandom functional connectivity. In summary, our study provides new insights into the microcircuit organization of the presubiculum by revealing area- and layer-specific connectivity rules and sets new constraints for future models of the parahippocampal navigation system.

KCTD12 Auxiliary Proteins Modulate Kinetics of GABAB Receptor-Mediated Inhibition in Cholecystokinin-Containing Interneurons.

Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABAB receptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.

GABA(B) autoreceptor-mediated cell type-specific reduction of inhibition in epileptic mice.

GABA(B) receptors (GABA(B)Rs) mediate slow inhibitory effects on neuronal excitability and synaptic transmission in the brain. However, the GABA(B)R agonist baclofen can also promote excitability and seizure generation in human patients and animals models. Here we show that baclofen has concentration-dependent effects on the hippocampal network in a mouse model of mesial temporal lobe epilepsy. Application of baclofen at a high dose (10 mg/kg i.p.) reduced the power of γ oscillations and the frequency of pathological discharges in the Cornu Ammonis area 3 (CA3) area of freely moving epileptic mice. Unexpectedly, at a lower dose (1 mg/kg), baclofen markedly increased γ activity accompanied by a higher incidence of pathological discharges. Intracellular recordings from CA3 pyramidal cells in vitro further revealed that, although at a high concentration (10 µM), baclofen invariably resulted in hyperpolarization, at low concentrations (0.5 µM), the drug had divergent effects, producing depolarization and an increase in firing frequency in epileptic but not control mice. These excitatory effects were mediated by the selective muting of inhibitory cholecystokinin-positive basket cells (CCK(+) BCs), through enhanced inhibition of GABA release via presynaptic GABA(B)Rs. We conclude that cell type-specific up-regulation of GABA(B)R-mediated autoinhibition in CCK(+) BCs promotes aberrant high frequency oscillations and hyperexcitability in hippocampal networks of chronic epileptic mice.

Synaptic properties of SOM- and CCK-expressing cells in dentate gyrus interneuron networks.

Hippocampal GABAergic cells are highly heterogeneous, but the functional significance of this diversity is not fully understood. By using paired recordings of synaptically connected interneurons in slice preparations of the rat and mouse dentate gyrus (DG), we show that morphologically identified interneurons form complex neuronal networks. Synaptic inhibitory interactions exist between cholecystokinin (CCK)-expressing hilar commissural associational path (HICAP) cells and among somatostatin (SOM)-containing hilar perforant path-associated (HIPP) interneurons. Moreover, both interneuron types inhibit parvalbumin (PV)-expressing perisomatic inhibitory basket cells (BCs), whereas BCs and HICAPs rarely target HIPP cells. HICAP and HIPP cells produce slow, weak, and unreliable inhibition onto postsynaptic interneurons. The time course of inhibitory signaling is defined by the identity of the presynaptic and postsynaptic cell. It is the slowest for HIPP-HIPP, intermediately slow for HICAP-HICAP, but fast for BC-BC synapses. GABA release at interneuron-interneuron synapses also shows cell type-specific short-term dynamics, ranging from multiple-pulse facilitation at HICAP-HICAP, biphasic modulation at HIPP-HIPP to depression at BC-BC synapses. Although dendritic inhibition at HICAP-BC and HIPP-BC synapses appears weak and slow, channelrhodopsin 2-mediated excitation of SOM terminals demonstrates that they effectively control the activity of target interneurons. They markedly reduce the discharge probability but sharpen the temporal precision of action potential generation. Thus, dendritic inhibition seems to play an important role in determining the activity pattern of GABAergic interneuron populations and thereby the flow of information through the DG circuitry.

Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons.

Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABA(B) receptors (GABA(B)Rs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABA(B)Rs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABA(B)Rs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABA(B)R-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABA(B)R activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABA(B) IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABA(B)Rs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABA(B)Rs may shift the balance between perisomatic and dendritic inhibition.

Morpho-physiological criteria divide dentate gyrus interneurons into classes.

GABAergic inhibitory interneurons control fundamental aspects of neuronal network function. Their functional roles are assumed to be defined by the identity of their input synapses, the architecture of their dendritic tree, the passive and active membrane properties and finally the nature of their postsynaptic targets. Indeed, interneurons display a high degree of morphological and physiological heterogeneity. However, whether their morphological and physiological characteristics are correlated and whether interneuron diversity can be described by a continuum of GABAergic cell types or by distinct classes has remained unclear. Here we perform a detailed morphological and physiological characterization of GABAergic cells in the dentate gyrus, the input region of the hippocampus. To achieve an unbiased and efficient sampling and classification we used knock-in mice expressing the enhanced green fluorescent protein (eGFP) in glutamate decarboxylase 67 (GAD67)-positive neurons and performed cluster analysis. We identified five interneuron classes, each of them characterized by a distinct set of anatomical and physiological parameters. Cross-correlation analysis further revealed a direct relation between morphological and physiological properties indicating that dentate gyrus interneurons fall into functionally distinct classes which may differentially control neuronal network activity.

Correction: Opportunities within the meat supply chain in Africa-The case of beef production in Northern Ghana.

[This corrects the article DOI: 10.1371/journal.pone.0260668.].