Putative Contacts Research Articles

Examining sleep modulation by Drosophila ellipsoid body neurons.

Recent work in Drosophila has uncovered several neighboring classes of sleep-regulatory neurons within the central complex. However, the logic of connectivity and network motifs remains limited by the incomplete examination of relevant cell types. Using a recent genetic-anatomical classification of ellipsoid body ring neurons, we conducted a thermogenetic screen in female flies to assess sleep/wake behavior and identified two wake-promoting drivers that label ER3d neurons and two sleep-promoting drivers that express in ER3m cells. We then used intersectional genetics to refine driver expression patterns. Activation of ER3d cells shortened sleep bouts, suggesting a key role in sleep maintenance. While sleep-promoting drivers from our miniscreen label overlapping ER3m neurons, intersectional strategies cannot rule out sleep regulatory roles for additional neurons in their expression patterns. Suppressing GABA synthesis in ER3m neurons prevents post-injury sleep, and GABAergic ER3d cells are required for thermogenetically induced wakefulness. Finally, we use an activity-dependent fluorescent reporter for putative synaptic contacts to embed these neurons within the known sleep-regulatory network. ER3m and ER3d neurons may receive connections from wake-active Helicon/ExR1 cells, and ER3m neurons likely inhibit ER3d neurons. Together, these data suggest a neural mechanism by which previously uncharacterized circuit elements stabilize sleep-wake states.Significance StatementNeural circuits that control sleep must be stable to ensure therapeutic rest but readily adaptable to a variety of experiences. The Drosophila ellipsoid body (EB) regulates many cognitive processes, and recent studies show that certain EB neurons can intensify sleepiness, but the contributions of other EB neuron subclasses in sleep regulation remain unclear. Thus, we searched for sleep-regulatory neurons across the EB to better understand sleep control. Our studies indicate that ER3d neurons promote wakefulness by reducing the persistence of sleep episodes and suggest that ER3m neuron activity may increase sleep. Understanding the stability and flexibility of sleep, then, may emerge from investigating the connectivity and conductivity of sleep-regulatory neurons in the EB.

Visuomotor interactions in the mouse forebrain mediated by extrastriate cortico-cortical pathways.

The mammalian visual system can be broadly divided into two functional processing pathways: a dorsal stream supporting visually and spatially guided actions, and a ventral stream enabling object recognition. In rodents, the majority of visual signaling in the dorsal stream is transmitted to frontal motor cortices via extrastriate visual areas surrounding V1, but exactly where and to what extent V1 feeds into motor-projecting visual regions is not well known. We employed a dual labeling strategy in male and female mice in which efferent projections from V1 were labeled anterogradely, and motor-projecting neurons in higher visual areas were labeled with retrogradely traveling adeno-associated virus (rAAV-retro) injected in M2. We characterized the labeling in both flattened and coronal sections of dorsal cortex and made high-resolution 3D reconstructions to count putative synaptic contacts in different extrastriate areas. The most pronounced colocalization V1 output and M2 input occurred in extrastriate areas AM, PM, RL and AL. Neurons in both superficial and deep layers in each project to M2, but high resolution volumetric reconstructions indicated that the majority of putative synaptic contacts from V1 onto M2-projecting neurons occurred in layer 2/3. These findings support the existence of a dorsal processing stream in the mouse visual system, where visual signals reach motor cortex largely via feedforward projections in anteriorly and medially located extrastriate areas.

Physical and Chemical Properties of Water in the Cytoplasma of Plant Cells

A study is performed of the physicochemical properties of the aqueous component of the cytoplasm of a living cell and the intercellular fluid associated with their structural organization. As a single coherent system, the structure of the water component of a plant plays a crucial role in its water regime, and therefore determines the development and adaptability of the plant to environmental conditions. Low-frequency L-dielcometry is used for the first time in experimental studies of the water regime of plants. The change in the peak intensities in the spectrum of the dependence of the dielectric loss tangent on the EMF frequency under conditions of artificial drought for different phytotest objects is used to show there are two types of water in living plants: free and self-organized bound. Free water is responsible for the mode of evaporation, while bound water participates in the organization of a complex network of intercellular communications, including the transmission of different signals through putative synapse-like contacts. Results correlate fully with others from plant phytomonitoring with pulsed NMR at the Kazan Scientific School of Phytophysiologist. The conclusion that there are two types of water in a living plant simultaneously is substantiated theoretically on the basis of generalized crystallography using Bulienkov’s modular computer design. It is the most important component of the concept of quantum bioenergetics.

Structural analysis of the microglia-interneuron interactions in the CA1 hippocampal area of the APP/PS1 mouse model of Alzheimer's disease.

Microglia can interact with glutamatergic neurons and, through control of synaptic elements, regulate their physiological function. Much less is known about the partnership between microglia and GABAergic inhibitory interneurons. Here, we compared the interactions between microglia and parvalbumin (PV+) and somatostatin (SOM+) expressing interneurons in the CA1 hippocampal area of APP/PS1 transgenic mice that mimic certain aspects of the Alzheimer's disease (AD). We first uncovered a high level of interactions between microglia and two types of interneurons, with 98% of SOM+ and 90% of PV+ cells receiving different types of putative microglial contacts. The latter included the microglia soma to the interneuron soma (SomaMG -to-SomaIN ), the microglia process to the interneuron soma (ProcessMG -to-SomaIN ) and the microglia process to the interneuron dendrite (ProcessMG -to-DendIN ) interactions. Moreover, we found significantly larger areas of interaction for the SomaMG -to-SomaIN and the ProcessMG -to-DendIN type of contacts between microglia and SOM+ cells. In contrast, PV+ cells exhibited larger areas for the ProcessMG -to-SomaIN interactions. Second, in APP/PS1 mice, although the overall microglia interactions with interneurons remained preserved, the fraction of interneurons receiving putative microglia contacts on their dendrites was reduced, and larger areas of interactions were observed for somatic contacts, suggesting a stronger modulation of the interneuron output by microglia in AD. In summary, these results reveal microglia as important partners of hippocampal PV+ and SOM+ GABAergic cells, with interneuron type-specific pattern of interactions. Thus, microglia may play an essential role in the operation of interneurons under normal conditions and their dysfunction in disease.

Structure and function of a neocortical synapse.

In 1986, electron microscopy was used to reconstruct by hand the entire nervous system of a roundworm, the nematode Caenorhabditis elegans1. Since this landmark study, high-throughput electron-microscopic techniques have enabled reconstructions of much larger mammalian brain circuits at synaptic resolution2,3. Nevertheless, it remains unknown how the structure of a synapse relates to its physiological transmission strength-a key limitation for inferring brain function from neuronal wiring diagrams. Here we combine slice electrophysiology of synaptically connected pyramidal neurons in the mouse somatosensory cortex with correlated light microscopy and high-resolution electron microscopy of all putative synaptic contacts between the recorded neurons. We find a linear relationship between synapse size and strength, providing the missing link in assigning physiological weights to synapses reconstructed from electron microscopy. Quantal analysis also reveals that synapses contain at least 2.7 neurotransmitter-release sites on average. This challenges existing release models and provides further evidence that neocortical synapses operate with multivesicular release4-6, suggesting that they are more complex computational devices than thought, and thereforeexpanding the computational power of the canonical cortical microcircuitry.

Serotonin distribution in the brain of the plainfin midshipman: Substrates for vocal-acoustic modulation and a reevaluation of the serotonergic system in teleost fishes.

Serotonin (5-HT) is a modulator of neural circuitry underlying motor patterning, homeostatic control, and social behavior. While previous studies have described 5-HT distribution in various teleosts, serotonergic raphe subgroups in fish are not well defined and therefore remain problematic for cross-species comparisons. Here we used the plainfin midshipman fish, Porichthys notatus, a well-studied model for investigating the neural and hormonal mechanisms of vertebrate vocal-acoustic communication, to redefine raphe subgroups based on both stringent neuroanatomical landmarks as well as quantitative cell measurements. In addition, we comprehensively characterized 5-HT-immunoreactive (-ir) innervation throughout the brain, including well-delineated vocal and auditory nuclei. We report neuroanatomical heterogeneity in populations of the serotonergic raphe nuclei of the brainstem reticular formation, with three discrete subregions in the superior raphe, an intermediate 5-HT-ir cell cluster, and an extensive inferior raphe population. 5-HT-ir neurons were also observed within the vocal motor nucleus (VMN), forming putative contacts on those cells. In addition, three major 5-HT-ir cell groups were identified in the hypothalamus and one group in the pretectum. Significant 5-HT-ir innervation was found in components of the vocal pattern generator and cranial motor nuclei. All vocal midbrain nuclei showed considerable 5-HT-ir innervation, as did thalamic and hindbrain auditory and lateral line areas and vocal-acoustic integration sites in the preoptic area and ventral telencephalon. This comprehensive atlas offers new insights into the organization of 5-HT nuclei in teleosts and provides neuroanatomical evidence for serotonin as a modulator of vocal-acoustic circuitry and behavior in midshipman fish, consistent with findings in vocal tetrapods.

Adult-Born Hippocampal Neurons Undergo Extended Development and Are Morphologically Distinct from Neonatally-Born Neurons.

During immature stages, adult-born neurons pass through critical periods for survival and plasticity. It is generally assumed that by 2 months of age adult-born neurons are mature and equivalent to the broader neuronal population, raising questions of how they might contribute to hippocampal function in old age when neurogenesis has declined. However, few have examined adult-born neurons beyond the critical period or directly compared them to neurons born in infancy. Here, we used a retrovirus to visualize functionally relevant morphological features of 2- to 24-week-old adult-born neurons in male rats. From 2 to 7 weeks, neurons grew and attained a relatively mature phenotype. However, several features of 7-week-old neurons suggested a later wave of growth: these neurons had larger nuclei, thicker dendrites, and more dendritic filopodia than all other groups. Indeed, between 7 and 24 weeks, adult-born neurons gained additional dendritic branches, formed a second primary dendrite, acquired more mushroom spines, and had enlarged mossy fiber presynaptic terminals. Compared with neonatal-born neurons, old adult-born neurons had greater spine density, larger presynaptic terminals, and more putative efferent filopodial contacts onto inhibitory neurons. By integrating rates of cell birth and growth across the life span, we estimate that adult neurogenesis ultimately produces half of the cells and the majority of spines in the dentate gyrus. Critically, protracted development contributes to the plasticity of the hippocampus through to the end of life, even after cell production declines. Persistent differences from neonatal-born neurons may additionally endow adult-born neurons with unique functions even after they have matured.SIGNIFICANCE STATEMENT Neurogenesis occurs in the hippocampus throughout adult life and contributes to memory and emotion. It is generally assumed that new neurons have the greatest impact on behavior when they are immature and plastic. However, since neurogenesis declines dramatically with age, it is unclear how they might contribute to behavior later in life when cell proliferation has slowed. Here we find that newborn neurons mature over many months in rats and may end up with distinct morphological features compared with neurons born in infancy. Using a mathematical model, we estimate that a large fraction of neurons is added in adulthood. Moreover, their extended growth produces a reserve of plasticity that persists even after neurogenesis has declined to low rates.

Neurotransmitters and Motoneuron Contacts of Multifunctional and Behaviorally Specialized Turtle Spinal Cord Interneurons.

The spinal cord can appropriately generate diverse movements, even without brain input and movement-related sensory feedback, using a combination of multifunctional and behaviorally specialized interneurons. The adult turtle spinal cord can generate motor patterns underlying forward swimming, three forms of scratching, and limb withdrawal (flexion reflex). We previously described turtle spinal interneurons activated during both scratching and swimming (multifunctional interneurons), interneurons activated during scratching but not swimming (scratch-specialized interneurons), and interneurons activated during flexion reflex but not scratching or swimming (flexion reflex-selective interneurons). How multifunctional and behaviorally specialized turtle spinal interneurons affect downstream neurons was unknown. Here, we recorded intracellularly from spinal interneurons activated during these motor patterns in turtles of both sexes in vivo and filled each with dyes. We labeled motoneurons using choline acetyltransferase antibodies or earlier intraperitoneal FluoroGold injection and used immunocytochemistry of interneuron axon terminals to identify their neurotransmitter(s) and putative synaptic contacts with motoneurons. We found that multifunctional interneurons are heterogeneous with respect to neurotransmitter, with some glutamatergic and others GABAergic or glycinergic, and can directly contact motoneurons. Also, scratch-specialized interneurons are heterogeneous with respect to neurotransmitter and some directly contact motoneurons. Thus, scratch-specialized interneurons might directly excite motoneurons that are more strongly activated during scratching than forward swimming, such as hip-flexor motoneurons. Finally, and surprisingly, we found that some motoneurons are behaviorally specialized, for scratching or flexion reflex. Thus, either some limb muscles are only used for a subset of limb behaviors or some limb motoneurons are only recruited during certain limb behaviors.SIGNIFICANCE STATEMENT Both multifunctional and behaviorally specialized spinal cord interneurons have been described in turtles, but their outputs are unknown. We studied responses of multifunctional interneurons (activated during swimming and scratching) and scratch-specialized interneurons, filled each with dyes, and used immunocytochemistry to determine their neurotransmitters and contacts with motoneurons. We found that both multifunctional and scratch-specialized interneurons are heterogeneous with respect to neurotransmitter, with some excitatory and others inhibitory. We found that some multifunctional and some scratch-specialized interneurons directly contact motoneurons. Scratch-specialized interneurons may excite motoneurons that are more strongly activated during scratching than swimming, such as hip-flexor motoneurons, or inhibit their antagonists, hip-extensor motoneurons. Surprisingly, we also found that some motoneurons are behaviorally specialized, for scratching or for flexion reflex.

GABAergic neurons in the olfactory cortex projecting to the lateral hypothalamus in mice

Olfaction guides goal-directed behaviours including feeding. To investigate how central olfactory neural circuits control feeding behaviour in mice, we performed retrograde tracing from the lateral hypothalamus (LH), an important feeding centre. We observed a cluster of retrogradely labelled cells distributed in the posteroventral region of the olfactory peduncle. Histochemical analyses revealed that the majority of these retrogradely labelled projection neurons expressed glutamic acid decarboxylase 65/67 (GAD65/67), but not vesicular glutamate transporter 1 (VGluT1). We named this region containing GABAergic projection neurons the ventral olfactory nucleus (VON) to differentiate it from the conventional olfactory peduncle. VON neurons were less immunoreactive for DARPP-32, a striatal neuron marker, compared to neurons in the olfactory tubercle and nucleus accumbens, which distinguished the VON from the ventral striatum. Fluorescent labelling confirmed putative synaptic contacts between VON neurons and olfactory bulb projection neurons. Rabies-virus-mediated trans-synaptic labelling revealed that VON neurons received synaptic inputs from the olfactory bulb, other olfactory cortices, horizontal limb of the diagonal band, and prefrontal cortex. Collectively, these results identify novel GABAergic projection neurons in the olfactory cortex that may integrate olfactory sensory and top-down inputs and send inhibitory output to the LH, which may modulate odour-guided LH-related behaviours.

Targets of olivocochlear collaterals in cochlear nucleus of rat and guinea pig.

Descending auditory pathways can modify afferent auditory input en route to cortex. One component of these pathways is the olivocochlear system which originates in brainstem and terminates in cochlea. Medial olivocochlear (MOC) neurons also project collaterals to cochlear nucleus and make synaptic contacts with dendrites of multipolar neurons. Two broadly distinct populations of multipolar cells exist: T-stellate and D-stellate neurons, thought to project to inferior colliculus and contralateral cochlear nucleus, respectively. It is unclear which of these neurons receive direct MOC collateral input due to conflicting results between in vivo and in vitro studies. This study used anatomical techniques to identify which multipolar cell population receives synaptic innervation from MOC collaterals. The retrograde tracer Fluorogold was injected into inferior colliculus or cochlear nucleus to label T-stellate and D-stellate neurons, respectively. Axonal branches of MOC neurons were labeled by biocytin injections at the floor of the fourth ventricle. Fluorogold injections resulted in labeled cochlear nucleus multipolar neurons. Biocytin abundantly labeled MOC collaterals which entered cochlear nucleus. Microscopic analysis revealed that MOC collaterals made some putative synaptic contacts with the retrogradely labeled neurons but many more putative contacts were observed on unidentified neural targets. This suggest that both T- and D-stellate neurons receive synaptic innervation from the MOC collaterals on their somata and proximal dendrites. The prevalence of these contacts cannot be stated with certainty because of technical limitations, but the possibility exists that the collaterals may also make contacts with neurons not projecting to inferior colliculus or the contralateral cochlear nucleus.

Characterization of calbindin D28k expressing interneurons in the ventral horn of the mouse spinal cord.

Expression of the calcium binding protein, calbindin (CB), is well established as a hallmark of Renshaw cells, a class of interneurons found in spatially restricted areas in the ventral spinal cord that directly modulate motor neuron activity. CB expression, however, is not restricted only to Renshaw cells in the ventral horn, and within this population other interneuron subtypes may be identifiable on the basis of cell position and the potential for coexpression of other calcium binding proteins. Here we have quantified the changing CB expression pattern in the ventral spinal cord across postnatal development in the mouse. Fewer neurons express CB as postnatal development progresses, and those neurons frequently coexpress other calcium binding proteins (calretinin and parvalbumin) in subpopulations with distinct spatial distributions. We also found a significant portion of CB-expressing interneurons receive putative synaptic contacts from primary sensory afferents. These findings suggest CB labels a heterogeneous group of interneurons in the ventral horn, some of which may process sensory information. Based on cellular position, CB expression may be a shared feature of subsets of interneurons arising from multiple ventral progenitor domains. Developmental Dynamics 247:185-193, 2018. © 2017 Wiley Periodicals, Inc.

Connectomics of synaptic microcircuits: lessons from the outer retina.

Photoreceptors form a sophisticated synaptic complex with bipolar and horizontal cells, transmitting the signals generated by the phototransduction cascade to downstream retinal circuitry. The cone photoreceptor synapse shows several characteristic anatomical connectivity motifs that shape signal transfer: typically, ON-cone bipolar cells receive photoreceptor input through invaginating synapses; OFF-cone bipolar cells form basal synapses with photoreceptors. Both ON- and OFF-cone bipolar cells are believed to sample from all cone photoreceptors within their dendritic span. Electron microscopy and immunolabelling studies have established the robustness of these motifs, but have been limited by trade-offs in sample size and spatial resolution, respectively, constraining precise quantitative investigation to a few individual cells. 3D-serial electron microscopy overcomes these limitations and has permitted complete sets of neurons to be reconstructed over a comparatively large section of retinal tissue. Although the published mouse dataset lacks labels for synaptic structures, the characteristic anatomical motifs at the photoreceptor synapse can be exploited to identify putative synaptic contacts, which has enabled the development of a quantitative description of outer retinal connectivity. This revealed unexpected exceptions to classical motifs, including substantial interaction between rod and cone pathways at the photoreceptor synapse, sparse photoreceptor sampling and atypical contacts. Here, we summarize what was learned from this study in a more general context: we consider both the implications and limitations of the study and identify promising avenues for future research.

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.

The Circuitry of Olfactory Projection Neurons in the Brain of the Honeybee, Apis mellifera.

In the honeybee brain, two prominent tracts – the medial and the lateral antennal lobe tract – project from the primary olfactory center, the antennal lobes (ALs), to the central brain, the mushroom bodies (MBs), and the protocerebral lobe (PL). Intracellularly stained uniglomerular projection neurons were reconstructed, registered to the 3D honeybee standard brain atlas, and then used to derive the spatial properties and quantitative morphology of the neurons of both tracts. We evaluated putative synaptic contacts of projection neurons (PNs) using confocal microscopy. Analysis of the patterns of axon terminals revealed a domain-like innervation within the MB lip neuropil. PNs of the lateral tract arborized more sparsely within the lips and exhibited fewer synaptic boutons, while medial tract neurons occupied broader regions in the MB calyces and the PL. Our data show that uPNs from the medial and lateral tract innervate both the core and the cortex of the ipsilateral MB lip but differ in their innervation patterns in these regions. In the mushroombody neuropil collar we found evidence for ALT boutons suggesting the collar as a multi modal input site including olfactory input similar to lip and basal ring. In addition, our data support the conclusion drawn in previous studies that reciprocal synapses exist between PNs, octopaminergic-, and GABAergic cells in the MB calyces. For the first time, we found evidence for connections between both tracts within the AL.

Synaptic Remodeling of Entorhinal Input Contributes to an Aberrant Hippocampal Network in Temporal Lobe Epilepsy.

The hippocampus is reciprocally connected with the entorhinal cortex. Although several studies emphasized a role for the entorhinal cortex in mesial temporal lobe epilepsy (MTLE), it remains uncertain whether its synaptic connections with the hippocampus are altered. To address this question, we traced hippocampo-entorhinal and entorhino-hippocampal projections, assessed their connectivity with the respective target cells and examined functional alterations in a mouse model for MTLE. We show that hippocampal afferents to the dorsal entorhinal cortex are lost in the epileptic hippocampus. Conversely, entorhino-dentate projections via the medial perforant path (MPP) are preserved, but appear substantially altered on the synaptic level. Confocal imaging and 3D-reconstruction revealed that new putative contacts are established between MPP fibers and dentate granule cells (DGCs). Immunohistochemical identification of pre- and postsynaptic elements indicated that these contacts are functionally mature synapses. On the ultrastructural level, pre- and postsynaptic compartments of MPP synapses were strongly enlarged. The length and complexity of postsynaptic densities were also increased pointing to long-term potentiation-related morphogenesis. Finally, whole-cell recordings of DGCs revealed an enhancement of evoked excitatory postsynaptic currents. In conclusion, the synaptic rearrangement of excitatory inputs to DGCs from the medial entorhinal cortex may contribute to the epileptogenic circuitry in MTLE.

Replacement of Asymmetric Synaptic Profiles in the Molecular Layer of Dentate Gyrus Following Cycloheximide in the Pilocarpine Model in Rats

Mossy fiber sprouting is among the best-studied forms of post-lesional synaptic plasticity and is regarded by many as contributory to seizures in both humans and animal models of epilepsy. It is not known whether mossy fiber sprouting increases the number of synapses in the molecular layer or merely replaces lost contacts. Using the pilocarpine (Pilo) model of status epilepticus to induce mossy fiber sprouting, and cycloheximide (CHX) to block this sprouting, we evaluated at the ultrastructural level the number and type of asymmetric synaptic contacts in the molecular layer of the dentate gyrus. As expected, whereas Pilo-treated rats had dense silver grain deposits in the inner molecular layer (IML) (reflecting mossy fiber sprouting), pilocarpine + cycloheximide (CHX + Pilo)-treated animals did not differ from controls. Both groups of treated rats (Pilo group and CHX + Pilo group) had reduced density of asymmetric synaptic profiles (putative excitatory synaptic contacts), which was greater for CHX-treated animals. For both treated groups, the loss of excitatory synaptic contacts was even greater in the outer molecular layer than in the best-studied IML (in which mossy fiber sprouting occurs). These results indicate that mossy fiber sprouting tends to replace lost synaptic contacts rather than increase the absolute number of contacts. We speculate that the overall result is more consistent with restored rather than with increased excitability.

Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity

Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132-713) and STAT3 (127-722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

Identification of Inhibitory Premotor Interneurons Activated at a Late Phase in a Motor Cycle during Drosophila Larval Locomotion.

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). Our knowledge of how CPG networks generate motor patterns in complex nervous systems remains incomplete, despite decades of work in a variety of model organisms. Substrate borne locomotion in Drosophila larvae is driven by waves of muscular contraction that propagate through multiple body segments. We use the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. Our recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. Here, we report on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). We used calcium imaging to search for interneurons that show rhythmic activity and identified GVLIs as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs’ wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. We propose that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.

Excitatory orexinergic innervation of rat nucleus incertus – Implications for ascending arousal, motivation and feeding control

Orexin/hypocretin peptides play a central role in the integrated control of feeding/reward and behavioural activation, principally via interactions with other neural systems. A brainstem area involved in behavioural activation is the nucleus incertus (NI), located in the posterior ventromedial central grey. Several studies have implicated NI in control of arousal/stress and reward/feeding responses. Orexin receptor mRNA expression identifies NI as a putative target of orexin modulation. Therefore, in this study we performed neural tract-tracing and immunofluorescence staining to characterise the orexinergic innervation of NI. Our results indicate a convergent innervation of the NI area by different orexin neuron populations, with an abundance of orexin-A-containing axons making putative synaptic contacts with relaxin-3-positive NI neurons. The influence of orexin-A on NI neuron activity was investigated using patch-clamp recordings. Orexin-A depolarised the majority (64%) of recorded neurons and this effect was maintained in the presence of tetrodotoxin and glutamate and GABA receptor antagonists, indicating a likely postsynaptic action. Voltage-clamp experiments revealed that in ‘type I’ NI neurons comprising relaxin-3-positive cells, orexin-A acted via L-type calcium channels, whereas in ‘type II’ relaxin-3-negative neurons, activation of a sodium/calcium exchanger was involved. A majority of the orexin-A sensitive neurons tested for the presence of orexin receptor mRNA, were OX2 mRNA-positive. Immunohistochemical staining for putative orexin receptors on NI neurons, confirmed stronger expression of OX2 than OX1 receptors. Our data demonstrate a strong influence of orexin-A on NI neurons, consistent with an important role for this hypothalamic/tegmental circuit in the regulation of arousal/vigilance and motivated behaviours.

Olfactory and cortical projections to bulbar and hippocampal adult-born neurons.

New neurons are continually generated in the subependymal layer of the lateral ventricles and the subgranular zone of dentate gyrus during adulthood. In the subventricular zone, neuroblasts migrate a long distance to the olfactory bulb where they differentiate into granule or periglomerular interneurons. In the hippocampus, neuroblasts migrate a short distance from the subgranular zone to the granule cell layer of the dentate gyrus to become granule neurons. In addition to the short-distance inputs, bulbar interneurons receive long-distance centrifugal afferents from olfactory-recipient structures. Similarly, dentate granule cells receive differential inputs from the medial and lateral entorhinal cortices through the perforant pathway. Little is known concerning these new inputs on the adult-born cells. In this work, we have characterized afferent inputs to 21-day old newly-born neurons. Mice were intraperitoneally injected with bromodeoxyuridine. Two weeks later, rhodamine-labeled dextran-amine was injected into the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral and medial entorhinal cortices. One week later, animals were perfused and immunofluorescences were carried out. The data show that projection neurons from the mentioned structures, establish putative synaptic contacts onto 21-day-old neurons in the olfactory bulb and dentate gyrus, in some cases even before they start to express specific subpopulation proteins. Long-distance afferents reach middle and outer one-third portions of the molecular layer of the dentate gyrus and granule and, interestingly, periglomerular layers of the olfactory bulb. In the olfactory bulb, these fibers appear to establish presumptive axo-somatic contacts onto newly-born granule and periglomerular cells.