High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis.

Two-photon (TP) uncaging of neurotransmitter molecules is the method of choice to mimic and study the subtleties of neuronal communication either in the intact brain or in slice preparations. However, the currently available caged materials are just at the limit of their usability and have several drawbacks. The local and focal nature of their use may for example be jeopardized by a high spontaneous hydrolysis rate of the commercially available compounds with increased photochemical release rate. Here, using quantum chemical modelling we show the mechanisms of hydrolysis and two-photon activation, and synthesized more effective caged compounds. Furthermore, we have developed a new enzymatic elimination method removing neurotransmitters inadvertently escaping from their compound during experiment. This method, usable both in one and two-photon experiments, allows for the use of materials with an increased rate of photochemical release. The efficiency of the new compound and the enzymatic method and of the new compound are demonstrated in neurophysiological experiments.

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons.

Inhibitory interneurons are considered to be the controlling units of neural networks, despite their sparse number and unique morphological characteristics compared with excitatory pyramidal cells. Although pyramidal cell dendrites have been shown to display local regenerative events--dendritic spikes (dSpikes)--evoked by artificially patterned stimulation of synaptic inputs, no such studies exist for interneurons or for spontaneous events. In addition, imaging techniques have yet to attain the required spatial and temporal resolution for the detection of spontaneously occurring events that trigger dSpikes. Here we describe a high-resolution 3D two-photon laser scanning method (Roller Coaster Scanning) capable of imaging long dendritic segments resolving individual spines and inputs with a temporal resolution of a few milliseconds. By using this technique, we found that local, NMDA receptor-dependent dSpikes can be observed in hippocampal CA1 stratum radiatum interneurons during spontaneous network activities in vitro. These NMDA spikes appear when approximately 10 spatially clustered inputs arrive synchronously and trigger supralinear integration in dynamic interaction zones. In contrast to the one-to-one relationship between computational subunits and dendritic branches described in pyramidal cells, here we show that interneurons have relatively small (∼14 µm) sliding interaction zones. Our data suggest a unique principle as to how interneurons integrate synaptic information by local dSpikes.

Expression of GCaMP6s in the dentate gyrus induces tonic-clonic seizures.

GCaMP is a genetically encoded calcium indicator (GECI) widely used in neuroscience research. It measures intracellular Ca2+ level by fluorescence changes as it directly binds to Ca2+. In this process, the effect of this calcium buffer on the intracellular calcium signaling and cell physiology is often not taken into consideration. However, growing evidence from calcium imaging studies shows GCaMP expression under certain conditions can generate aberrant activity, such as seizures. In this study, we examined the effect of GCaMP6 expression in the dentate gyrus (DG) on epileptogenesis. We found that viral expression of GCaMP6s but not GCaMP6f in the DG induces tonic-clonic seizures several weeks after viral injection. Cell-type specific expression of GCaMP6s revealed the granule cells (GCs) as the key player in GCaMP6s-induced epilepsy. Finally, by using slice electrophysiology, we demonstrated that GCaMP6s expression increases neuronal excitability in the GCs. Together, this study highlights the ability of GCaMP6s in DG-associated epileptogenesis.

Serotonin modulates infraslow oscillation in the dentate gyrus during Non-REM sleep.

Synchronous neuronal activity is organized into neuronal oscillations with various frequency and time domains across different brain areas and brain states. For example, hippocampal theta, gamma and sharp wave oscillations are critical for memory formation and communication between hippocampal subareas and the cortex. In this study, we investigated the neuronal activity of the dentate gyrus (DG) with electrophysiological and optical imaging tools during sleep-wake cycles. We found that the activity of major glutamatergic cell populations in the DG is organized into in-fraslow oscillations (0.01 - 0.03 Hz) during NREM sleep. Although the DG is considered a sparsely active network during wakefulness, we found that 50% of granule cells and about 25% of mossy cells exhibit increased activity during NREM sleep. Further experiments revealed that the infraslow oscillation in the DG is modulated by rhythmic serotonin release during sleep, which oscillates at the same frequency but in an opposite phase. Genetic manipulation of 5-HT receptors revealed that this neuromodulatory regulation is mediated by 5-HT1a receptors and the knockdown of these receptors leads to memory impairment. Together, our results provide novel mechanistic insights into how the 5-HT system can influence hippocampal activity patterns during sleep.

An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice.

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and optical imaging tools while delivering precise sensory stimuli to a behaving animal. Recently, human and rodent studies using virtual reality (VR) environments have shown VR to be an important tool for uncovering the neural mechanisms underlying spatial learning in the hippocampus and cortex, due to the extremely precise control over parameters such as spatial and contextual cues. Setting up virtual environments for rodent spatial behaviors can, however, be costly and require an extensive background in engineering and computer programming. Here, we present a simple yet powerful system based upon inexpensive, modular, open-source hardware and software that enables researchers to study spatial learning in head-restrained mice using a VR environment. This system uses coupled microcontrollers to measure locomotion and deliver behavioral stimuli while head-restrained mice run on a wheel in concert with a virtual linear track environment rendered by a graphical software package running on a single-board computer. The emphasis on distributed processing allows researchers to design flexible, modular systems to elicit and measure complex spatial behaviors in mice in order to determine the connection between neural circuit activity and spatial learning in the mammalian brain.

Parallel processing of sensory cue and spatial information in the dentate gyrus.

During exploration, animals form an internal map of an environment by combining information about landmarks and the animal's movement, a process that depends on the hippocampus. The dentate gyrus (DG) is the first stage of the hippocampal circuit where self-motion ("where") and sensory cue information ("what") are integrated, but it remains unknown how DG neurons encode this information during cognitive map formation. Using two-photon calcium imaging in mice running on a treadmill along with online cue manipulation, we identify robust sensory cue responses in DG granule cells. Cue cell responses are stable, stimulus-specific, and accompanied by inhibition of nearby neurons. This demonstrates the existence of "cue cells" in addition to better characterized "place cells" in the DG. We hypothesize that the DG supports parallel channels of spatial and non-spatial information that contribute distinctly to downstream computations and affect roles of the DG in spatial navigation and episodic memory.

Enhanced dendritic action potential backpropagation in parvalbumin-positive basket cells during sharp wave activity.

In this study two-photon imaging and single cell electrophysiological measurements were carried out in PV+ hippocampal interneurons to compare the dendritic calcium dynamics of somatically evoked backpropagating action potentials (BAPs) and in vitro sharp wave oscillation (SPW) activated BAPs at different distances from the soma. In the case of 300 µm thick, non-oscillating slices, the BAP-evoked Ca(2+) (BAP-Ca(2+)) influx propagated along the dendritic tree in a non-uniform manner and its amplitude gradually reduced when measured at more distal regions. In contrast to the evoked BAP-Ca(2+)s, the spontaneous SPW-induced Ca(2+) influx had only a small distance-dependent decrement. Our results suggest that similarly to nicotinic acetylcholine receptor activation, synaptic activity during hippocampal SPWs increases AP backpropagation into distant dendritic segments. Bath application of Nimodipine, a specific Ca(2+) channel blocker and tetrodotoxine decreased the amplitude of the somatically evoked Ca(2+) influx, which suggests that L-type Ca(2+) channels play an important role both during somatically evoked and SPW-induced BAPs.

Functional Access to Neuron Subclasses in Rodent and Primate Forebrain.

Viral vectors enable foreign proteins to be expressed in brains of non-genetic species, including non-human primates. However, viruses targeting specific neuron classes have proved elusive. Here we describe viral promoters and strategies for accessing GABAergic interneurons and their molecularly defined subsets in the rodent and primate. Using a set intersection approach, which relies on two co-active promoters, we can restrict heterologous protein expression to cortical and hippocampal somatostatin-positive and parvalbumin-positive interneurons. With an orthogonal set difference method, we can enrich for subclasses of neuropeptide-Y-positive GABAergic interneurons by effectively subtracting the expression pattern of one promoter from that of another. These methods harness the complexity of gene expression patterns in the brain and significantly expand the number of genetically tractable neuron classes across mammals.

Cholinergic afferents to gonadotropin-releasing hormone neurons of the rat.

Gonadotropin-releasing hormone-synthesizing neurons represent the final common pathway in the hypothalamic regulation of reproduction and their secretory activity is influenced by a variety of neurotransmitters and neuromodulators acting centrally in synaptic afferents to gonadotropin-releasing hormone neurons. The present study examined the anatomical relationship of cholinergic neuronal pathways and gonadotropin-releasing hormone neurons of the preoptic area. The immunocytochemical detection of choline acetyltransferase or vesicular acetylcholine transporter revealed a fine network of cholinergic fibers in this region. At the light microscopic level, the cholinergic axons formed appositions to the gonadotropin-releasing hormone immunoreactive cell bodies and dendrites. Results of electron microscopic studies confirmed the absence of glial interpositions in many of these neuronal contacts. Classical cholinergic synapses, which belonged to the asymmetric category, were only observed rarely on gonadotropin-releasing hormone neurons. The lack of synaptic density in most contacts corroborates previous observations on the cholinergic system elsewhere in the brain. Further, it suggests a dominantly non-synaptic route also in this cholinergic neuronal communication. This study provides direct neuromorphological evidence for the involvement of the cholinergic system in the afferent neuronal regulation of gonadotropin-releasing hormone neurons. The sources of cholinergic afferents and the receptorial mechanisms underlying this interaction will require further clarification.

Multimodal Characterization of Neural Networks Using Highly Transparent Electrode Arrays.

Transparent and flexible materials are attractive for a wide range of emerging bioelectronic applications. These include neural interfacing devices for both recording and stimulation, where low electrochemical electrode impedance is valuable. Here the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is used to fabricate electrodes that are small enough to allow unencumbered optical access for imaging a large cell population with two-photon (2P) microscopy, yet provide low impedance for simultaneous high quality recordings of neural activity in vivo. To demonstrate this, pathophysiological activity was induced in the mouse cortex using 4-aminopyridine (4AP), and the resulting electrical activity was detected with the PEDOT:PSS-based probe while imaging calcium activity directly below the probe area. The induced calcium activity of the neuronal network as measured by the fluorescence change in the cells correlated well with the electrophysiological recordings from the cortical grid of PEDOT:PSS microelectrodes. Our approach provides a valuable vehicle for complementing classical high temporal resolution electrophysiological analysis with optical imaging.

In Vivo Imaging of Dentate Gyrus Mossy Cells in Behaving Mice.

Mossy cells in the hilus of the dentate gyrus constitute a major excitatory principal cell type in the mammalian hippocampus; however, it remains unknown how these cells behave in vivo. Here, we have used two-photon Ca2+ imaging to monitor the activity of mossy cells in awake, behaving mice. We find that mossy cells are significantly more active than dentate granule cells in vivo, exhibit spatial tuning during head-fixed spatial navigation, and undergo robust remapping of their spatial representations in response to contextual manipulation. Our results provide a functional characterization of mossy cells in the behaving animal and demonstrate their active participation in spatial coding and contextual representation.

Expression of vesicular glutamate transporter-2 in gonadotrope and thyrotrope cells of the rat pituitary. Regulation by estrogen and thyroid hormone status.

Immunocytochemical studies of the rat adenohypophysis identified a cell population that exhibits immunoreactivity for type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype. The in situ hybridization detection of VGLUT2 mRNA expression in adenohypophysial cells verified that VGLUT2 immunoreactivity is due to local synthesis of authentic VGLUT2. Dual-immunofluorescent studies of the hypophyses from male rats showed the presence of VGLUT2 in high percentages of LH (93.3 +/- 1.3%)-, FSH (44.7 +/- 3.9%)-, and TSH (70.0 +/- 5.6%)-immunoreactive cells and its much lower incidence in cells of the prolactin, GH, and ACTH phenotypes. Quantitative in situ hybridization studies have established that the administration of a single dose of 17-beta-estradiol (20 microg/kg; sc) to ovariectomized rats significantly elevated VGLUT2 mRNA in the adenohypophysis 16 h postinjection. Thyroid hormone dependence of VGLUT2 expression was addressed by the comparison of hybridization signals in animal models of hypo- and hyperthyroidism to those in euthyroid controls. Although hyperthyroidism had no effect on VGLUT2 mRNA, hypothyroidism increased adenohypophysial VGLUT2 mRNA levels. This coincided with a decreased ratio of VGLUT2-immunoreactive TSH cells, regarded as a sign of enhanced secretion. The presence of the glutamate marker VGLUT2 in gonadotrope and thyrotrope cells, and its up-regulation by estrogen or hypothyroidism, address the possibility that endocrine cells of the adenohypophysis may cosecrete glutamate with peptide hormones in an estrogen- and thyroid status-regulated manner. The exact roles of endogenous glutamate observed primarily in gonadotropes and thyrotropes, including its putative involvement in autocrine/paracrine regulatory mechanisms, will require clarification.

Progressive Decrease of Mitochondrial Motility during Maturation of Cortical Axons In Vitro and In Vivo.

The importance of mitochondria for neuronal function is evident by the large number of neurodegenerative diseases that have been associated with a disruption of mitochondrial function or transport (reviewed in [1, 2]). Mitochondria are essential for proper biological function as a result of their ability to produce ATP through oxidative phosphorylation, buffer cytoplasmic calcium, regulate lipid biosynthesis, and trigger apoptosis (reviewed in [2]). Efficient transport of mitochondria is thought to be particularly important in neurons in light of their compartmentalization, length of axonal processes, and high-energy requirements (reviewed in [3]). However, the majority of these results were obtained using short-term, in vitro neuronal culture models, and very little is currently known about mitochondrial dynamics in mature axons of the mammalian CNS in vitro or in vivo. Furthermore, recent evidence has demonstrated that mitochondrial immobilization at specific points along the axon, such as presynaptic boutons, play critical roles in axon morphogenesis [4, 5]. We report that as cortical axons mature, motility of mitochondria (but not other cargoes) is dramatically reduced and this coincides with increased localization to presynaptic sites. We also demonstrate using photo-conversion that in vitro mature axons display surprisingly limited long-range mitochondrial transport. Finally, using in vivo two-photon microscopy in anesthetized or awake-behaving mice, we document for the first time that mitochondrial motility is also remarkably low in distal cortical axons in vivo. These results argue that mitochondrial immobilization and presynaptic localization are important hallmarks of mature CNS axons both in vitro and in vivo.

Rabies Virus CVS-N2c(ΔG) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability.

Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19(ΔG) transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2c(ΔG), and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19(ΔG), the CVS-N2c(ΔG) strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2c(ΔG) strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS.

Histamine H3 receptors inhibit serotonin release in substantia nigra pars reticulata.

The substantia nigra pars reticulata (SNr) plays a key role in basal ganglia function. Projections from multiple basal ganglia nuclei converge at the SNr to regulate nigrothalamic output. The SNr is also characterized by abundant aminergic input, including dopaminergic dendrites and axons containing 5-hydroxytryptamine (5-HT) or histamine (HA). The functions of HA in the SNr include motor control via HA H3 receptors (H3Rs), although the mechanism remains far from elucidated. In Parkinson's disease, there is an increase in H3Rs and the density of HA-immunoreactive axons in the SN. We explored the role of H3Rs in the regulation of 5-HT release in SNr using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in rat midbrain slices. Immunohistochemistry identified a similar distribution for histaminergic and serotonergic processes in the SNr: immunoreactive varicosities were observed in the vicinity of dopaminergic dendrites. Electrically evoked 5-HT release was dependent on extracellular Ca2+ and prevented by NaV+-channel blockade. Extracellular 5-HT concentration was enhanced by inhibition of uptake transporters for 5-HT but not dopamine. Selective H3R agonists (R)-(-)-alpha-methyl-histamine or immepip inhibited evoked 5-HT release by up to 60%. This inhibition was prevented by the H3R antagonist thioperamide but not by the 5-HT1B receptor antagonist isamoltane. H3R inhibition of 5-HT release prevailed in the presence of GABA or glutamate receptor antagonists (ionotropic and metabotropic), suggesting minimal involvement of GABA or glutamate synapses. The potent regulation of 5-HT by H3Rs reported here not only elucidates HA function in the SNr but also raises the possibility of novel targets for basal ganglia therapies.

Hypophysiotropic thyrotropin-releasing hormone and corticotropin-releasing hormone neurons of the rat contain vesicular glutamate transporter-2.

TRH and CRH are secreted into the hypophysial portal circulation by hypophysiotropic neurons located in parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVH). Recently these anatomical compartments of the PVH have been shown to contain large numbers of glutamatergic neurons expressing type 2 vesicular glutamate transporter (VGLUT2). In this report we presented dual-label in situ hybridization evidence that the majority (>90%) of TRH and CRH neurons in the PVH of the adult male rat express the mRNA encoding VGLUT2. Dual-label immunofluorescent studies followed by confocal laser microscopic analysis of the median eminence also demonstrated the occurrence of VGLUT2 immunoreactivity within TRH and CRH axon varicosities, suggesting terminal glutamate release from these neuroendocrine systems. These data together indicate that the hypophysiotropic TRH and CRH neurons possess glutamatergic characteristics. Future studies will need to address the physiological significance of the endogenous glutamate content in these neurosecretory systems in the neuroendocrine regulation of thyroid and adrenal functions.

Ambient GABA modulates septo-hippocampal inhibitory terminals via presynaptic GABAb receptors.

The septo-hippocampal GABAergic pathway connects inhibitory neurons in the medial septum with hippocampal interneurons. Phasic release of GABA from septo-hippocampal terminals is thought to play an important role in shaping hippocampal network activity during behavior. Here, we found that GABA release from septo-hippocampal terminals is under negative feedback from the hippocampal local inhibitory network. We found that the strength of septo-hippocampal GABAergic inhibition is constrained by presynaptic GABAb receptors that are activated by ambient GABA during states of increased hippocampal network activity.

Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone neurons of the adult male rat.

Isoforms of the recently cloned vesicular glutamate transporters (VGLUT1-3) selectively accumulate glutamic acid into synaptic vesicles in excitatory axon terminals and are viewed as reliable markers for glutamatergic neurons. Our present studies provided dual-label in situ hybridization evidence that virtually all (99.5%) GnRH neurons express VGLUT2 mRNA in the preoptic region of the adult male rat. Dual-label immunofluorescent experiments were carried out to examine the presence of VGLUT2 protein in GnRH axon terminals. Confocal laser microscopic analysis of the organum vasculosum of the lamina terminalis and the external zone of the median eminence, the major termination fields for GnRH-secreting axons, demonstrated the frequent occurrence of VGLUT2 immunoreactivity in GnRH axon terminals. Together these mRNA hybridization and immunocytochemical data indicate that GnRH neurons of the adult male rat possess marked glutamatergic characteristics. The physiological significance of endogenous glutamate in the regulation of gonadotropin secretion requires clarification.

Origin of neuropeptide Y-containing afferents to gonadotropin-releasing hormone neurons in male mice.

The origin of neuropeptide Y (NPY) afferents to GnRH neurons was investigated in male mice. Neonatal lesioning of the hypothalamic arcuate nuclei (ARC) with monosodium glutamate markedly reduced the number of NPY fibers in the preoptic area as well as the frequency of their contacts with perikarya and proximal dendrites of GnRH neurons. Dual-label immunofluorescence studies to determine the precise contribution of the ARC to the innervation of GnRH neurons by NPY axons were carried out on transgenic mice in which enhanced green fluorescent protein was expressed under the control of the GnRH promoter (GnRH-enhanced green fluorescent protein mice). The combined application of red Cy3 and blue AMCA fluorochromogenes established that 49.1 +/- 7.3% of NPY axons apposed to green GnRH neurons also contained agouti-related protein (AGRP), a selective marker for NPY axons arising from the ARC. Immunoelectronmicroscopic analysis detected symmetric synapses between AGRP fibers and GnRH-immunoreactive perikarya. Additional triple-fluorescence experiments revealed the presence of dopamine-beta-hydroxylase immunoreactivity within 25.4 +/- 3.3% of NPY afferents to GnRH neurons. This enzyme marker enabled the selective labeling of NPY pathways ascending from noradrenergic/adrenergic cell populations of the brain stem, thus defining a second important source for NPY-containing fibers regulating GnRH cells. The absence of both topographic markers (AGRP and dopamine-beta-hydroxylase) within 26% of NPY contacts suggests that additional sources of NPY fibers to GnRH neurons exist. Future studies will address distinct functions of the two identified NPY systems in the afferent neuronal regulation of the GnRH system.

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves.

Sharp-wave ripples are transient oscillatory events in the hippocampus that are associated with the reactivation of neuronal ensembles within specific circuits during memory formation. Fast-spiking, parvalbumin-expressing interneurons (FS-PV INs) are thought to provide fast integration in these oscillatory circuits by suppressing regenerative activity in their dendrites. Here, using fast 3D two-photon imaging and a caged glutamate, we challenge this classical view by demonstrating that FS-PV IN dendrites can generate propagating Ca(2+) spikes during sharp-wave ripples. The spikes originate from dendritic hot spots and are mediated dominantly by L-type Ca(2+) channels. Notably, Ca(2+) spikes were associated with intrinsically generated membrane potential oscillations. These oscillations required the activation of voltage-gated Na(+) channels, had the same frequency as the field potential oscillations associated with sharp-wave ripples, and controlled the phase of action potentials. Furthermore, our results demonstrate that the smallest functional unit that can generate ripple-frequency oscillations is a segment of a dendrite.