Differential temporal filtering in the fly optic lobe.

There is a growing consensus that brain development in Huntington's disease (HD) is abnormal, leading to the idea that HD is not only a neurodegenerative but also a neurodevelopmental disorder. Indeed, structural and functional abnormalities have been observed during brain development in both humans and animal models of HD. However, a concurrent study of cortical and striatal development in a genetic model of HD is still lacking. Here we report significant alterations of corticostriatal development in the R6/2 mouse model of juvenile HD. We examined wildtype (WT) and R6/2 mice at postnatal (P) days 7, 14, and 21. Morphological examination demonstrated early structural and cellular alterations reminiscent of malformations of cortical development, and ex vivo electrophysiological recordings of cortical pyramidal neurons (CPNs) demonstrated significant age- and genotype-dependent changes of intrinsic membrane and synaptic properties. In general, R6/2 CPNs had reduced cell membrane capacitance and increased input resistance (P7 and P14), along with reduced frequency of spontaneous excitatory and inhibitory synaptic events during early development (P7), suggesting delayed cortical maturation. This was confirmed by increased occurrence of GABA A receptor-mediated giant depolarizing potentials at P7. At P14, the rheobase of CPNs was significantly reduced, along with increased excitability. Altered membrane and synaptic properties of R6/2 CPNs recovered progressively, and by P21 they were similar to WT CPNs. In striatal medium-sized spiny neurons (MSNs), a different picture emerged. Intrinsic membrane properties were relatively normal throughout development, except for a transient increase in membrane capacitance at P14. The first alterations in MSNs synaptic activity were observed at P14 and consisted of significant deficits in GABAergic inputs, however, these also were normalized by P21. In contrast, excitatory inputs began to decrease at this age. We conclude that the developing HD brain is capable of compensating for early developmental abnormalities and that cortical alterations precede and are a main contributor of striatal changes. Addressing cortical maldevelopment could help prevent or delay disease manifestations.

Corticostriatal maldevelopment in the R6/2 mouse model of juvenile Huntington's disease.

Type I spiral ganglion cells provide the afferent innervation to the inner hair cells of the mammalian organ of Corti and project centrally to the cochlear nucleus. While single-unit studies conducted over the past several decades have provided a wealth of information concerning the response characteristics of these neurons and, to some extent, their receptor targets, little is known about the neuron's intrinsic electrical properties. These properties undeniably will contribute to the firing patterns induced by acoustic stimuli. Type I spiral ganglion cell somata from the guinea pig inner ear were acutely isolated and the voltage-dependent conductances were analyzed with the whole-cell voltage clamp. Under conditions that mimic the normal intra- and extracellular ionic environments, type I spiral ganglion cells demonstrate fast inward TTX-sensitive Na currents (whose current density varied markedly among cells) and somewhat more slowly developing outward K currents. Resting potentials averaged -67.3 mV. Under current clamp, no spontaneous spike activity was noted, but short current injections produced graded action potentials with after hyperpolarizations lasting several milliseconds. The nondecaying outward K current activated at potentials near rest and was characterized by a pronounced rectification. The kinetics of the Na and K currents were rapid. Maximum peak inward Na currents occurred within 400 microseconds, between a voltage range of -10 and 0 mV, and inactivated within 4 msec. Recovery from inactivation was also rapid. At a holding potential of -80 mV, the time constant for recovery from an inactivating voltage step to -10 mV was 2.16 msec. Above -50 mV outward K currents reach half-maximal amplitude within 1.5 msec. In addition to these currents, a slow noninactivating TTX-sensitive inward current was observed that was blockable with Cd2+ or Gd3+. Problems encountered with blocking the tremendous outward K current hampered the characterization of this inward current. Similarities between the kinetics of ganglion cell currents and some of the rapid temporal characteristics of eighth nerve single-unit activity confirm the notion that intrinsic membrane properties help shape auditory neuron responses to sound.

Tiling of R7 Axons in the Drosophila Visual System Is Mediated Both by Transduction of an Activin Signal to the Nucleus and by Mutual Repulsion

Under awake and idling conditions, spontaneous intracellular membrane voltage is characterized by large, synchronous, low-frequency fluctuations. Although these properties reflect correlations in synaptic inputs, intrinsic membrane properties often indicate voltage-dependent changes in membrane resistance and time constant values that can amplify and help to generate low-frequency voltage fluctuations. The specific contribution of intrinsic and synaptic factors to the generation of spontaneous fluctuations, however, remains poorly understood. Using visually guided intracellular recordings of somatosensory layer 2/3 pyramidal cells and interneurons in awake male and female mice, we measured the spectrum and size of voltage fluctuation and intrinsic cellular properties at different voltages. In both cell types, depolarizing neurons increased the size of voltage fluctuations. Amplitude changes scaled with voltage-dependent changes in membrane input resistance. Because of the small membrane time constants observed in both pyramidal cells and interneuron cell bodies, the low-frequency content of membrane fluctuations reflects correlations in the synaptic current inputs rather than significant filtering associated with membrane capacitance. Further, blocking synaptic inputs minimally altered somatic membrane resistance and time constant values. Overall, these results indicate that spontaneous synaptic inputs generate a low-conductance state in which the amplitude, but not frequency structure, is influenced by intrinsic membrane properties.SIGNIFICANCE STATEMENT In the absence of sensory drive, cortical activity in awake animals is associated with self-generated and seemingly random membrane voltage fluctuations characterized by large amplitude and low frequency. Partially, these properties reflect correlations in synaptic input. Nonetheless, neurons express voltage-dependent intrinsic properties that can potentially influence the amplitude and frequency of spontaneous activity. Using visually guided intracellular recordings of cortical neurons in awake mice, we measured the voltage dependence of spontaneous voltage fluctuations and intrinsic membrane properties. We show that voltage-dependent changes in membrane resistance amplify synaptic activity, whereas the frequency of voltage fluctuations reflects correlations in synaptic inputs. Last, synaptic activity has a small impact on intrinsic membrane properties in both pyramidal cells and interneurons.

Molecular and circuit analysis of stable contrast processing in the visual system

Visual motion is an essential cue for many sighted animals. This can either be caused by the movement of an object, or the relative movement of the entire world caused by self-motion of the animal. Accordingly, the brain must compute both local motion cues, corresponding to spatiotemporal changes in luminance, and global motion patterns composed of many local motion vectors. In the fly eye, which is composed of hexagonally arranged visual units, the first-direction selective cells, the T4 and T5 neurons, are known as local motion detectors. Global motion was thought to be computed downstream, in large wide-field cells that sample information from many local motion detectors. Despite many years of research, the detailed mechanisms underlying local motion tuning in T4 and T5 cells, as well as the transformation of local into global motion information is not fully understood. In this thesis, I first studied the mechanisms of local motion detection and how local motion information is transferred into a global information about self-motion. First, blocking GABAergic signaling in the whole brain leads to a loss of direction-selectivity in T4 and T5 cells, arguing for a significant role of GABAergic circuits for local motion computation (Fisher et al., 2015a). However, GABAergic cell types and their potential interactions with the neuronal circuit responsible for motion detection had not yet been identified. Based on a behavioral genetic screen, in vivo calcium imaging and genetic analyses, we propose a GABAergic feedback mechanism, implemented by the two columnar C2 and C3 cells, to be required for directional tuning of T4 and T5 cells. Both neurons mainly interact with neurons upstream of T4 and T5 cells, indirectly affecting motion processing. While our data suggest a specific role of C2 for suppressing responses into the neuron’s non-preferred direction in T4 cells, C3 silencing affected the temporal properties of T4 and T5. T4 and T5 cells have been classified anatomically into four subtypes, ostensibly responding to the four cardinal directions of visual motion (Fisher et al., 2015a; Maisak et al., 2013). How these four motion axes arise, given the hexagonal arrangement of visual units in the fly eye, was not clear. Furthermore, it was not known how local cardinal motion from T4/T5 inputs can be transformed into complex optic flow fields encoded downstream. To understand how global motion is represented by the population of T4 and T5 cells, I used in vivo two-photon calcium imaging to characterize the direction tuning of T4 and T5 cells across visual space and the extent of the lobula plate. In contrast to the four anatomically subtypes described previously, we found six functional subtypes of local motion detectors at the population level / across the lobula plate. On average, tuning of these six subtypes matches the hexagonal structure of the eye. Tuning of neighboring motion detectors gradually changes, such that all T4/T5 cells of one subtype encode global motion patterns induced by translational and rotational self-movements of the fly. Together, the T4/T5 population represents six types of self-motion encountered during flight. Thus, downstream LPTCs can simply pool information from the local motion detectors, T4 and T5, to compute diverse complex flow fields. This population code for optic flow is reminiscent of coding of retinal ganglion cells in the vertebrate retina where only four directions of self-motion faced during walking are represented (Sabbah et al., 2017). While the number of motion dimensions encoded by the local motion detectors differ, this suggests a general coding strategy of visual systems to extract self-motion of the animal, adapted to the complexity of maneuvers encountered during locomotion. Taken together, the data presented in this thesis provide new insights about local as well as global mechanisms of visual motion processing in the fly and suggest striking parallels but also highlight differences between the vertebrate and invertebrate visual system. This is critical not only for understanding computational principles of visual systems but also for understanding how evolution adapts neuronal coding strategies to the animal’s behavioral constraints.

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO(2) avoidance, proboscis extension and giant-fiber mediated startle response. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-β, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-β, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Intracellular recording and staining of directionally selective motion detecting neurons in fly optic lobe

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

The morphology of visual interneurons in the tiger beetle larva was identified after recording their responses. Stained neurons were designated as either medulla or protocerebral neurons according to the location of their cell bodies. Medulla neurons were further subdivided into three groups. Afferent medulla neurons extended processes distally in the medulla neuropil and a single axon to the brain through the optic nerve. They received their main input from stemmata on the ipsilateral side. Two distance-sensitive neurons, near-by sensitive and far-sensitive neurons, were also identified. Atypical medulla neurons extended their neurites distally in the medulla and proximally to the brain, as afferent medulla neurons, but their input patterns and the shapes of their spikes differed from afferent neurons. Protocerebral neurons sent a single axon to the medulla neuropil. They spread collateral branches in the posterior region of the protocerebrum on its way to the medulla neuropil. They received main input from stemmata on the contralateral side. Medulla intrinsic neurons did not extend an axon to the brain, and received either bilateral or contralateral stemmata input only. The input patterns and discharge patterns of medulla neurons are discussed with reference to their morphology.

Photoreceptors of the fly's compound eye generally show no very obvious daily or circadian rhythms, a lack which prompted us to examine whether their function might be regulated not in the retina, but at the site of transmission in the first visual neuropile, or lamina. Here, photoreceptor terminals (R1-R6) are reciprocally interconnected with one class of lamina monopolar cell, L2: L2 receives input from R1-R6 at so-called tetrad synapses, and in turn is presynaptic to R1-R6 at feedback synapses. We have calculated the mean frequencies of these synaptic profiles in electron micrographs of single lamina sections. L2 feedback synapses were more numerous at night than during the day, whereas the number of tetrads showed only small modulations between day and night. These changes persisted amongst feedback synapses in flies held in constant darkness, and are thus circadian. In contrast to the slow modulations during a 24 h cycle, the number of L2 feedback synapses after 1 h light pulse in flies held in constant darkness showed no clear change, whereas it increased the number of tetrad profiles. These findings support the occurrence of cyclical daily and circadian changes amongst the two lamina synaptic populations, with tetrads showing rather weak modulations in frequency, but more pronounced responses to the light pulse than feedback synapses.

The algorithms and neural circuits that process spatio-temporal changes in luminance to extract visual motion cues have been the focus of intense research. An influential model, the Hassenstein-Reichardt correlator, relies on differential temporal filtering of two spatially separated input channels, delaying one input signal with respect to the other. Motion in a particular direction causes these delayed and non-delayed luminance signals to arrive simultaneously at a subsequent processing step in the brain; these signals are then nonlinearly amplified to produce a direction-selective response. Recent work in Drosophila has identified two parallel pathways that selectively respond to either moving light or dark edges. Each of these pathways requires two critical processing steps to be applied to incoming signals: differential delay between the spatial input channels, and distinct processing of brightness increment and decrement signals. Here we demonstrate, using in vivo patch-clamp recordings, that four medulla neurons implement these two processing steps. The neurons Mi1 and Tm3 respond selectively to brightness increments, with the response of Mi1 delayed relative to Tm3. Conversely, Tm1 and Tm2 respond selectively to brightness decrements, with the response of Tm1 delayed compared with Tm2. Remarkably, constraining Hassenstein-Reichardt correlator models using these measurements produces outputs consistent with previously measured properties of motion detectors, including temporal frequency tuning and specificity for light versus dark edges. We propose that Mi1 and Tm3 perform critical processing of the delayed and non-delayed input channels of the correlator responsible for the detection of light edges, while Tm1 and Tm2 play analogous roles in the detection of moving dark edges. Our data show that specific medulla neurons possess response properties that allow them to implement the algorithmic steps that precede the correlative operation in the Hassenstein-Reichardt correlator, revealing elements of the long-sought neural substrates of motion detection in the fly.

The metamorphic fate of larval visual interneurons in the swallowtail butterfly Papilio xuthus has been examined by using reduced silver impregnation and immunocytochemistry of gamma-amino butyric acid (GABA). Visual interneurons in the second larval optic neuropile (medulla) can be followed throughout metamorphosis because of large somata located in the anterior edge of the imaginal medulla. Ten to 12 neurons in the larval medulla were immunoreactive to a GABA-antiserum. They arborize in the larval medulla and extend dendritic processes to the first larval optic neuropile (lamina). After pupation, the medulla neurons lose GABA-immunoreactive larval processes and differentiate new processes that enter tangentially into the developing imaginal medulla. Axons of the surviving larval neurons follow an optic tract between the lobula and the lobula plate and extend to the lateral area of the protocerebrum. Thus, larval visual interneurons are incorporated into the imaginal optic lobe and may play a new role in the visual processing of the butterfly.