Andrew S. Lowe

Parametric Functional Maps of Visual Inputs to the Tectum

SUMMARY How features of the visual scene are encoded in the population activity of retinal ganglion cells (RGCs) targeting specific regions of the brain is not well understood. To address this, we have used a genetically encoded reporter of presynaptic function (SyGCaMP3) to record visually evoked activity in the population of RGC axons innervating the zebrafish tectum. Using unbiased voxel-wise analysis of SyGCaMP3 signals, we identify three subtypes of direction-selective and two subtypes of orientationselective retinal input. Composite parametric functional maps generated across many larvae show laminar segregation of direction- and orientationselective responses and unexpected retinotopic biases in the distribution of functional subtypes. These findings provide a systematic description of the form, organization, and dimensionality of visual inputs to the brain and will serve as a platform for understanding emergent properties in tectal circuits associated with visually driven behavior.How features of the visual scene are encoded in the population activity of retinal ganglion cells (RGCs) targeting specific regions of the brain is not well understood. To address this, we have used a genetically encoded reporter of presynaptic function (SyGCaMP3) to record visually evoked activity in the population of RGC axons innervating the zebrafish tectum. Using unbiased voxel-wise analysis of SyGCaMP3 signals, we identify three subtypes of direction-selective and two subtypes of orientation-selective retinal input. Composite parametric functional maps generated across many larvae show laminar segregation of direction- and orientation-selective responses and unexpected retinotopic biases in the distribution of functional subtypes. These findings provide a systematic description of the form, organization, and dimensionality of visual inputs to the brain and will serve as a platform for understanding emergent properties in tectal circuits associated with visually driven behavior.

Small animal, whole brain fMRI: innocuous and nociceptive forepaw stimulation

Supra-spinal pain processing involves a number of extensive networks. An examination of these networks using small animal functional magnetic resonance imaging (fMRI) is difficult. While prior studies have successfully delineated regions consistent with known pain processing pathways, they have been restricted to acquisitions of limited spatial extent with coarse in-plane resolution to achieve a high temporal resolution. An isotropic, whole brain fMRI protocol has been developed for the examination of the supra-spinal consequences of innocuous and nociceptive electrical stimulation of the rat forepaw. Innocuous electrical stimulation of the rat forepaw delineated BOLD contrast responses consistent with known somatosensory processing pathways (contralateral primary somatosensory cortex (S1), a region consistent with secondary somatosensory cortex, the ventral posterolateral thalamic nucleus and ipsilateral cuneate nucleus), providing face validity for the technique. The putative noxious stimulus delineated additional regions consistent with the classical lateral and medial pain systems as well as secondarily associated areas: the aversion and descending inhibition systems. These included the ipsilateral inferior colliculus, anterior pretectal nucleus, mediodorsal thalamic nucleus, with regions in the pre-frontal, cingulated, ventral orbital and infra-limbic cortices, nucleus accumbens all exhibiting negative BOLD changes. Such regions are in agreement with, and extend, those previously reported. Acquisition, post-processing and analysis methodologies undertaken in this study constitute a marked extension of previous fMRI in the rat, enabling whole brain coverage at a spatial resolution sufficient to delineate regional changes in BOLD contrast consistent with somatosensory and nociceptive networks.

Emergent Properties of the Optic Tectum Revealed by Population Analysis of Direction and Orientation Selectivity

How local circuits within the brain process visual information has classically been addressed at the single neuron level. Such reductionist approaches, however, struggle to capture the full scope of functional properties associated with even “simple” brain nuclei. Using population functional calcium imaging, we aim to describe how local circuits within the zebrafish optic tectum process visual information. Specifically, how are previously identified direction-selective (DS) and orientation-selective (OS) retinal ganglion cell (RGC) inputs (Nikolaou et al., 2012) represented in tectal cells? First, we identify an emergent population of DS tectal cell with a direction preference not explicitly present in any one of the RGC inputs. Second, this is associated with a striking shift from a tiled and triangular representation of directional space (RGC inputs) into an overlapping cardinal representation by tectal cell populations. Third, and in contrast, we find that orientation space is represented similarly in both the RGC input and tectal cell populations illustrating feature-dependent differences in how tectal circuits process their inputs. Finally, we identify OS and two populations of DS cells at the superficial border of the tectal neuropil, one of which is an emergent population. This study, together with our previous one (Nikolaou et al., 2012), demonstrate that direction-selectivity is established in both the retina and tectum.

BDNF Promotes Axon Branching of Retinal Ganglion Cells via miRNA-132 and p250GAP

A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

Imaging localised dynamic changes in the nucleus accumbens following nicotine withdrawal in rats

This study utilises pharmacological functional magnetic resonance imaging (fMRI) to examine the neurobiological mechanisms through which nicotine produces dependence. Using an established regime to induce physical dependence to nicotine in rats (osmotic minipumps delivering 3.16 mg/kg/day nicotine for 7 days SC), animals were subsequently anaesthetised under urethane and positioned in a stereotaxic frame to allow collection of gradient echo whole brain images with a 4.7-T MRI spectrometer. Rats were initially scanned for 34 min (40 baseline image volumes, 1 volume per 51 s) then challenged with mecamylamine (1.0 mg/kg SC) or saline (1 ml/kg) and scanned for a further 68 min (80 image volumes). Mecamylamine precipitated highly significant positive changes in fMRI blood oxygen level dependent (BOLD) contrast that were predominantly localised to the NAc of nicotine-dependent rats. Saline-treated rats challenged with the same dose of mecamylamine exhibited similar but smaller increases in BOLD contrast although such changes were less defined around the NAc. Precipitated withdrawal also elicited statistically significant negative BOLD contrast changes in widespread cortical regions. These findings are consistent with previous neurochemical reports on decreases in dopamine in the NAc during nicotine withdrawal. This fMRI study further highlights the potential and power to image the neurobiological events during nicotine dependence.

Functional magnetic resonance neuroimaging of drug dependence: naloxone-precipitated morphine withdrawal.

This study investigated the potential utility of fMRI as a neuroimaging technique to examine drug dependence using a robust animal model of drug withdrawal. Two groups of rats chronically pretreated with incremental doses of morphine sulfate (2, 7, 15, 30, 40, 50, 50, and 50 mg/kg--subcutaneous injection) were subjected to opioid precipitated withdrawal (using the opioid antagonist, naloxone) and subsequently behaviorally assessed or gradient-echo imaged under urethane anesthesia. Whole brain, group statistical parametric maps revealed statistically significant changes in signal intensity following administration of 1 mg/kg naloxone (corrected for multiple comparisons: P < 0.05, T > 5.03). Control groups within the fully crossed designs did not exhibit any statistically significant changes in behavior or signal intensity changes. Regional patterns of modulated activity include the retrosplenial, piriform, insular, entorhinal, cingulate, visual and auditory cortices, posterior fields of the hippocampus, and in particular the dentate gyrus. Such areas are consistent with biochemical correlates of morphine withdrawal and time profiles derived from our behavioral observations (P < 0.02). A notable lack of signal intensity changes in a number of subcortical areas suggests a possible confound associated with fMRI under anesthesia. This paper reports the first whole brain fMRI examination of an animal model of drug withdrawal, we believe there is considerable scope for extrapolation of our methods to a multitude of pharmacological applications-most notably in conjunction with other techniques in the development of potential therapeutic agents for drug dependence.

Multi-modal characterisation of the neocortical clip model of focal cerebral ischaemia by MRI, behaviour and immunohistochemistry

The neocortical clip model of focal cerebral ischaemia has previously been used with success in neuroprotection studies. To further improve its translational qualities, we have characterised this model using a combination of serial Magnetic Resonance Imaging (MRI), neurological assessment, the bilateral asymmetry test (BAT) and immunohistochemistry. The right MCA was occluded in spontaneously hypertensive rats for 0, 60 and 120 min. MRI was performed pre-surgery, 1, 3 and 7 days post-surgery. Behavioural assessment was performed 2 days before and 3 and 7 days post-surgery whilst neurological deficits were monitored daily. Neuroimaging results showed that 0 min of MCA occlusion did not produce a lesion, whereas occlusion for 60 min produced a lesion that remained stable over time. Occlusion for 120 min caused a more severe lesion 1 day post-surgery, but decreased by 7 days. Behaviour, neurological scores and histological lesion volumes correlated strongly with MRI lesion volume. Immunohistochemistry revealed neuronal loss, astrogliosis and macrophage infiltration in lesioned cortices. The neocortical clip model produced ischaemic lesions that are restricted to cortical territories of the MCA. The duration of occlusion dictates lesion severity which may prove useful for probing therapeutic interventions at different stages of stroke progression. The correlation of MRI with two different behavioural measures and post-mortem histology strengthens the basis for MRI providing an in vivo surrogate marker for structural and behavioural deficits caused by a cortical stroke.

Mapping the effects of the selective dopamine D2/D3 receptor agonist quinelorane using pharmacological magnetic resonance imaging

Dopamine agonists with a high affinity for D2 and D3 receptors have a biphasic effect on rodent locomotion, inducing hypolocomotion at low doses and hyperlocomotion at higher doses. Controversy surrounds the role of the D3 receptor in mediating the hypolocomotor response to low agonist doses. This study examines patterns of neuronal activation induced by varying doses of the D2/D3 receptor agonist quinelorane using blood oxygen level dependent (BOLD) pharmacological magnetic resonance imaging (phMRI), and compares them with corresponding behavioural responses. Quinelorane (3 microg/kg) induced hypolocomotion in rats naive to the testing environment, and in phMRI experiments increased neuronal activity within the anterior olfactory nuclei, nucleus accumbens and islets of Calleja, regions containing a high density of D3 receptors. A 30 microg/kg dose of quinelorane resulted in biphasic locomotor effects, with initial hypolocomotion followed by sustained hyperlocomotion. phMRI indicated that this higher dose increased cerebral activity within limbic and olfactory regions, as did the lower drug dose, but induced additional activation in the caudate-putamen and globus pallidus, areas dense in D2 receptors but containing few D3 receptors. The more restricted pattern of activation at low agonist doses and close temporal relationship between behavioural and BOLD signal responses to quinelorane suggest that those nuclei most dense in D3 receptors play a key role in mediating the hypolocomotor effects of quinelorane. However, the presence of D3 receptors in activated brain regions may be coincidental, and further studies are required to show definitively which class of receptors mediates agonist-induced hypolocomotion. In contrast, the activation of D2 receptors within the striatum appears necessary for quinelorane-induced hyperlocomotion.

A Systems-Based Dissection of Retinal Inputs to the Zebrafish Tectum Reveals Different Rules for Different Functional Classes during Development

We have examined the form, diversity, and organization of three functional classes of retinal inputs to the zebrafish optic tectum during development. Our systems-based approach was to analyze data from populations of retinal ganglion cells labeled with a presynaptic targeted calcium indicator, synaptophysin GCaMP3 (SyGCaMP3). Collectively, our findings provide an insight as to the degree of visual encoding during retino-tectal development and how it dynamically evolves from a nascent and noisy presynaptic neural-scape to an increasingly complex and refined representation. We report five key features: (1) direction-selective inputs are developmentally invariant; (2) orientation-selective inputs exhibit highly dynamic properties over the same period, with changes in their functional characteristics and spatial organization; (3) inputs defined as anisotropic are an early dominant functional class, with heterogeneous response profiles, which progressively diminish in incidence and spatial extent; (4) dark rearing selectively affects the orientation-selective responses: both functional characteristics and relative spatial distributions; and (5) orientation-selective inputs exhibit four subtypes, two more than previously identified in any species. Our approach was to label RGC axon terminals with an indicator of activity and quantitatively characterize coherent response properties to different visual stimuli. Its application in the zebrafish, given its small size and the accessibility of the tectum, has enabled a quick yet robust assessment of multiple functional populations of responses.

Quantitative manganese tract tracing: dose-dependent and activity-independent terminal labelling in the mouse visual system

At concentrations sufficient for visualisation using MRI, manganese (Mn) is believed to behave as a calcium analogue. This study examines different concentrations of Mn for enhanced MR tract tracing. The premise of activity‐dependent axonal transport was also examined by partial or complete blockade of retinal ganglion cell activity. Quantitative T1 maps and semi‐quantitative normalised signal intensities in the superior colliculi facilitated assessment of applied intraocular concentrations and activity dependence, respectively. Varying the concentration of applied Mn revealed a non‐monotonic profile, with optimal, unfavourable and undesirable effects noted: 25 mM proved optimal, showing a maximal decrease in T1, whereas 400 mM was associated with no terminal‐field enhancement. The estimated vitreal concentration for optimal transport of Mn (2 mM) is substantially lower than that used in previous studies of the mouse. Both the partial blockade of inputs to 50% of retinal ganglion cells by a mGluR6 glutamate agonist and the complete blockade of all retinal ganglion cell activity with tetrodotoxin failed to decrease the relative enhancement in the superior colliculus. The failure to prevent axonal transport of Mn by blocking activity (and therefore theoretically the intracellular influx) appeared to be paradoxical. The optimal vitreal concentration of Mn has previously been shown to facilitate massive intracellular uptake of Mn, competitively blocking calcium, and 1 mM Mn blocks neurotransmission pre‐synaptically. These results suggest that, at concentrations required for optimal Mn‐enhanced MRI tract tracing in the visual system of the mouse, the uptake and transport of Mn may be dominated by passive mechanisms, which may also block neurotransmission. Copyright