Single Synapse Indicators of Impaired Glutamate Clearance Derived from Fast iGluu Imaging of Cortical Afferents in the Striatum of Normal and Huntington (Q175) Mice

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single-synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGluu. We report findings from individual corticostriatal synapses in acute slices prepared from mice of either sex that were >1 year of age. Contrasting patterns of short-term plasticity and a size criterion identified two classes of terminals, presumably corresponding to the previously defined IT (intratelencephalic) and PT (pyramidal tract) synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGluu single-synapse study in Q175 mice, a model of Huntington's disease (HD). In HD mice, the decay time constant of the perisynaptic Glu concentration (TauD), as an indicator of uptake, and the peak iGluu amplitude, as an indicator of release, were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nm) mimicked the TauD changes in homozygotes. Considering the largest TauD values encountered in WT, ∼40% of PT synapses tested in Q175 heterozygotes can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGluu. Finally, EAAT2 (excitatory amino acid transport protein 2) immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention

Altered balance of glutamatergic/GABAergic synaptic input and associated changes in dendrite morphology after BDNF expression in BDNF-deficient hippocampal neurons

Cultured neurons from bdnf-/- mice display reduced densities of synaptic terminals, although in vivo these deficits are small or absent. Here we aimed at clarifying the local responses to postsynaptic brain-derived neurotrophic factor (BDNF). To this end, solitary enhanced green fluorescent protein (EGFP)-labeled hippocampal neurons from bdnf-/- mice were compared with bdnf-/- neurons after transfection with BDNF, bdnf-/- neurons after transient exposure to exogenous BDNF, and bdnf+/+ neurons in wild-type cultures. Synapse development was evaluated on the basis of presynaptic immunofluorescence and whole-cell patch-clamp recording of miniature postsynaptic currents. It was found that neurons expressing BDNF::EGFP for at least 16 h attracted a larger number of synaptic terminals than BDNF-deficient control neurons. Transfected BDNF formed clusters in the vicinity of glutamatergic terminals and produced a stronger upregulation of synaptic terminal numbers than high levels of ambient BDNF. Glutamatergic and GABAergic synapses reacted differently to postsynaptic BDNF: glutamatergic input increased, whereas GABAergic input decreased. BDNF::EGFP-expressing neurons also differed from BDNF-deficient neurons in their dendrite morphology: they exhibited weaker dendrite elongation and stronger dendrite initiation. The upregulation of glutamatergic synaptic input and the BDNF-induced downregulation of GABAergic synaptic terminal numbers by postsynaptic BDNF depended on tyrosine receptor kinase B activity, as deduced from the blocking effects of K252a. The suppression of dendrite elongation was also prevented by block of tyrosine receptor kinase B but required, in addition, glutamate receptor activity. Dendritic length decreased with the number of glutamatergic contacts. These results illuminate the role of BDNF as a retrograde synaptic regulator of synapse development and the dependence of dendrite elongation on glutamatergic input

Uncoupling the excitatory amino acid transporter 2 from its C-terminal interactome restores synaptic glutamate clearance at corticostriatal synapses and alleviates mutant huntingtin-induced hypokinesia

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. However, despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have not been fully uncovered. This limits the options for pharmacological treatment of glutamate-related motor disorders, including Huntington’s disease (HD). We therefore investigated the possible binding partners of transgenic EAAT2 and their alterations under the influence of mutant huntingtin (mHTT). Mass spectrometry analysis after pull-down of striatal YFP-EAAT2 from wild-type (WT) mice and heterozygote (HET) Q175 mHTT-knock-in mice identified a total of 148 significant (FDR < 0.05) binders to full-length EAAT2. Of them 58 proteins exhibited mHTT-related differences. Most important, in 26 of the 58 mHTT-sensitive cases, protein abundance changed back toward WT levels when the mice expressed a C-terminal-truncated instead of full-length variant of EAAT2. These findings motivated new attempts to clarify the role of astrocytic EAAT2 regulation in cortico-basal movement control. Striatal astrocytes of Q175 HET mice were targeted by a PHP.B vector encoding EAAT2 with different degree of C-terminal modification, i.e., EAAT2-S506X (truncation at S506), EAAT2-4KR (4 lysine to arginine substitutions) or EAAT2 (full-length). The results were compared to HET and WT injected with a tag-only vector (CTRL). It was found that the presence of a C-terminal-modified EAAT2 transgene (i) increased the level of native EAAT2 protein in striatal lysates and perisynaptic astrocyte processes, (ii) enhanced the glutamate uptake of transduced astrocytes, (iii) stimulated glutamate clearance at individual corticostriatal synapses, (iv) increased the glutamate uptake of striatal astrocytes and (iv) alleviated the mHTT-related hypokinesia (open field indicators of movement initiation). In contrast, over-expression of full-length EAAT2 neither facilitated glutamate uptake nor locomotion. Together, our results support the new hypothesis that preventing abnormal protein-protein interactions at the C-terminal of EAAT2 could eliminate the mHTT-related deficits in corticostriatal synaptic glutamate clearance and movement initiation

Optimal Control of Saccades by Spatial-Temporal Activity Patterns in the Monkey Superior Colliculus

A major challenge in computational neurobiology is to understand how populations of noisy, broadly-tuned neurons produce accurate goal-directed actions such as saccades. Saccades are high-velocity eye movements that have stereotyped, nonlinear kinematics; their duration increases with amplitude, while peak eye-velocity saturates for large saccades. Recent theories suggest that these characteristics reflect a deliberate strategy that optimizes a speed-accuracy tradeoff in the presence of signal-dependent noise in the neural control signals. Here we argue that the midbrain superior colliculus (SC), a key sensorimotor interface that contains a topographically-organized map of saccade vectors, is in an ideal position to implement such an optimization principle. Most models attribute the nonlinear saccade kinematics to saturation in the brainstem pulse generator downstream from the SC. However, there is little data to support this assumption. We now present new neurophysiological evidence for an alternative scheme, which proposes that these properties reside in the spatial-temporal dynamics of SC activity. As predicted by this scheme, we found a remarkably systematic organization in the burst properties of saccade-related neurons along the rostral-to-caudal (i.e., amplitude-coding) dimension of the SC motor map: peak firing-rates systematically decrease for cells encoding larger saccades, while burst durations and skewness increase, suggesting that this spatial gradient underlies the increase in duration and skewness of the eye velocity profiles with amplitude. We also show that all neurons in the recruited population synchronize their burst profiles, indicating that the burst-timing of each cell is determined by the planned saccade vector in which it participates, rather than by its anatomical location. Together with the observation that saccade-related SC cells indeed show signal-dependent noise, this precisely tuned organization of SC burst activity strongly supports the notion of an optimal motor-control principle embedded in the SC motor map as it fully accounts for the straight trajectories and kinematic nonlinearity of saccades

Diminution of Voltage Threshold Plays a Key Role in Determining Recruitment of Oculomotor Nucleus Motoneurons during Postnatal Development

The size principle dictates the orderly recruitment of motoneurons (Mns). This principle assumes that Mns of different sizes have a similar voltage threshold, cell size being the crucial property in determining neuronal recruitment. Thus, smaller neurons have higher membrane resistance and require a lower depolarizing current to reach spike threshold. However, the cell size contribution to recruitment in Mns during postnatal development remains unknown. To investigate this subject, rat oculomotor nucleus Mns were intracellularly labeled and their electrophysiological properties recorded in a brain slice preparation. Mns were divided into 2 age groups: neonatal (1–7 postnatal days, n = 14) and adult (20–30 postnatal days, n = 10). The increase in size of Mns led to a decrease in input resistance with a strong linear relationship in both age groups. A well-fitted inverse correlation was also found between input resistance and rheobase in both age groups. However, input resistance versus rheobase did not correlate when data from neonatal and adult Mns were combined in a single group. This lack of correlation is due to the fact that decrease in input resistance of developing Mns did not lead to an increase in rheobase. Indeed, a diminution in rheobase was found, and it was accompanied by an unexpected decrease in voltage threshold. Additionally, the decrease in rheobase co-varied with decrease in voltage threshold in developing Mns. These data support that the size principle governs the recruitment order in neonatal Mns and is maintained in adult Mns of the oculomotor nucleus; but during postnatal development the crucial property in determining recruitment order in these Mns was not the modifications of cell size-input resistance but of voltage threshold

The origin of horizontal gaze signals in neck muscles during orienting.

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Anatomical evidence for ipsilateral collicular projections to the spinal cord in the cat.

Injections of WGA-HRP were made within the C1 segment of spinal cord in cats with a midsagittal section of the midbrain. A small number of labelled cells were found in the latero-caudal part of the deeper layers of the superior colliclus (SC) ipsilateral to the injection sites. Because of the complete section of the dorsal tegmental decussation, these results definitively demonstrate the existence of an ipsilateral tecto-spinal pathway projecting to upper cervical segments in the cat. Ipsilaterally projecting tecto-reticulo-spinal neurons represent about 5% of the total population of tecto-spinal neurons. They were exclusively located in the deeper collicular layers and most of them were found in the latero-caudal part of the SC. Comparison with our previous studies suggests that more ipsilateral tecto-spinal projections that found after the section of the dorsal tegmental decussation probably exist. They may arise from tecto-reticulo-spinal neurons recrossing the midline in the brainstem or in the rostral part of C1. By analogy with the cortico-spinal tract, we suggest that the existence of an ipsilateral tecto-spinal pathway can be regarded as evidence for a substantial development of the cat tecto-spinal system as compared with other mammals

Rostrocaudal and lateromedial density distributions of superior colliculus neurons projecting in the predorsal bundle and to the spinal cord: a retrograde HRP study in the cat

Efferent neurons of the cat superior colliculus (SC) which project in the predorsal bundle (PDB) and to the spinal cord (PDB neurons) form a major pathway by which the SC controls the changes of the direction of gaze in response to stimuli of visual and other modalities. Knowledge of rostrocaudal and lateromedial density distributions of different groups of PDB neurons within the SC is necessary to analyse their relationships with the topography of sensory and motor maps. Density gradients may also bear on the efficacy of connections originating from topographically different collicular regions. In the present study, large injections of HRP/WGA-HRP were made in the C1 segment of the spinal cord and in the pontobulbar tegmentum. Judged by several morphological criteria, axons of passage, including those not subjected to a direct mechanical damage, were participating in the uptake of tracers. Therefore, labeled SC neurons corresponded to the nearly total populations of contralaterally projecting tectospinal neurons (TSNs) and neurons projecting in the PDB, respectively. Subtraction of the TSN density map from that of the whole PDB population was used to infer the distribution of tectal neurons terminating in the rhombencephalic tegmentum (TRhN). This subtotal labeling method proved useful in resolving the contradictions between the earlier HRP studies on the TSN and TRhN topography. The following density distributions were obtained for different groups of PDB neurons: 1) The mean TSN density is more than two times higher in the lateral half of the SC, representing the lower visual field. In this region the density remains constant from rostral to caudal, i.e., from the representation of vertical meridian to large contralateral azimuths. In the medial half, the average density decreases from rostral to caudal. Consequently, TSNs do not show the caudalward increment predicted by the higher efficacy of caudal stimulation points in eliciting head movements. 2) The distribution of PDB neurons is symmetrical with respect to the representation of the horizontal meridian. It is close to homogeneous at all azimuths of the retinotopic map and within the zone limited by small (10-15 degrees) upward and downward elevations.(ABSTRACT TRUNCATED AT 400 WORDS