Synaptic Distance Research Articles

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a "cytokine window" defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.

Postsynaptic cell type and synaptic distance do not determine efficiency of monosynaptic rabies virus spread measured at synaptic resolution.

Retrograde monosynaptic tracing using glycoprotein-deleted rabies virus is an important component of the toolkit for investigation of neural circuit structure and connectivity. It allows for the identification of first-order presynaptic connections to cell populations of interest across both the central and peripheral nervous system, helping to decipher the complex connectivity patterns of neural networks that give rise to brain function. Despite its utility, the factors that influence the probability of transsynaptic rabies spread are not well understood. While it is well established that expression levels of rabies glycoprotein used to trans-complement G-deleted rabies can result in large changes in numbers of inputs labeled per starter cell (convergence index [CI]), it is not known how typical values of CI relate to the proportions of synaptic contacts or input neurons labeled. And it is not known whether inputs to different cell types, or synaptic contacts that are more proximal or distal to the cell body, are labeled with different probabilities. Here, we use a new rabies virus construct that allows for the simultaneous labeling of pre- and postsynaptic specializations to quantify the proportion of synaptic contacts labeled in mouse primary visual cortex. We demonstrate that with typical conditions about 40% of first-order presynaptic excitatory synapses to cortical excitatory and inhibitory neurons are labeled. We show that using matched tracing conditions there are similar proportions of labeled contacts onto L4 excitatory pyramidal, somatostatin (Sst) inhibitory, and vasoactive intestinal peptide (Vip) starter cell types. Furthermore, we find no difference in the proportions of labeled excitatory contacts onto postsynaptic sites at different subcellular locations.

An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales

Astrocytes are complex bushy cells that serve important functions through close contacts between theirprocesses and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report agenetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following invivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT.

Sensory Neuroscience: Taste Responses in Primary Olfactory Cortex

A new electrophysiological study in rodents demonstrates that taste-odor convergence occurs in posterior piriform olfactory cortex and calls for a reformulation of classic models of the central representation of flavor.

Hierarchical computation in the canonical auditory cortical circuit

Sensory cortical anatomy has identified a canonical microcircuit underlying computations between and within layers. This feed-forward circuit processes information serially from granular to supragranular and to infragranular layers. How this substrate correlates with an auditory cortical processing hierarchy is unclear. We recorded simultaneously from all layers in cat primary auditory cortex (AI) and estimated spectrotemporal receptive fields (STRFs) and associated nonlinearities. Spike-triggered averaged STRFs revealed that temporal precision, spectrotemporal separability, and feature selectivity varied with layer according to a hierarchical processing model. STRFs from maximally informative dimension (MID) analysis confirmed hierarchical processing. Of two cooperative MIDs identified for each neuron, the first comprised the majority of stimulus information in granular layers. Second MID contributions and nonlinear cooperativity increased in supragranular and infragranular layers. The AI microcircuit provides a valid template for three independent hierarchical computation principles. Increases in processing complexity, STRF cooperativity, and nonlinearity correlate with the synaptic distance from granular layers.

Dendritic amplification of inhibitory postsynaptic potentials in a model Purkinje cell

In neurons with large dendritic arbors, the postsynaptic potentials interact in a complex manner with active and passive membrane properties, causing not easily predictable transformations during the propagation from synapse to soma. Previous theoretical and experimental studies in both cerebellar Purkinje cells and neocortical pyramidal neurons have shown that voltage-dependent ion channels change the amplitude and time-course of postsynaptic potentials. We investigated the mechanisms involved in the propagation of inhibitory postsynaptic potentials (IPSPs) along active dendrites in a model of the Purkinje cell. The amplitude and time-course of IPSPs recorded at the soma were dependent on the synaptic distance from the soma, as predicted by passive cable theory. We show that the effect of distance on the amplitude and width of the IPSP was significantly reduced by the dendritic ion channels, whereas the rise time was not affected. Somatic IPSPs evoked by the activation of the most distal synapses were up to six times amplified owing to the presence of voltage-gated channels and the IPSP width became independent of the covered distance. A transient deactivation of the Ca(2+) channels and the Ca(2+)-dependent K(+) channels, triggered by the hyperpolarization following activation of the inhibitory synapse, was found to be responsible for these dynamics. Nevertheless, the position of activated synapses had a marked effect on the Purkinje cell firing pattern, making stellate cells and basket cells most suitable for controlling the firing rate and spike timing, respectively, of their target Purkinje cells.

Pharmacology and Physiology of Perivascular Nerves Regulating Vascular Function: Sympathetic Modulation of Nitrergic Neurogenic Vasodilation

The presence of close apposition between the adrenergic and the non-adrenergic or nitrergic nerve terminals in large cerebral arteries in several species is well documented. The axo-axonal distance between these different types of nerve terminals is substantially closer than the synaptic distance between the adventitial nerve terminals and the outermost layer of smooth muscle in the media. This feature suggests that a functional axo-axonal interaction between nerve terminals is more likely to occur than that between the nerve and muscle. Thus, transmitters released from one nerve terminal may modulate release of transmitters from the neighboring nerve terminals, resulting in a neurogenic response. We have reported that nicotine-induced nitric oxide (NO)-mediated neurogenic vasodilation is dependent on intact sympathetic innervation in porcine and cat cerebral arteries. Evidence also has been presented to indicate that nicotine acts on alpha7-nicotinic receptors located on sympathetic nerve terminals, resulting in release of norepinephrine which then diffuses to act on beta2-adrenoceptos located on the neighboring nitrergic nerve terminals to release NO and therefore vasodilation. The predominant facilitatory effect of beta2-adrenoceptors in releasing NO is compromised by presynaptic alpha2-adrenoceptors located on the same nerves. Activation of cerebral sympathetic nerves may cause NO-mediated dilation in large cerebral arteries at the base of the brain.

Presynaptic contributions to response shape in an auditory neuron of the grasshopper

Neuron 714 is morphologically one of the most prominent neurons in the central auditory pathway of the grasshopper with arborizations extending from the abdominal neuromeres of the metathoracic ganglion to the brain. The aim of this study is to explore auditory information flow involving neuron 714 at the level of the ventral nerve cord. Paired intracellular recordings were made from neuron 714 in the mesothorax on the one hand, and from candidate presynaptic auditory neurons of the metathorax on the other. Electrical stimulation of the tympanal nerves provides an estimate of the synaptic distance between these interneurons and auditory afferents. Four, including neuron 714, are monosynaptically connected to afferents, the remainder disynaptically. Current-injection and spike-triggered averaging reveal that of nine neurons examined, seven make either monosynaptic, disynaptic or polysynaptic connections onto neuron 714. All connections are excitatory. Paired recordings show that response duration and response amplitude in synaptically linked cells vary according to the frequency of the stimulus. Measurements of the latency of the first excitatory post-synaptic potential evoked in neuron 714 by afferents and by metathoracic interneurons show how the synaptic drive from these sources shapes the auditory response of neuron 714.

The constructive nature of vision: direct evidence from functional magnetic resonance imaging studies of apparent motion and motion imagery.

Echoplanar functional magnetic resonance imaging was used to monitor activation changes of brain areas while subjects viewed apparent motion stimuli and while they were engaged in motion imagery. Human cortical areas MT (V5) and MST were the first areas of the 'dorsal' processing stream which responded with a clear increase in signal intensity to apparent motion stimuli as compared with flickering control conditions. Apparent motion of figures defined by illusory contours evoked greater activation in V2 and MT/MST than appropriate control conditions. Several areas of the dorsal pathway (V3A, MT/MST, areas in the inferior and superior parietal lobule) as well as prefrontal areas including FEF and BA 9/46 responded strongly when subjects merely imagined moving stimuli which they had seen several seconds before. The activation during motion imagery increased with the synaptic distance of an area from V1 along the dorsal processing stream. Area MT/MST was selectively activated during motion imagery but not during a static imagery control condition. The comparison between the results obtained with objective motion, apparent motion and imagined motion provides further insights into a complex cortical network of motion-sensitive areas driven by bottom-up and top-down neural processes.

Elicitation, modification, and conditioning of the rabbit nictitating membrane response by electrical stimulation in the spinal trigeminal nucleus, inferior olive, interpositus nucleus, and red nucleus.

Elicitation of responses by electrical brain stimulation (EBS) was related to the synaptic distance of the target nucleus from the accessory abducens. Specifically, responses to EBS in the spinal trigeminal nucleus (TRIG) and red nucleus (RN) increased as a positive function of stimulation parameters. Responding to EBS in the interpositus nucleus (IP) was lower, and responding to EBS in the inferior olive (IO) was negligible. EBS in the TRIG, IP, and RN nuclei was then paired with a tone conditioned stimulus (CS). The CS modified responses for EBS in RN and TRIG but not IP. CS-EBS pairings yielded conditioned response (CR) acquisition, in which Groups TRIG, IP, and RN reached asymptotes of 90%, 70%, and 43% CRs, respectively. Thus, contrary to previous findings, EBS in the efferent pathway can support CR acquisition. The results are discussed with respect to the role of projections from the RN to the cerebellar cortex and the TRIG nucleus.

Long-term elevation of cyclooxygenase-2, but not lipoxygenase, in regions synaptically distant from spreading depression.

Eicosanoids, produced from arachidonic acid by cyclooxygenases (COXs) and lipoxygenases (LIPOXs), are involved in numerous brain processes. To explore if brief and noninjurious stimuli chronically alter expression of these enzymes, we examined the induction of COX-2 and LIPOX expression following unilateral neocortical spreading depression (SD). Expression was examined over time and in regions not experiencing SD (hippocampus) but synaptically connected to the site of stimulation (cortex). One hundred six male Wistar rats had SD induced via microinjection of 0.5 M KCl (0.5 M NaCl for sham) into left parietal cortex every 9 minutes for 1 or 3 hours. One hour before SD some animals received dexamethasone (Dex), mepacrine (Mep), indomethacin (Indo), nordihydroguaiaretic acid (Ndga), phenylephrine (Pe), sodium nitroprusside (Snp) with Pe, or N omega-nitro-L-arginine methyl ester (Lnam). Animals survived for 0, 3, or 6 hours, or 1, 2, 3, 7, 14, 21, or 28 days. Brains were processed immunohistochemically for COX-2 and LIPOX, and the optical density (OD) of the left and right cortex, dentate gyrus (DG), CA3, and CA1 immunoreactivity (IR) were measured. Induction was expressed as the log of left divided by right side OD for each region. COX-2 IR in the left cortex was elevated rapidly and was sustained for 21 days following SD. COX-2 IR was also elevated in the ipsilateral hippocampus not experiencing SD, with the rank order of induction as follows: DG > CA3 > CA1. Dex, Snp, and/or Pe significantly reduced the induction of COX-2. No changes in LIPOX IR were observed. These results show that long-term changes in COX-2 expression are induced by SD and these changes decrease with synaptic distance. Benign stimuli increase COX-2 expression and thus may influence brain function for extended periods and at distant locations.

Homologue pairing and synaptic behavior at zygotene in maize.

A new fixation technique has permitted more detailed light microscope observations of the leptotene to pachytene meiotic progression of maize microsporocyte chromosomes than has been possible following use of traditional fixatives. With use of this new procedure, squash preparation produces excellent spreading of chromosomes at stages where the tightness of the synizetic knot has previously prohibited analysis. Using this technique, a novel finding was that at zygotene stage, extensive homologue alignment, which can be either loose or closely parallel, but at greater than synaptic distance, apparently precedes synapsis. Synaptic initiation is frequently intercalary and there may be a number of sites of initiation per bivalent. Telomeres and conspicuous heterochromatic regions are often among the last parts to synapse. The clear alignment of homologues in advance of synapsis is regarded as strongly suggestive of the existence of multiple chromosome sites which are active at homologue pairing and function in advance of synapsis.

Identification of excitatory and inhibitory neuromuscular synapses in the crayfish thoracic deep flexor musculature

Excitatory and inhibitory neuromuscular synapses in the thoracic deep flexors of the crayfish were examined using electron microscopy. Excitatory nerve terminals were found to have larger synaptic vesicles and a greater synaptic cleft distance than did inhibitory terminals. These results were confirmed by fixation enhancement of differences in synaptic vesicle morphology and the experimental depletion of vesicles in the excitatory nerve terminals.

Localization of synaptic input on dendrites of a lamprey spinal cord neurone from physiological measurements of membrane properties.

1. Composite excitatory post-synaptic potentials (e.p.s.p.s) resulting from electrotonic and chemical synaptic junctions were recorded from eighteen interneurones following stimulation of the I2 burster axon in the isolated lamprey spinal cord. 2. In each cell, the half-width of the electrotonic e.p.s.p. was measured and used, together with the cable parameters estimated for the same neurone, to locate the position of synaptic contact made by the I2 axon on the dendrites of the interneurone. The synaptic location ranged from 0.05 to 1.35 space constants with a mean of 0.46. 3. The synaptic potential was simulated using the Rall model of the neurone. When compared with the experimentally recorded e.p.s.p. with the same half-width, the rise-time of the simulated synaptic potential was found to be faster. By changing the value of synaptic distance and/or synaptic current duration the half-width, rise-time, and decay of the simulated synaptic potential fit closely the experimental e.p.s.p. The range of synaptic distance estimated from the simulation decreased considerably (0.2--0.7 space constants; mean 0.52). 4. Direct comparison of synaptic location estimated from histological tracings of dendritic trees from these same cells injected with horseradish peroxidase compared favourably with synaptic location estimated from the simulations. 5. These results support the hypothesis that functionally similar presynaptic axons make synaptic connexions at the same electrotonic distance from the soma on functionally similar post-synaptic cells. This occurs in the face of large variations in physical distance for these same synaptic contacts.

Organization of cat striate cortex: a correlation of receptive-field properties with afferent and efferent connections.

The purposes of this study were 1) to relate the receptive-field characteristics of area 17 cells to their afferent and efferent connections, and 2) to obtain quantitative data from area 17 neurons for later comparison with area 18 cells. Intra- and extracellular recordings were obtained in paralyzed preparations which were anesthetized with nitrous oxide. The connectivities of the recorded cells were determined from responses to electrical stimulation of afferent and efferent pathways. In parallel to the classification of units as simple and complex cells, the receptive fields were grouped in four classes according to the spatial arrangement of on- and off-areas; class I, fields with exclusive on- or off-areas; class II, fields with spatially separate on- and off-areas; class III, fields with mixed on-off areas; class IV, fields which could not be mapped with stationary stimuli. The results from electrical stimulation suggest two major classes of cells: cells in the first group are driven mainly or exclusively by LGN afferents. They rarely receive additional excitation from intrinsic or callosal afferents and rarely possess corticofugal axons. Cells in the second group receive either converging inputs from LGN afferents and further intrinsic afferents or only from intrinsic afferents. They frequently received additional input from callosum and from recurrent collaterals of corticofugal axons. They project subcortically more often than cells in the first group. Cells in both groups can be driven either by X- or Y-type afferents. Cells in the first group have mainly class I and class II fields or simple fields, whereas the neurons in the second group have mainly class III and class IV fields or complex fields. Thus, simple and complex cells differ in their connectivity patterns, but the discriminative parameter is neither the selective connection to the X- or the Y-system nor, in a strict sense, the synaptic distance from subcortical input. From the combined consideration of receptive-field properties and connectivity patterns it is concluded that class I and class II cells or simple cells are concerned mainly with the primary analysis of subcortical activity, whereas class III and class IV cells or complex cells perform a correlative analysis between highly convergent activity from extrinsic and intrinsic afferents.

Ultrastructural evidence for adrenergic innervation of the interstitial gland in the guinea pig ovary.

Fluorescence microscopy has shown that the guinea pig ovary contains not only vasomotor adrenergic nerves but also many nerve terminals running within endocrine structures. Electron microscopy of the interstitial gland confirmed the presence of adrenergic nerves distinguished by administration of the false transmitter, 5-hydroxydropamine. Axon varicosities, denuded of their Schwann cell ensheathing, innervated the endocrine cells in the interstitial gland, with a synaptic distance of approximately 400 A. This suggests that the interstitial gland participates in a neuroendocrine transducer mechanism.