Brain mapping across 16 autism mouse models reveals a spectrum of functional connectivity subtypes.

Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.

In-Depth Characterization of Layer 5 Output Neurons of the Primary Somatosensory Cortex Innervating the Mouse Dorsal Spinal Cord.

Neuronal circuits of the spinal dorsal horn integrate sensory information from the periphery with inhibitory and facilitating input from higher central nervous system areas. Most previous work focused on projections descending from the hindbrain. Less is known about inputs descending from the cerebral cortex. Here, we identified cholecystokinin (CCK) positive layer 5 pyramidal neurons of the primary somatosensory cortex (CCK + S1-corticospinal tract [CST] neurons) as a major source of input to the spinal dorsal horn. We combined intersectional genetics and virus-mediated gene transfer to characterize CCK+ S1-CST neurons and to define their presynaptic input and postsynaptic target neurons. We found that S1-CST neurons constitute a heterogeneous population that can be subdivided into distinct molecular subgroups. Rabies-based retrograde tracing revealed monosynaptic input from layer 2/3 pyramidal neurons, from parvalbumin positive cortical interneurons, and from thalamic relay neurons in the ventral posterolateral nucleus. Wheat germ agglutinin-based anterograde tracing identified postsynaptic target neurons in dorsal horn laminae III and IV. About 60% of these neurons were inhibitory and about 60% of all spinal target neurons expressed the transcription factor c-Maf. The heterogeneous nature of both S1-CST neurons and their spinal targets suggest complex roles in the fine-tuning of sensory processing.

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals.

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.

In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons.

Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+ ] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca 2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.

Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo.

In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two-photon imaging of dendritic [Ca2+] dynamics with dendritic membrane potential measurements. We found that sodium action potentials attenuated and broadened rapidly with distance from the soma. However, dendrites of layer 2/3 cells were electrically excitable, and direct current injections could evoke large [Ca2+] transients. The restricted pattern of dendritic [Ca2+] transients is therefore due to a failure of sodium action-potential propagation into dendrites. Also, stimulating subcortical activating systems by tail pinch can enhance dendritic [Ca2+] influx induced by a sensory stimulus by increasing cellular excitability, consistent with the importance of these systems in plasticity and learning.

Optogenetically stimulating intact rat corticospinal tract post-stroke restores motor control through regionalized functional circuit formation.

Current neuromodulatory strategies to enhance motor recovery after stroke often target large brain areas non-specifically and without sufficient understanding of their interaction with internal repair mechanisms. Here we developed a novel therapeutic approach by specifically activating corticospinal circuitry using optogenetics after large strokes in rats. Similar to a neuronal growth-promoting immunotherapy, optogenetic stimulation together with intense, scheduled rehabilitation leads to the restoration of lost movement patterns rather than induced compensatory actions, as revealed by a computer vision-based automatic behavior analysis. Optogenetically activated corticospinal neurons promote axonal sprouting from the intact to the denervated cervical hemi-cord. Conversely, optogenetically silencing subsets of corticospinal neurons in recovered animals, results in mistargeting of the restored grasping function, thus identifying the reestablishment of specific and anatomically localized cortical microcircuits. These results provide a conceptual framework to improve established clinical techniques such as transcranial magnetic or transcranial direct current stimulation in stroke patients.

Neuronal repair. Asynchronous therapy restores motor control by rewiring of the rat corticospinal tract after stroke.

The brain exhibits limited capacity for spontaneous restoration of lost motor functions after stroke. Rehabilitation is the prevailing clinical approach to augment functional recovery, but the scientific basis is poorly understood. Here, we show nearly full recovery of skilled forelimb functions in rats with large strokes when a growth-promoting immunotherapy against a neurite growth-inhibitory protein was applied to boost the sprouting of new fibers, before stabilizing the newly formed circuits by intensive training. In contrast, early high-intensity training during the growth phase destroyed the effect and led to aberrant fiber patterns. Pharmacogenetic experiments identified a subset of corticospinal fibers originating in the intact half of the forebrain, side-switching in the spinal cord to newly innervate the impaired limb and restore skilled motor function.

Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons.

The effect of the fluorescent Ca2+ indicator dye Fura-2 on Ca2+ dynamics was studied in proximal apical dendrites of neocortical layer V and hippocampal CA1 pyramidal neurons in rat brain slices using somatic whole-cell recording and a charge-coupled device camera. A single action potential evoked a transient increase of intradendritic calcium concentration ([Ca2+]i) that was reduced in size and prolonged when the Fura-2 concentration was increased from 20 to 250 microM. Extrapolation to zero Fura-2 concentration suggests that "physiological" transients at 37 degrees C have large amplitudes (150-300 nM) and fast decays (time constant < 100 ms). Assuming a homogeneous compartment model for the dendrite, 0.5-1% of the total Ca2+ entering during an action potential was estimated to remain free. Washout of cytoplasmic Ca2+ buffers was not detectable, suggesting that they are relatively immobile. During trains of action potentials, [Ca2+]i increased and rapidly reached a steady state (time constant < 200 ms), fluctuating around a plateau level which depended linearly on the action potential frequency. Thus, the mean dendritic [Ca2+]i encodes the action potential frequency during physiological patterns of electrical activity and may regulate Ca(2+)-dependent dendritic functions in an activity-dependent way.

Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones.

1. Simultaneous measurements of intracellular free calcium concentration ([Ca2+]i) and intrasomatic and intradendritic membrane potential (Vm) were performed using fura-2 fluorimetry and whole-cell recording in neocortical layer V pyramidal neurones in rat brain slices. 2. Back-propagating action potentials (APs) evoked [Ca2+]i transients in the entire neurone including the soma, the axon initial segment, the apical dendrite up to the distal tuft branches, and the oblique and basal dendrites, indicating that following suprathreshold activation the entire dendritic tree is depolarized sufficiently to open voltage-dependent calcium channels (VDCCs). 3. The [Ca2+]i transient peak evoked by APs showed large differences between various compartments of the neurone. Following a single AP, up to 6-fold differences were measured, ranging from 43 +/- 14 nM in the soma to 267 +/- 109 nM in the basal dendrites. 4. Along the main apical dendrite, the [Ca2+]i transients evoked by single APs or trains of APs had the largest amplitude and the fastest decay in the proximal region; the [Ca2+]i transient peak and decay time constant following a single AP were 128 +/- 25 nM and 420 +/- 150 ms, respectively, and following a train of five APs (at 10-12 Hz), 710 +/- 214 nM and 390 +/- 150 ms, respectively. The [Ca2+]i transients gradually decreased in amplitude and broadened in more distal portions of the apical dendrite up to the main bifurcation. 5. In the apical tuft branches, the profile of the [Ca2+]i transients was dependent on AP frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive calcium binding: implications for dendritic calcium signaling.

Action potentials evoke calcium transients in dendrites of neocortical pyramidal neurons with time constants of < 100 ms at physiological temperature. This time period may not be sufficient for inflowing calcium ions to equilibrate with all present Ca2+-binding molecules. We therefore explored nonequilibrium dynamics of Ca2+ binding to numerous Ca2+ reaction partners within a dendritelike compartment using numerical simulations. After a brief Ca2+ influx, the reaction partner with the fastest Ca2+ binding kinetics initially binds more Ca2+ than predicted from chemical equilibrium, while companion reaction partners bind less. This difference is consolidated and may result in bypassing of slow reaction partners if a Ca2+ clearance mechanism is active. On the other hand, slower reaction partners effectively bind Ca2+ during repetitive calcium current pulses or during slower Ca2+ influx. Nonequilibrium Ca2+ distribution can further be enhanced through strategic placement of the reaction partners within the compartment. Using the Ca2+ buffer EGTA as a competitor of fluo-3, we demonstrate competitive Ca2+ binding within dendrites experimentally. Nonequilibrium calcium dynamics is proposed as a potential mechanism for differential and conditional activation of intradendritic targets.

Calcium dynamics associated with a single action potential in a CNS presynaptic terminal.

Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the basis of a single compartment model. A single action potential led to a total Ca2+ influx of 0.8-1 pC. The accessible volume of the terminal was about 0.4 pl; thus the total calcium concentration increased by 10-13 microM. The Ca(2+)-binding ratio of the endogenous buffer was about 40, as estimated from the competition with Fura-2, indicating that 2.5% of the total calcium remained free. This is consistent with the peak increase in free calcium concentration of about 400 nM, which was measured directly with MagFura-2. The decay of the [Ca2+]i transients was fast, with time constants of 100 ms at 23 degrees C and 45 ms at 35 degrees C, indicating Ca2+ extrusion rates of 400 and 900 s-1, respectively. The combination of the relatively low endogenous Ca(2+)-binding ratio and the high rate of Ca2+ extrusion provides an efficient mechanism for rapidly removing the large Ca2+ load of the terminal evoked by an action potential.

Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat.

1. Simultaneous whole-cell recordings in a rat brain slice preparation are described from presynaptic terminals (calyces of Held) and postsynaptic somata which form an axosomatic synapse in the medial nucleus of the trapezoid body (MNTB). 2. Presynaptic action potentials evoked suprathreshold excitatory postsynaptic potentials (EPSPs). The minimum synaptic delay was around 0.4 ms at 36 degrees C and 0.9 ms at 23-24 degrees C. The amplitude of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated component of the excitatory postsynaptic currents (EPSCs) was 2-13 nA (at -80 mV). 3. Current-voltage relations showed that presynaptic Ca2+ channels were of the high voltage-activated type. 4. A single action potential evoked a presynaptic fluorescence transient that decayed with a time constant of 0.3-0.7 s, depending on the concentration (60-200 microM) of the Ca2+ indicator Calcium Green-5N (CG-5N). The peak amplitude of the [Ca2+]i transient was severalfold larger in the terminal than in the preterminal axon. 5. EPSC peak amplitudes were stable for more than 30 min after establishing the whole-cell configuration in the presynaptic terminal when the pipette contained 50 microM BAPTA. In contrast, with 1 mM BAPTA, peak amplitudes of EPSCs were reduced to one-third. 6. Trains of presynaptic action potentials evoked EPSCs with progressively smaller amplitudes. Little change was observed in the depression when the terminals were dialysed with 50 microM BAPTA, whereas depression was reduced with 1 mM BAPTA. 7. In low (1 mM) [Ca2+]o, facilitation instead of depression of EPSCs was observed. 8. The effects of presynaptic BAPTA suggest that the endogenous mobile Ca2+ buffer capacity of giant presynaptic terminals in the MNTB is lower than in other terminals of fast transmitting synapses.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 micrometers below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of approximately 70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

Postsynaptic Ca2+ influx mediated by three different pathways during synaptic transmission at a calyx-type synapse.

Whole-cell recordings and Ca2+ flux measurements were made at a giant calyx-type synapse in rat brainstem slices to determine the contribution of glutamate receptor (GluR) channels and voltage-dependent Ca2+ channels (VDCCs) to postsynaptic Ca2+ influx during synaptic transmission. A single presynaptic action potential (AP) evoked an EPSP, followed by a single AP. The EPSP-AP sequence caused a postsynaptic Ca2+ influx of approximately 3.0 pC, primarily through VDCCs ( approximately 70%) and NMDA-type (up to 30%) channels but also through AMPA-type (<5%) GluR channels. At -80 mV, the fractional Ca2+ current (Pf) mediated by AMPA receptor (AMPAR) and NMDA receptor (NMDAR) channels was 1.3 and 11-12%, respectively. Simulations of the time course of Ca2+ influx through GluR channels suggested that the small contribution of AMPAR channels occurred only during the first few milliseconds of an EPSP, whereas influx through NMDAR channels dominated later. The NMDAR-mediated Ca2+ influx was localized in regions covered by the presynaptic terminal, whereas the Ca2+ influx mediated by VDCCs was more homogeneously distributed. Because of the temporal and spatial differences, calcium ions entering through the three different pathways are likely to activate different intracellular targets in the postsynaptic cell.