Kerry R. Delaney

Extensive Turnover of Dendritic Spines and Vascular Remodeling in Cortical Tissues Recovering from Stroke

Recovery of function after stroke is thought to be dependent on the reorganization of adjacent, surviving areas of the brain. Macroscopic imaging studies (functional magnetic resonance imaging, optical imaging) have shown that peri-infarct regions adopt new functional roles to compensate for damage caused by stroke. To better understand the process by which these regions reorganize, we used in vivo two-photon imaging to examine changes in dendritic and vascular structure in cortical regions recovering from stroke. In adult control mice, dendritic arbors were relatively stable with very low levels of spine turnover (<0.5% turnover over 6 h). After stroke, however, the organization of dendritic arbors in peri-infarct cortex was fundamentally altered with both apical dendrites and blood vessels radiating in parallel from the lesion. On a finer scale, peri-infarct dendrites were exceptionally plastic, manifested by a dramatic increase in the rate of spine formation that was maximal at 1–2 weeks (5–8-fold increase), and still evident 6 weeks after stroke. These changes were selective given that turnover rates were not significantly altered in ipsilateral cortical regions more distant to the lesion (>1.5 mm). These data provide a structural framework for understanding functional and behavioral changes that accompany brain injury and suggest new targets that could be exploited by future therapies to rebuild and rewire neuronal circuits lost to stroke.

Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes

We have identified large‐scale patterns of electrical activity in cortical circuits that occur in response to stimulation of peripheral receptors. Our focus was on primary (S1) vibrissal cortex of anesthetized rat, and we used optical techniques in conjunction with voltage‐sensitive dyes to measure depolarization of the upper layers of cortex. Displacement of one vibrissa produced a field of activity that extends over very many cortical columns in S1. There are multiple, focal maxima within this field. A global maximum is located near the center of the field of activity, and, as determined electrically and histologically, this site maps to the cortical column appropriate for the deflected vibrissa. The amplitude of this component attains a steady‐state value under continuous stimulation. Additional temporal characteristics are revealed by the response to a single displacement; the signal was triphasic and began with a prompt depolarization that was followed by a transient phase of inhibition and a final phase of long‐lasting depolarization. The somatotopy of the other, satellite maxima in the field of activity were established through the reconstruction of the fields of activity produced by individual stimulation of other vibrissae. Local maxima for one vibrissa were seen to overlie the global maximum found for stimulation of nearest‐ and next‐nearest‐neighbor vibrissae. In contrast to the amplitude of the global maxima, the amplitude associated with the local maxima was not maintained with either continuous or infrequent but repetitive stimulation. Finally, the field of activity induced by alternate deflection of two neighboring vibrissae was suppressed in amplitude in comparison to the summed amplitudes of the signals elicited by deflection of each vibrissa alone. We suggest that these patterns of activity are a manifestation of the dynamic interaction among neighboring cortical columns.

Rapid Reversible Changes in Dendritic Spine Structure In Vivo Gated by the Degree of Ischemia

Current therapeutic windows for effective application of thrombolytic agents are within 3-6 h of stroke. Although treatment can improve outcome, it is unclear what happens to synaptic fine structure during this critical period in vivo. The relationship between microcirculation and dendritic spine structure was determined in mouse somatosensory neurons during stroke. Spines were, on average, 13 μm from a capillary and were supplied by ∼100 red blood cells per second. Moderate ischemia (∼50% supply) did not significantly affect spines within 5 h; however, severe ischemia (<10% supply) caused a rapid loss of spine and dendrite structure within as little as 10 min. Surprisingly, if reperfusion occurred within 20-60 min, dendrite and spine structure was mostly restored. These data suggest that the basic dendritic wiring diagram remains mostly intact during moderate ischemia and that affected synapses could potentially contribute to functional recovery. With severe ischemia, markedly deformed dendritic structure can partially recover if reperfusion occurs early.

Competition between Phasic and Asynchronous Release for Recovered Synaptic Vesicles at Developing Hippocampal Autaptic Synapses

Developing hippocampal neurons in microisland culture undergo rapid and extensive transmitter release-dependent depression of evoked (phasic) excitatory synaptic activity in response to 1 sec trains of 20 Hz stimulation. Although evoked phasic release was attenuated by repeated stimuli, asynchronous (miniature like) release continued at a high rate equivalent to ∼2.8 readily releasable pools (RRPs) of quanta/sec. Asynchronous release reflected the recovery and immediate release of quanta because it was resistant to sucrose-induced depletion of the RRP. Asynchronous and phasic release appeared to compete for a common limited supply of release-ready quanta because agents that block asynchronous release, such as EGTA-AM, led to enhanced steady-state phasic release, whereas prolongation of the asynchronous release time course by LiCl delayed recovery of phasic release from depression. Modeling suggested that the resistance of asynchronous release to depression was associated with its ability to out-compete phasic release for recovered quanta attributable to its relatively low release rate (up to 0.04/msec per vesicle) stimulated by bulk intracellular Ca2+ concentration ([Ca2+]i) that could function over prolonged intervals between successive stimuli. Although phasic release was associated with a considerably higher peak rate of release (0.4/msec per vesicle), the [Ca2+]i microdomains that trigger it are brief (1 msec), and with asynchronous release present, relatively few quanta can accumulate within the RRP to be available for phasic release. We conclude that despite depression of phasic release during train stimulation, transmission can be maintained at a near-maximal rate by switching to an asynchronous mode that takes advantage of a bulk presynaptic [Ca2+]i.

Ca2+ from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

Neurotransmitter release is triggered by the cooperative action of approximately five Ca2+ ions entering the presynaptic terminal through Ca2+ channels. Depending on the organization of the active zone (AZ), influx through one or many channels may be needed to cause fusion of a vesicle. Using a combination of experiments and modeling, we examined the number of channels that contribute Ca2+ for fusion of a single vesicle in a frog neuromuscular AZ. We compared Ca2+ influx to neurotransmitter release by measuring presynaptic action potential-evoked (AP-evoked) Ca2+ transients simultaneously with postsynaptic potentials. Ca2+ influx was manipulated by changing extracellular [Ca2+] (Caext) to alter the flux per channel or by reducing the number of open Ca2+ channels with ω-conotoxin GVIA (ω-CTX). When Caext was reduced, the exponent of the power relationship relating release to Ca2+ influx was 4.16 ± 0.62 (SD; n = 4), consistent with a biochemical cooperativity of ∼5. In contrast, reducing influx with ω-CTX yielded a power relationship of 1.7 ± 0.44 (n = 5) for Caext of 1.8 mm and 2.12 ± 0.44 for Caext of 0.45 mm (n = 5). Using geometrically realistic Monte Carlo simulations, we tracked Ca2+ ions as they entered through each channel and diffused in the terminal. Experimental and modeling data were consistent with two to six channel openings per AZ per AP; the Ca2+ that causes fusion of a single vesicle originates from one or two channels. Channel cooperativity depends mainly on the physical relationship between channels and vesicles and is insensitive to changes in the non-geometrical parameters of our model.

Branch-Specific Ca2+ Influx from Na+-Dependent Dendritic Spikes in Olfactory Granule Cells

Two-photon laser scanning microscopy was used to correlate electrical events detected with whole-cell somatic recordings to Ca2+ transients in dendrites of olfactory bulb granule cells. A subset of spontaneous subthreshold depolarizing events recorded at the soma were shown to correspond to suprathreshold dendritic, Na-dependent action potentials [APs; dendritic spikes (D-spikes)]. These potentials were blocked by intracellular QX-314 (lidocaine N-ethyl bromide), hyperpolarizing current injection at the soma, and by partial inhibition of AMPA/kainate receptors with 0.75 μm DNQX. They were affected only slightly by 100 μm NiCl2. The majority of D-spikes recorded at the soma had a time to peak of <4 ms, comparable with somatic APs, a nonexponential decay, and amplitudes between 3 and 21 mV. Somatically recorded APs produced Ca2+ transients that were observed in spines and dendrites in all parts of the cell. Ca2+ transients from D-spikes were restricted to subsets of distal dendrites and their associated spines but were absent from the soma and dendrite within ∼50–80 μm of the soma. Ca2+ transients in different branches could be correlated with different-sized D-spikes. D-spike and backpropagating AP-induced Ca2+ transients summed in dendrites, provided the interval between them was >5–6 ms. Generation of a D-spike in a particular dendrite <5–6 ms before a somatic AP blocked backpropagation of the somatic AP into that dendrite. The temporally specific interplay between D-spikes and backpropagating APs may play a role in regulating feedback and feedforward inhibition of groups of mitral cells synapsing on different granule cell dendrites.

MeCP2 Mutation Results in Compartment-Specific Reductions in Dendritic Branching and Spine Density in Layer 5 Motor Cortical Neurons of YFP-H Mice

Rett Syndrome (RTT) is a neurodevelopmental disorder predominantly caused by mutations in the X-linked gene MECP2. A primary feature of the syndrome is the impaired maturation and maintenance of excitatory synapses in the central nervous system (CNS). Different RTT mouse models have shown that particular Mecp2 mutations have highly variable effects on neuronal architecture. Distinguishing MeCP2 mutant cellular phenotypes therefore demands analysis of specific mutations in well-defined neuronal subpopulations. We examined a transgenically labeled subset of cortical neurons in YFP-H mice crossed with the Mecp2tm1.1Jae mutant line. YFP+ Layer 5 pyramidal neurons in the motor cortex of wildtype and hemizygous mutant male mice were examined for differences in dendrite morphology and spine density. Total basal dendritic length was decreased by 18.6% due to both shorter dendrites and reduced branching proximal to the soma. Tangential dendrite lengths in the apical tuft were reduced by up to 26.6%. Spine density was reduced by 47.4% in the apical tuft and 54.5% in secondary apical dendrites, but remained unaffected in primary apical and proximal basal dendrites. We also found that MeCP2 mutation reduced the number of YFP+ cells in YFP-H mice by up to 72% in various cortical regions without affecting the intensity of YFP expression in individual cells. Our results support the view that the effects of MeCP2 mutation are highly context-dependent and cannot be generalized across mutation types and cell populations.

An in vitro preparation of frog nose and brain for the study of odour-evoked oscillatory activity

An in vitro preparation is described that consists of frog brain rostral to the brainstem connected to the nasal epithelium by the olfactory nerves. Field potential and intracellular recordings from various brain structures can be obtained while stimulating the nasal epithelium with air-borne odours for at least 12 h after removal of the brain. Power spectra, amplitude and duration of odour-evoked and spontaneous field potentials in vitro are similar to those obtained from paralyzed, spinal cord pithed frogs. A brief puff of odorant applied to the olfactory epithelium produces a 1-2 s bout of 7-13 Hz oscillations in the field potential recorded from the ipsilateral bulb and various ventral, lateral and medial telencephalic structures. Odour evoked bulbar oscillations are maintained after removal of the telencephalon. Electrical stimulation of the olfactory nerves will not elicit oscillations like those evoked by odour stimulation. High-pressure puffs of non-odorised, moist air, elicit olfactory bulb oscillations similar to those evoked by lower pressure puffs of odorised air. Intracellular recordings from most mitral cells reveal oscillations in membrane potential that are phase-locked to the field potential. The extent to which these phase-locked oscillations produce action potentials varies, apparently as a function of the strength and duration of a long-lasting inhibitory potential that is superimposed upon the 7-13 Hz oscillations. This preparation is well-suited for the study of the cellular basis of oscillatory activity in vertebrate brain, and the function of sensory-evoked oscillatory responses in processing of sensory information.

Contribution of a calcium-activated non-specific conductance to NMDA receptor-mediated synaptic potentials in granule cells of the frog olfactory bulb

We studied granule cells (GCs) in the intact frog olfactory bulb (OB) by combining whole‐cell recordings and functional two‐photon Ca2+ imaging in an in vitro nose‐brain preparation. GCs are local interneurones that shape OB output via distributed dendrodendritic inhibition of OB projection neurones, the mitral‐tufted cells (MTCs). In contrast to MTCs, GCs exhibited a Ca2+‐activated non‐specific cation conductance (ICAN) that could be evoked through strong synaptic stimulation or suprathreshold current injection. Photolysis of the caged Ca2+ chelator o‐nitrophenol‐EGTA resulted in activation of an inward current with a reversal potential within the range ‐20 to +10 mV. ICAN in GCs was suppressed by the intracellular Ca2+ chelator BAPTA (0.5–5.0 mM), but not by EGTA (up to 5 mM). The current persisted in whole‐cell recordings for up to 1.5 h post‐breakthrough, was observed during perforated‐patch recordings and was independent of ionotropic glutamate and GABAA receptor activity. In current‐clamp mode, GC responses to synaptic stimulation consisted of an initial AMPA‐mediated conductance followed by a late‐phase APV‐sensitive plateau (100–500 ms). BAPTA‐mediated suppression of ICAN resulted in a selective reduction of the late component of the evoked synaptic potential, consistent with a positive feedback relationship between NMDA receptor (NMDAR) current and ICAN. ICAN requires Ca2+ influx either through voltage‐gated Ca2+ channels or possibly NMDARs, both of which have a high threshold for activation in GCs, predicting a functional role for this current in the selective enhancement of strong synaptic inputs to GCs.

Synaptic Activation of T-Type Ca2+ Channels Via mGluR Activation in the Primary Dendrite of Mitral Cells

Mitral cells are the primary output of the olfactory bulb, projecting to many higher brain areas. Understanding how mitral cells process and transmit information is key to understanding olfactory perception. Mitral dendrites possess high densities of voltage-gated channels, are able to initiate and propagate orthodromic and antidromic action potentials, and release neurotransmitter. We show that mitral cells also possess a low-voltage-activated T-type Ca(2+) current. Immunohistochemistry shows strong Cav3.3 labeling in the primary dendrite and apical tuft with weaker staining in basal dendrites and no staining in somata. A low-voltage-activated Ca(2+) current activates from -68 mV, is blocked by 500 microM Ni(2+) and 50 microM NNC 55-0396, but is insensitive to 50 microM Ni(2+) and 500 microM isradipine. 2-photon Ca(2+) imaging shows that T channels are functionally expressed in the primary dendrite where their activity determines the resting [Ca(2+)] and are responsible for subthreshold voltage-dependent Ca(2+) changes previously observed in vivo. Application of the group 1 mGluR agonist dihydroxyphenylglycine (DHPG) (50 microM) robustly upregulates T-channel current in the primary and apical tuft dendrite. Olfactory nerve stimulation generates a long-lasting depolarization, and we show that mGluRs recruit T channels to contribute approximately 36% of the voltage integral of this depolarization. The long-lasting depolarization results in sustained firing and block of T channels decreased action potential firing by 84.1 +/- 4.6%. Therefore upregulation of T channels by mGluRs is required for prolonged firing in response to olfactory nerve input.