Biomedical subjects

Yves De Koninck

Publications and source records attributed to Yves De Koninck.

16 recordsLinked to original sources

PubMed2006

Two-photon excitation fluorescence microscopy with a high depth of field using an axicon.

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.

Equipment Design↗

PubMed2006

Altered chloride homeostasis in neurological disorders: a new target.

Other than modifying either neurotransmitter release or receptor expression and/or properties, one way to modulate gamma-aminobutyric acid (GABA)A and glycine receptor-mediated signalling is to alter the transmembrane gradient for chloride ions. Because intracellular chloride concentration is low in neurons, and the reversal potential for chloride currents is close to the resting membrane potential, small changes in intracellular chloride concentration can dramatically affect the strength and even polarity of GABA/glycine-mediated transmission. It now appears that chloride homeostasis is actively regulated in the adult brain and affected by endogenous neuromodulators such as brain-derived neurotrophic factor. Modulating chloride gradients even emerges as a mechanism by which microglia can control neuronal excitability. These findings, together with the observation of altered chloride homeostasis in several neurological disorders, point to new targets for therapeutic agents.

Animals↗

PubMed2006

Dendritic spine viscoelasticity and soft-glassy nature: balancing dynamic remodeling with structural stability.

Neuronal dendritic spines are a key component of brain circuitry, implicated in many mechanisms for plasticity and long-term stability of synaptic communication. They can undergo rapid actin-based activity-dependent shape fluctuations, an intriguing biophysical property that is believed to alter synaptic transmission. Yet, because of their small size (approximately 1 microm or less) and metastable behavior, spines are inaccessible to most physical measurement techniques. Here we employ atomic force microscopy elasticity mapping and novel dynamic indentation methods to probe the biomechanics of dendritic spines in living neurons. We find that spines exhibit 1), a wide range of rigidities, correlated with morphological characteristics, axonal association, and glutamatergic stimulation, 2), a uniquely large viscosity, four to five times that of other cell types, consistent with a high density of solubilized proteins, and 3), weak power-law rheology, described by the soft-glassy model for cellular mechanics. Our findings provide a new perspective on spine functionality and identify key mechanical properties that govern the ability of spines to rapidly remodel and regulate internal protein trafficking but also maintain structural stability.

Animals↗

PubMed2006

Reduction of anion reversal potential subverts the inhibitory control of firing rate in spinal lamina I neurons: towards a biophysical basis for neuropathic pain.

BACKGROUND: Reduction of the transmembrane chloride gradient in spinal lamina I neurons contributes to the cellular hyperexcitability producing allodynia and hyperalgesia after peripheral nerve injury. The resultant decrease in anion reversal potential (i.e. shift in Eanion to less negative potentials) reduces glycine/GABAA receptor-mediated hyperpolarization, but the large increase in membrane conductance caused by inhibitory input can nonetheless shunt concurrent excitatory input. Without knowing the relative contribution of hyperpolarization and shunting to inhibition's modulation of firing rate, it is difficult to predict how much net disinhibition results from reduction of Eanion. We therefore used a biophysically accurate lamina I neuron model to investigate quantitatively how changes in Eanion affect firing rate modulation. RESULTS: Simulations reveal that even a small reduction of Eanion compromises inhibitory control of firing rate because reduction of Eanion not only decreases glycine/GABAA receptor-mediated hyperpolarization, but can also indirectly compromise the capacity of shunting to reduce spiking. The latter effect occurs because shunting-mediated modulation of firing rate depends on a competition between two biophysical phenomena: shunting reduces depolarization, which translates into reduced spiking, but shunting also shortens the membrane time constant, which translates into faster membrane charging and increased spiking; the latter effect predominates when average depolarization is suprathreshold. Disinhibition therefore occurs as both hyperpolarization- and shunting-mediated modulation of firing rate are subverted by reduction of Eanion. Small reductions may be compensated for by increased glycine/GABAA receptor-mediated input, but the system decompensates (i.e. compensation fails) as reduction of Eanion exceeds a critical value. Hyperexcitability necessarily develops once disinhibition becomes incompensable. Furthermore, compensation by increased glycine/GABAA receptor-mediated input introduces instability into the system, rendering it increasingly prone to abrupt decompensation and even paradoxical excitation. CONCLUSION: Reduction of Eanion dramatically compromises the inhibitory control of firing rate and, if compensation fails, is likely to contribute to the allodynia and hyperalgesia associated with neuropathic pain. These data help explain the relative intractability of neuropathic pain and illustrate how it is important to choose therapies not only based on disease mechanism, but based on quantitative understanding of that mechanism.

Animals↗

PubMed2006

Nonlinear interaction between shunting and adaptation controls a switch between integration and coincidence detection in pyramidal neurons.

The membrane conductance of a pyramidal neuron in vivo is substantially increased by background synaptic input. Increased membrane conductance, or shunting, does not simply reduce neuronal excitability. Recordings from hippocampal pyramidal neurons using dynamic clamp revealed that adaptation caused complete cessation of spiking in the high conductance state, whereas repetitive spiking could persist despite adaptation in the low conductance state. This behavior was reproduced in a phase plane model and was explained by a shunting-induced increase in voltage threshold. The increase in threshold allows greater activation of the M current (I(M)) at subthreshold potentials and reduces the minimum adaptation required to stabilize the system; in contrast, activation of the afterhyperpolarization current is unaffected by the increase in threshold and therefore remains unable to stop repetitive spiking. The nonlinear interaction between shunting and I(M) has other important consequences. First, timing of spikes elicited by brief stimuli is more precise when background spikes elicited by sustained input are prohibited, as occurs exclusively with I(M)-mediated adaptation in the high conductance state. Second, activation of I(M) at subthreshold potentials, which is increased in the high conductance state, hyperpolarizes average membrane potential away from voltage threshold, allowing only large, rapid fluctuations to reach threshold and elicit spikes. These results suggest that the shift from a low to high conductance state in a pyramidal neuron is accompanied by a switch from encoding time-averaged input with firing rate to encoding transient inputs with precisely timed spikes, in effect, switching the operational mode from integration to coincidence detection.

Action Potentials↗

PubMed2006

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury.

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.

Activating Transcription Factor 3↗

PubMed2006

[Two-photon laser scanning fluorescence microscopy for functional cellular imaging: Advantages and challenges or One photon is good... but two is better!].

One of the main challenges of modern biochemistry and cell biology is to be able to observe molecular dynamics in their functional context, i.e. in live cells in situ. Thus, being able to track ongoing molecular events with maximal spatial and temporal resolution (within subcellular compartments), while minimizing interference with tissue biology, is key to future developments for in situ imaging. The recent use of non-linear optics approaches in tissue microscopy, made possible in large part by the availability of femtosecond pulse lasers, has allowed major advances on this front that would not have been possible with conventional linear microscopy techniques. Of these approaches, the one that has generated most advances to date is two-photon laser scanning fluorescence microscopy. While this approach does not really provide improved resolution over linear microscopy in non absorbing media, it allows us to exploit a window of low absorbance in live tissue in the near infrared range. The end result is much improved tissue penetration, minimizing unwanted excitation outside the focal area, which yields an effective improvement in resolution and sensitivity. The optical system is also simplified and, more importantly, phototoxicity is reduced. These advantages are at the source of the success of two-photon microscopy for functional cellular imaging in situ. Yet, we still face further challenges, reaching the limits of resolution that conventional optics can offer. Here we review some recent advances in optics/photonics approaches that hold promises to improve our ability to probe the tissue in finer areas, at faster speed, and deeper into the tissue. These include super-resolution techniques, introduction of non paraxial optics in microscopy and use of amplified femtosecond lasers, yielding enhanced spatial and temporal resolution as well as tissue penetration.

Cells↗

PubMed2005

Imbalance towards inhibition as a substrate of aging-associated cognitive impairment.

The number of synapses in the cerebral cortex decreases with aging. However, how this structural change translates into the cognitive impairment observed in aged animals remains unknown. Aged animals are not a homogenous group with respect to their cognitive performances; but instead, they can be separated into aged cognitively unimpaired ("normal") and aged cognitively impaired groups using a spatial memory task such as the Morris water maze. These two aged groups provide an unprecedented opportunity to isolate synaptic properties that relate to cognitive impairment from unrelated factors associated with normal aging. Using such classification, we conducted whole-cell patch-clamp recordings to measure basal spontaneous miniature excitatory (mEPSCs) and inhibitory synaptic currents (mIPSCs) bombarding layer V pyramidal neurons in the parietal cortex. We found that the frequencies of both mEPSC and mIPSC were lower in aged normal rats when compared with young rats. In contrast, aged cognitively impaired rats displayed a reduction in mEPSC frequency only. This results in an imbalance towards inhibition that may be an important substrate of the cognitive impairment in aged animals. We also found that pyramidal neurons in both aged normal and aged cognitively impaired rats exhibit similar structural attritions. Thus, cognitive impairment may be more related to an altered balance between different neurotransmitter systems than a mere reduction in synaptic structures.

Action Potentials↗

PubMed2005

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain.

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (E(anion)) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (gamma-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in E(anion). Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the E(anion) shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and E(anion). Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia-neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.

Adenosine Triphosphate↗

PubMed2005

Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity.

A deficit in inhibition in the spinal dorsal horn has been proposed to be an underlying cause of the exaggerated cutaneous sensory reflexes observed in newborn rats. However, the developmental shift in transmembrane anion gradient, potentially affecting the outcome of GABAA transmission, was shown to be completed within 1 week after birth in the spinal cord, an apparent disparity with the observation that reflex hypersensitivity persists throughout the first 2-3 postnatal weeks. To further investigate this issue, we used several approaches to assess the action of GABA throughout development in spinal lamina I (LI) neurons. GABA induced an entry of extracellular calcium in LI neurons from postnatal day 0 (P0) to P21 rats, which involved T- and N-type voltage-gated calcium channels. Gramicidin perforated-patch recordings revealed that the shift in anion gradient was completed by P7 in LI neurons. However, high chloride pipette recordings demonstrated that these neurons had not reached their adult chloride extrusion capacity by P10-P11. Simultaneous patch-clamp recordings and calcium imaging revealed that biphasic responses to GABA, consisting of a primary hyperpolarization followed by a rebound depolarization, produced a rise in [Ca2+]i. Thus, even if Eanion predicts GABAA-induced hyperpolarization from rest, a low chloride extrusion capacity can cause a rebound depolarization and an ensuing rise in [Ca2+]i. We demonstrate that GABA action in LI neurons matures throughout the first 3 postnatal weeks, therefore matching the time course of maturation of withdrawal reflexes. Immature spinal GABA signaling may thus contribute to the nociceptive hypersensitivity in infant rats.

Animals↗

PubMed2005

Morphology and neurokinin 1 receptor expression of spinothalamic lamina I neurons in the rat spinal cord.

Distinct morphological types of spinothalamic tract (STT) lamina I (LI) neurons have been identified in the cat and monkey spinal dorsal horn. Because these morphological types appear to differ in functional properties and receptor expression, we examined their distribution in the rat to test how their identification relates to earlier classification schemes. LI STT cells were retrogradely labeled with cholera toxin subunit b (CTb). Three types were recognized on the basis of cell body shape and proximal dendrites in the horizontal plane: fusiform, multipolar, and pyramidal. The relative distribution of these types was: 43, 26, and 28%, respectively, similar to that observed in the cat and monkey. 3D reconstructions were used to view each cell in all three major projection planes: horizontal, parasagittal, and transverse. Most LI STT neurons appeared fusiform in the parasagittal plane even though they belonged to different types based on their appearance in the horizontal plane, except in the most lateral portion of the dorsal horn, where LI curves ventrally. The proportion of STT neurons within LI was quantified by using the optical dissector method. To label all LI neurons, we used an anti-neuron-specific nuclear protein (NeuN) antibody. We found that approximately 9% of LI neurons projected to the thalamus. We also investigated neurokinin 1 receptor (NK-1r) expression in LI STT neurons. As in the monkey, most pyramidal STT neurons did not express NK-1r. These results provide further evidence that distinct morphological types of neurons differ in phenotype but not in their projection pattern.

Animals↗

PubMed2005

Integration time in a subset of spinal lamina I neurons is lengthened by sodium and calcium currents acting synergistically to prolong subthreshold depolarization.

Lamina I of the spinal dorsal horn plays an important role in processing and relaying nociceptive information to the brain. It comprises physiologically distinct cell types that process information in fundamentally different ways: tonic neurons fire repetitively during stimulation and display prolonged EPSPs, suggesting operation as integrators, whereas single-spike neurons act like coincidence detectors. Using whole-cell recordings from a rat spinal slice preparation, we set out to determine the basis for prolonged EPSPs in tonic cells and the implications for signal processing. Kinetics of synaptic currents could not explain differences in EPSP kinetics. Instead, tonic neurons were found to express a persistent sodium current, I(Na,P), that amplified and prolonged depolarization in response to brief stimulation. Tonic neurons also expressed a persistent calcium current, I(Ca,P), that contributed to prolongation but not to amplification. Simulations using NEURON software demonstrated that I(Na,P) was necessary and sufficient to explain amplification, whereas I(Na,P) and I(Ca,P) acted synergistically to prolong depolarization: initial activation of the slower current (I(Ca,P)) depended on the faster current (I(Na,P)) but maintained activation of the faster current likewise depended on the slower current. Additional investigation revealed that I(Na,P) and I(Ca,P) could dramatically increase integration time (>30x) and thereby encourage temporal summation but at the expense of spike time precision. Thus, by prolonging subthreshold depolarization, intrinsic inward currents allow tonic neurons in spinal lamina I to specialize as integrators that are optimally suited to encode stimulus intensity.

Animals↗

PubMed2003

Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain.

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.

Animals↗

PubMed2003

Gain control of firing rate by shunting inhibition: roles of synaptic noise and dendritic saturation.

Adjusting input-output gain is crucial for information processing by the brain. Gain control of subthreshold depolarization is commonly ascribed to increased membrane conductance caused by shunting inhibition. But contrary to its divisive effect on depolarization, shunting inhibition on its own fails to divisively modulate firing rate, apparently upsetting a critical tenet of neural models that use shunting inhibition to achieve gain control. Using a biophysically realistic neuron model, we show that divisive modulation of firing rate by shunting inhibition requires synaptic noise to smooth the relation between firing rate and somatic depolarization; although necessary, noise alone endows shunting inhibition with only a modest divisive effect on firing rate. In addition to introducing noise, synaptic input is associated with a nonlinear relation between somatic depolarization and excitation because of dendritic saturation; this nonlinearity dramatically enhances divisive modulation of firing rate by shunting inhibition under noisy conditions. Thus, shunting inhibition can act as a mechanism for firing rate gain control, but its modulatory effects (which include both divisive and subtractive components) are fully explained only when both synaptic noise and dendritic saturation are taken into account.

Animals↗

PubMed2002

Four cell types with distinctive membrane properties and morphologies in lamina I of the spinal dorsal horn of the adult rat.

Lamina I of the spinal dorsal horn plays an important role in the processing and relay of nociceptive information. Signal processing depends, in part, on neuronal membrane properties. Intrinsic membrane properties of lamina I neurons were therefore investigated using whole cell patch clamp recordings in a slice preparation of adult rat spinal cord. Based on responses to somatic current injection, four cell types were identified: tonic, which fire comparatively slowly but continuously throughout stimulation; phasic, which fire a high frequency burst of variable duration; delayed onset, which fire irregularly and with a marked delay to the first spike; and single spike, which typically fire only one action potential even when strongly depolarised. Classification by spiking pattern was further refined by identification of characteristic stimulus-response curves and quantification of several response parameters. Objectivity of the classification was confirmed by cluster analysis. Responses to stimulus trains and synaptic input as well as the kinetics of spontaneous synaptic events revealed differences in the signal processing characteristics of the cell types: tonic and delayed onset cells appeared to act predominantly as integrators whereas phasic and single spike cells acted as coincidence detectors. Intracellular labelling revealed a significant correlation between morphological and physiological cell types: tonic cells were typically fusiform, phasic cells were pyramidal, and delayed onset and single spike cells were multipolar. Thus, there are multiple physiological cells types in lamina I with specific morphological correlates and distinctive signal processing characteristics that confer significant differences in the transduction of input into spike trains.

Animals↗

PubMed2002

Aging causes a preferential loss of cholinergic innervation of characterized neocortical pyramidal neurons.

Aging is known to markedly affect the number and structural characteristics of both pre- and post-synaptic sites in the cerebral cortex. There is evidence that lamina V pyramidal neurons, and their basilar dendrites in particular, are affected by age-related decline. Furthermore, layer V is the area where the greatest overall age- related losses in the total population of synaptic boutons and of cholinergic boutons are observed. Since both pyramidal neurons and cortical cholinergic input are characteristically compromised in aging, we investigated whether aging altered the pattern of cholinergic boutons in apposition to the soma, proximal and distal basal dendrites of intracellularly labeled lamina V large pyramidal neurons in the parietal cortex of young and aged rats. We observed a significant age-related decrease in the population of both total and cholinergic boutons apposed to proximal and distal dendrites of layer V large pyramidal neurons. However, the age-related decreases of cholinergic presynaptic boutons were higher than those in the total bouton population apposed to the pyramidal neurons. The average decrease in cholinergic boutons in aged rats was 3.7-fold more pronounced than the diminution in the overall number of presynaptic boutons. Our results add important new evidence in support of the concept that the age-related learning and memory deficits are attributable, at least partially, to a decline in the functional integrity of the forebrain cholinergic systems.

Aging↗