Biomedical subjects

Greg J Stuart

Publications and source records attributed to Greg J Stuart.

14 recordsLinked to original sources

PubMed2006

Imaging membrane potential in dendrites and axons of single neurons.

This review focuses on the use of imaging techniques to record electrical signaling in the fine processes of neurons such as dendrites and axons. Voltage imaging began with the use and development of externally applied voltage-sensitive dyes. With the introduction of internally applied dyes and advances in detection technology, it is now possible to record supra-threshold action potential responses, as well as sub-threshold synaptic potentials, in fine neuronal processes including dendritic spines. The development of genetically coded sensors, as well as variants of laser scanning microscopy such as second harmonic generation, offers promise for further advances in this field. Through the use and further development of these methods, optical imaging of membrane potential will continue to be a valuable tool for investigators wishing to explore the electrical events underlying single neuronal computation.

Action Potentials↗

PubMed2006

Calcium spikes in basal dendrites of layer 5 pyramidal neurons during action potential bursts.

Patch-clamp recording from dendrites has lead to a significant increase in our understanding of the mechanisms underlying signal integration and propagation in neurons. The majority of synaptic input to neurons, however, is made onto small-diameter dendrites, currently beyond the scope of patch-clamp recording techniques. Here we use both calcium and voltage imaging to investigate propagation of action potentials (APs) in fine basal dendrites of cortical layer 5 pyramidal neurons. High-frequency (200 Hz) AP bursts caused supralinear increases in dendritic calcium at distal, but not proximal, basal locations. Supralinear increases in dendritic calcium were also observed at distal basal locations during AP trains above a critical frequency (approximately 100 Hz). Using voltage imaging, we show that single APs undergo significant attenuation as they propagate into basal dendrites, whereas AP bursts lead to generation of dendritic calcium spikes. Focal and bath application of 4-AP increased the amplitude of calcium transients evoked by APs at distal, but not proximal, locations, suggesting that A-type potassium channels regulate AP backpropagation into basal dendrites. Finally, we show that pairing EPSPs with AP bursts is an effective means of activating synaptic NMDA receptors in basal dendrites. The experimental observations on the role of A-type potassium channels in regulation of AP backpropagation in basal dendrites, as well as the generation of dendritic calcium spikes during AP bursts, were reproduced in a morphologically realistic neuronal model with uniform distributions of dendritic sodium, calcium, and potassium channels. Together, these findings have important implications for understanding dendritic integration and synaptic plasticity in cortical basal dendrites.

Action Potentials↗

PubMed2006

Requirement of dendritic calcium spikes for induction of spike-timing-dependent synaptic plasticity.

Spike-timing-dependent synaptic plasticity (STDP) by definition requires the temporal association of pre- and postsynaptic action potentials (APs). Yet, in cortical pyramidal neurons pairing unitary EPSPs with single APs at low frequencies is ineffective at generating plasticity. Using recordings from synaptically coupled layer 5 pyramidal neurons, we show here that high-frequency (200 Hz) postsynaptic AP bursts, rather than single APs, are required for both long-term potentiation (LTP) induction and NMDA channel activation during EPSP-AP pairing at low frequencies. Furthermore, we find that AP bursts can lead to LTP induction and NMDA channel activation during EPSP-AP pairing at both positive and negative times. High-frequency AP bursts generated supralinear calcium signals in basal dendrites suggesting the generation of dendritic calcium spikes, as has been observed previously in apical dendrites during AP burst firing at frequencies greater than 100 Hz. Consistent with a role of these dendritic calcium spikes in LTP induction, pairing EPSPs with low frequency (50 Hz) AP bursts was ineffective in generating LTP. Furthermore, supralinear calcium signals in basal dendrites during AP bursts were blocked by low concentrations of the T- and R-type calcium channel antagonist nickel, which also blocked LTP and NMDA channel activation. These data suggest an important role of dendritic calcium spikes during AP bursts in determining both the efficacy and time window for STDP induction.

Action Potentials↗

PubMed2006

Single Ih channels in pyramidal neuron dendrites: properties, distribution, and impact on action potential output.

The hyperpolarization-activated cation current (Ih) plays an important role in regulating neuronal excitability, yet its native single-channel properties in the brain are essentially unknown. Here we use variance-mean analysis to study the properties of single Ih channels in the apical dendrites of cortical layer 5 pyramidal neurons in vitro. In these neurons, we find that Ih channels have an average unitary conductance of 680 +/- 30 fS (n = 18). Spectral analysis of simulated and native Ih channels showed that there is little or no channel flicker below 5 kHz. In contrast to the uniformly distributed single-channel conductance, Ih channel number increases exponentially with distance, reaching densities as high as approximately 550 channels/microm2 at distal dendritic sites. These high channel densities generate significant membrane voltage noise. By incorporating a stochastic model of Ih single-channel gating into a morphologically realistic model of a layer 5 neuron, we show that this channel noise is higher in distal dendritic compartments and increased threefold with a 10-fold increased single-channel conductance (6.8 pS) but constant Ih current density. In addition, we demonstrate that voltage fluctuations attributable to stochastic Ih channel gating impact on action potential output, with greater spike-timing precision in models with the experimentally determined single-channel conductance. These data suggest that, in the face of high current densities, the small single-channel conductance of Ih is critical for maintaining the fidelity of action potential output.

Action Potentials↗

PubMed2006

Site of action potential initiation in layer 5 pyramidal neurons.

Fundamental to an understanding of how neurons integrate synaptic input is the knowledge of where within a neuron this information is converted into an output signal, the action potential. Although it has been known for some time that action potential initiation occurs within the axon of neurons, the precise location has remained elusive. Here, we provide direct evidence using voltage-sensitive dyes that the site of action potential initiation in cortical layer 5 pyramidal neurons is approximately 35 microm from the axon hillock. This was the case during action potential generation under a variety of conditions, after axonal inhibition, and at different stages of development. Once initiated action potentials propagated down the axon in a saltatory manner. Experiments using local application of low-sodium solution and TTX, as well as an investigation of the influence of axonal length on action potential properties, provided evidence that the initial 40 microm of the axon is essential for action potential generation. To morphologically identify the relationship between the site of action potential initiation and axonal myelination, we labeled oligodendrocytes supplying processes to the proximal region of the axon. These experiments indicated that the axon initial segment was approximately 40 mcirom in length, and the first node of Ranvier was approximately 90 microm from the axon hillock. Experiments targeting the first node of Ranvier suggested it was not involved in action potential initiation. In conclusion, these results indicate that, in layer 5 pyramidal neurons, action potentials are generated in the distal region of the axon initial segment.

Action Potentials↗

PubMed2005

Cholinergic inhibition of neocortical pyramidal neurons.

Acetylcholine (ACh) is a central neurotransmitter critical for normal cognitive function. Here we show that transient muscarinic acetylcholine receptor activation directly inhibits neocortical layer 5 pyramidal neurons. Using whole-cell and cell-attached recordings from neurons in slices of rat somatosensory cortex, we demonstrate that transient activation of M1-type muscarinic receptors induces calcium release from IP3-sensitive intracellular calcium stores and subsequent activation of an apamin-sensitive, SK-type calcium-activated potassium conductance. ACh-induced hyperpolarizing responses were blocked by atropine and pirenzepine but not by methoctramine or GABA receptor antagonists (picrotoxin, SR 95531 [2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide], and CGP 55845 [(2S)-3-[[(15)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid]). Responses were associated with a 31 +/- 5% increase in membrane conductance, had a reversal potential of -93 +/- 1 mV, and were eliminated after internal calcium chelation with BAPTA, blockade of IP3 receptors, or extracellular application of cadmium but not by sodium channel blockade with tetrodotoxin. Calcium-imaging experiments demonstrated that ACh-induced hyperpolarizing, but not depolarizing, responses were correlated with large increases in intracellular calcium. Surprisingly, transient increases in muscarinic receptor activation were capable of generating hyperpolarizing responses even during periods of tonic muscarinic activation sufficient to depolarize neurons to action potential threshold. Furthermore, eserine, an acetylcholinesterase inhibitor similar to those used therapeutically in the treatment of Alzheimer's disease, disproportionately enhanced the excitatory actions of acetylcholine while reducing the ability of acetylcholine to generate inhibitory responses during repeated applications of ACh. These data demonstrate that acetylcholine can directly inhibit the output of neocortical pyramidal neurons.

Animals↗

PubMed2005

Synaptic integration in dendritic trees.

Most neurons have elaborate dendritic trees that receive tens of thousands of synaptic inputs. Because postsynaptic responses to individual synaptic events are usually small and transient, the integration of many synaptic responses is needed to depolarize most neurons to action potential threshold. Over the past decade, advances in electrical and optical recording techniques have led to new insights into how synaptic responses propagate and interact within dendritic trees. In addition to their passive electrical and morphological properties, dendrites express active conductances that shape individual synaptic responses and influence synaptic integration locally within dendrites. Dendritic voltage-gated Na(+) and Ca(2+) channels support action potential backpropagation into the dendritic tree and local initiation of dendritic spikes, whereas K(+) conductances act to dampen dendritic excitability. While all dendrites investigated to date express active conductances, different neuronal types show specific patterns of dendritic channel expression leading to cell-specific differences in the way synaptic responses are integrated within dendritic trees. This review explores the way active and passive dendritic properties shape synaptic responses in the dendrites of central neurons, and emphasizes their role in synaptic integration.

Action Potentials↗

PubMed2004

Kinetics of Mg2+ unblock of NMDA receptors: implications for spike-timing dependent synaptic plasticity.

The time course of Mg(2+) block and unblock of NMDA receptors (NMDARs) determines the extent they are activated by depolarization. Here, we directly measure the rate of NMDAR channel opening in response to depolarizations at different times after brief (1 ms) and sustained (4.6 s) applications of glutamate to nucleated patches from neocortical pyramidal neurons. The kinetics of Mg(2+) unblock were found to be non-instantaneous and complex, consisting of a prominent fast component (time constant approximately 100 micros) and slower components (time constants 4 and approximately 300 ms), the relative amplitudes of which depended on the timing of the depolarizing pulse. Fitting a kinetic model to these data indicated that Mg(2+) not only blocks the NMDAR channel, but reduces both the open probability and affinity for glutamate, while enhancing desensitization. These effects slow the rate of NMDAR channel opening in response to depolarization in a time-dependent manner such that the slower components of Mg(2+) unblock are enhanced during depolarizations at later times after glutamate application. One physiological consequence of this is that brief depolarizations occurring earlier in time after glutamate application are better able to open NMDAR channels. This finding has important implications for spike-timing-dependent synaptic plasticity (STDP), where the precise (millisecond) timing of action potentials relative to synaptic inputs determines the magnitude and sign of changes in synaptic strength. Indeed, we find that STDP timing curves of NMDAR channel activation elicited by realistic dendritic action potential waveforms are narrower than expected assuming instantaneous Mg(2+) unblock, indicating that slow Mg(2+) unblock of NMDAR channels makes the STDP timing window more precise.

Action Potentials↗

PubMed2003

Action potential initiation and propagation in layer 5 pyramidal neurons of the rat prefrontal cortex: absence of dopamine modulation.

Somatic and dendritic whole-cell recording was used to examine action potential (AP) initiation and propagation in layer 5 pyramidal neurons of the rat prelimbic prefrontal cortex. APs generated by somatic current injection, or via antidromic stimulation, were reliably recorded at apical dendritic locations as far as 480 microm from the soma. Although the backpropagation of single APs into the apical dendrite was robust, frequency-dependent attenuation was observed during AP trains delivered at 10-100 Hz. APs were usually initiated close to the soma (presumably in the axon); however, strong depolarizing input to the apical dendrite could generate dendritic spikes that preceded somatic APs. AP backpropagation was dependent solely on activation of dendritic voltage-gated sodium channels and did not require activation of dendritic calcium channels. Despite not playing a role in AP backpropagation, calcium-imaging experiments demonstrated that dendritic calcium channels are activated by backpropagating APs, leading to transient increases in intracellular calcium. In addition, calcium imaging revealed that AP backpropagation into the distal apical tuft was frequency dependent. Finally, we tested whether dopamine, a prominent neuromodulator associated with prefrontal activity, could alter AP initiation or backpropagation. Bath-applied dopamine (10 or 100 microm) did not effect AP backpropagation, frequency-dependent depression, local dendritic spike initiation, or AP-induced calcium signaling. These data indicate that AP backpropagation in prefrontal layer 5 pyramidal neurons is robust but frequency dependent in the distal tuft, requires dendritic sodium rather than calcium channel activation, and, unlike other aspects of neuronal excitability, insensitive to modulation by dopamine.

Action Potentials↗

PubMed2003

Voltage- and site-dependent control of the somatic impact of dendritic IPSPs.

Inhibitory interneurons target specific subcellular compartments of cortical pyramidal neurons, where location-specific interactions of IPSPs with voltage-activated ion channels are likely to influence the inhibitory control of neuronal output. To investigate this, we simulated IPSPs as a conductance source at sites across the somato-apical dendritic axis (up to 750 microm) of neocortical layer 5 pyramidal neurons. Analysis revealed that the electrotonic architecture of cortical pyramidal neurons is highly voltage dependent, resulting in a significant site-dependent disparity between the amplitude, kinetics, and dendro-somatic attenuation of IPSPs generated from depolarized (-50 mV) and hyperpolarized (-80 mV) membrane potentials. At the soma, the time course of IPSPs evoked from depolarized potentials was greatest when generated from proximal dendritic sites and decreased as events were generated more distally, whereas the somatic time course of IPSPs evoked from hyperpolarized potentials was independent of the dendritic site of generation. This behavior resulted from the concerted actions of axo-somatic sodium channels that increased the duration of proximal dendritic IPSPs generated at depolarized potentials and distal dendritic hyperpolarization-activated channels that mediated site independence of somatic IPSP time course at hyperpolarized potentials. Functionally, this voltage-dependent control of IPSPs shaped the spatial and temporal profile of inhibition of axonal action potential firing and dendritic spike generation. Together, these findings demonstrate that the somatic impact of dendritic IPSPs is highly voltage dependent and controlled by classes of ion channels differentially distributed across axodendritic domains, directly revealing site-dependent inhibitory synaptic processing in cortical pyramidal neurons.

Action Potentials↗

PubMed2003

Excitatory actions of GABA in the cortex.

Little is known about how GABAergic inputs interact with excitatory inputs under conditions that maintain physiological concentrations of intracellular anions. Using extracellular and gramicidin perforated-patch recording, we show that somatic and dendritic GABA responses in mature cortical pyramidal neurons are depolarizing from rest and can facilitate action potential generation when combined with proximal excitatory input. Dendritic GABA responses were excitatory regardless of timing, whereas somatic GABA responses were inhibitory when coincident with excitatory input but excitatory at earlier times. These excitatory actions of GABA occur even though the GABA reversal potential is below action potential threshold and largely uniform across the somato-dendritic axis, and arise when GABAergic inputs are temporally or spatially isolated from concurrent excitation. Our findings demonstrate that under certain circumstances GABA will have an excitatory role in synaptic integration in the cortex.

Action Potentials↗

PubMed2003

Role of dendritic synapse location in the control of action potential output.

How effective are synaptic inputs made at different dendritic sites in the control of action potential output? Recent technical advances have allowed direct observation of the amplitude and time course of excitatory synaptic inputs generated at defined dendritic sites. These findings indicate that contrasting mechanisms exist to enhance the impact of dendritic excitatory inputs to pyramidal neurons of the neocortex and hippocampus. Furthermore, multiple sites of synaptic integration have been revealed, suggesting that proximal and distal inputs are likely to be integrated in different ways. Taken together, these data emphasize the cell- and input-specific strategies used by neurons to enhance the impact of dendritic inputs on action potential generation.

Action Potentials↗

PubMed2002

Dependence of EPSP efficacy on synapse location in neocortical pyramidal neurons.

Neurons receive thousands of synaptic inputs throughout elaborate dendritic trees. Here we determine the somatic impact of excitatory postsynaptic potentials (EPSPs) generated at known dendritic sites in neocortical pyramidal neurons. As inputs became more distal, somatic EPSP amplitude decreased, whereas use-dependent depression increased. Despite marked attenuation (>40-fold), when coactivated within a narrow time window (approximately 10 milliseconds), distal EPSPs could directly influence action potential output following dendritic spike generation. These findings reveal that distal EPSPs are ineffective sources of background somatic excitation, but through coincidence detection have a powerful transient signaling role.

Action Potentials↗

PubMed2002

Membrane potential bistability is controlled by the hyperpolarization-activated current I(H) in rat cerebellar Purkinje neurons in vitro.

We investigated the role of the hyperpolarization-activated mixed cation current, I(H), in the control of spontaneous action potential firing of rat cerebellar Purkinje neurons in brain slices. Extracellular recordings revealed that the continual action potential firing of Purkinje neurons was disrupted by the pharmacological blockade of I(H). Blockade of I(H) revealed spontaneous transitions between periods of tonic action potential firing and quiescence, without effects on the frequency or variance of action potential generation. Whole-cell recordings revealed that blockade of I(H) unmasked a form of membrane potential bistability, where transitions between tonic firing and quiescent states (separated by approximately 20 mV) were evoked by excitatory and inhibitory postsynaptic potentials, or by the delivery of brief (20 ms) somatic or dendritic positive and negative current pulses. The stable upper state of tonic action potential firing was maintained by the recruitment of axo-somatic voltage-activated sodium, but not calcium, channels. Negative modulation of I(H) by serotonin unmasked bistability, indicating that bistability of Purkinje neurons is likely to occur under physiological conditions. These data indicate that I(H) acts as a 'safety net', maintaining the membrane potential of Purkinje neurons within the range necessary for the generation of tonic action potential firing. Following the downregulation of I(H), synaptic inhibition can generate long periods (seconds) of quiescence, the duration of which can be controlled by climbing fibre activation and by the underlying 'tone' of parallel fibre activity.

Action Potentials↗