whole-cell-patch-clamp-for-investigating-mechanisms-infrared-neural

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

A direct commissural connection between cochlear nuclei provides a pathway by which binaural input can influence the processing of acoustic information through the ventral cochlear nucleus. Despite anatomical evidence to suggest the existence of such a pathway, its nature and behavior have not been investigated previously. This in vivo intracellular electrophysiological study provides direct evidence of monosynaptic (mean latency, 1.43 msec), inhibitory commissural input to T stellate cells. This inhibition is fast acting (duration, <10 msec), occurring with little synaptic delay ( approximately 0.3 msec). Electrical stimulation also revealed the initiation of antidromic responses in the onset chopper population, signifying D stellate neurons as a source of commissural inputs. Activation of the commissural connection was most evident in response to broadband stimuli. These results provide the first compelling evidence of a fast, monosynaptic commissural pathway arising from contralateral D stellate neurons providing broadband inhibitory input to T stellate cells.

Fast inhibition underlies the transmission of auditory information between cochlear nuclei.

Within the first processing site of the central auditory pathway, inhibitory neurons (D stellate cells) broadly tuned to tonal frequency project on narrowly tuned, excitatory output neurons (T stellate cells). The latter is thought to provide a topographic representation of sound spectrum, whereas the former is thought to provide lateral inhibition that improves spectral contrast, particularly in noise. In response to pure tones, the overall discharge rate in T stellate cells is unlikely to be suppressed dramatically by D stellate cells because they respond primarily to stimulus onset and provide fast, short-duration inhibition. In vivo intracellular recordings from the ventral cochlear nucleus (VCN) showed that, when tones were presented above or below the characteristic frequency (CF) of a T stellate neuron, they were inhibited during depolarization. This resulted in a delay in the initial action potential produced by T stellate cells. This ability of fast inhibition to alter the first spike timing of a T stellate neuron was confirmed by electrically activating the D stellate cell pathway that arises in the contralateral cochlear nucleus. Delay was also induced when two tones were presented: one at CF and one outside the frequency response area of the T stellate neuron. These findings suggest that the traditional view of lateral inhibition within the VCN should incorporate delay as one of its principle outcomes.

Fast inhibition alters first spike timing in auditory brainstem neurons.

Ventral cochlear nucleus stellate cells respond to characteristic frequency (CF) tones with sustained (C(S)), transient (C(T)) or onset chopping (O(C)) activity. The mechanisms underlying these different response patterns are not fully understood, and the present study used in vivo intracellular recordings (n = 42) in urethane-anaesthetized rats to examine the possible influence of inhibition on action potential regularity. Hyperpolarization following the offset of a CF tone burst was used as a measure of on-CF inhibition. A cluster analysis based on several membrane potential features, including on-CF inhibition, discriminated three groups in addition to the C(S) response type - two types of C(T) responses and the O(C) type. The different patterns of firing regularity exhibited by C(S/T) neurons reflected different thresholds or degrees of overlap between these cells' narrowly tuned excitatory and inhibitory inputs. C(T) cells with closely matched inhibitory and excitatory response areas showed substantial on-CF inhibition and the greatest decline in firing regularity during a CF tone, whereas those with a mismatch between their response areas showed lateral inhibition and a less marked decline in firing regularity. The presence of inhibition in C(S) neurons did not alter their firing regularity, possibly because of the lower threshold for excitation compared with inhibition. The latency, duration and frequency extent of sustained hyperpolarization in C(S/T) cells is inconsistent with the response properties of O(C) neurons, suggesting that another source(s) of inhibition influences firing regularity, and presumably response magnitude, in these neurons.

Balanced inhibition and excitation underlies spike firing regularity in ventral cochlear nucleus chopper neurons.

The ventral cochlear nucleus (VCN) stellate cell population comprises two clusters: narrowly-tuned, excitatory T stellate neurons, and D stellate neurons, a broadly-tuned population of inhibitory cells. These neurons respond to best frequency (BF) tone bursts in a chopper or onset manner, respectively. Through extensive local and commissural projections the D stellate population provides a source of fast inhibitory input to both intrinsic and contralateral T stellate neurons. Whilst the nature of interactions between intrinsic stellate populations is difficult to examine, our previous intracellular investigations of the commissural pathway have provided a means by which to study this relationship in the in vivo preparation. It is the aim of this paper to both review and extend our understanding of the link between stellate populations and their involvement in the commissural pathway by presenting an overview of the results attained in our recently expanded study. The sample of 17 intracellular and 34 extracellular onset chopper (O(C)) and late/ideal (On(L)/On(I)) neurons revealed antidromic activity in 31.4% of neurons following contralateral stimulation, providing physiological evidence that On(L)/On(I) neurons also contribute projections to the commissural connection. Alternatively, 64.7% of the 34 intracellularly-recorded chopper neurons displayed fast, monosynaptic inhibitory potentials. This commissural input was found to influence the timing of neural activity in chopper neurons, providing insight into the relationship that exists between T and D stellate neurons.

Neural timing, inhibition and the nature of stellate cell interaction in the ventral cochlear nucleus.

A direct commissural connection formed between cochlear nuclei allows information from the contralateral ear to rapidly influence the processing of the ascending auditory signal. Among the neuronal groups proposed to both receive, and contribute to, commissural input is the bushy cell population in the ventral cochlear nucleus (VCN). In this in vivo electrophysiological study we examine the intracellular recordings of bushy neurons during electrical stimulation of the contralateral cochlear nucleus (CN) for evidence of both their contribution to, and input from commissural projections. Activation of the commissural pathway revealed short-latency fast hyperpolarisation in 19.5% of the 41 bushy neurons examined. The hyperpolarising potentials were small in amplitude, displayed a highly variable time course between neurons, and in some cases were eliminated with injection of depolarising current. There was no indication of antidromic activity, or short-latency excitatory potentials. These results suggest that i) bushy neurons do not contribute projections to the commissural connection, and ii) a small portion of bushy neurons are hyperpolarised following commissural stimulation.

The commissural pathway and cochlear nucleus bushy neurons: an in vivo intracellular investigation.

The family of two-pore domain potassium (K(â‚‚P)) channels is important in setting and controlling the background potassium current of excitable cells. This study examines the localisation of the acid-sensitive channel, K(â‚‚P)9.1 (TASK3), in cells of the gastric mucosa. We observed K(â‚‚P)9.1 immunoreactivity in endocrine cells of the mucosal glands of the guinea-pig, rat and mouse but the channels were not detected in parietal, chief, or mucous cells. K(â‚‚P)9.1 channel immunoreactivity was consistently co-localised with histidine decarboxylase immunopositive enterochromaffin-like (ECL) cells, and with the majority of ghrelin immunoreactive X/A cells. Localisation in somatostatin immunoreactive D cells was rare in the guinea-pig, and did not occur in the stomach of rat, but, in the mouse, K(â‚‚P)9.1 channels were observed in the majority of somatostatin-immunoreactive D cells. Conversely, sections taken from the guinea-pig and mouse stomachs, but not rat stomach, revealed K(â‚‚P)9.1 in gastrin-containing G cells. These results demonstrate the presence of K(â‚‚P)9.1 channels in the entero-endocrine ECL, G and D cell populations of the stomach that regulate acid secretion through the release of histamine, gastrin and somatostatin. K(â‚‚P)9.1 channels were located in the ghrelin X/A cells that regulate food intake.

Identification of endocrine cells of the stomach that express acid-sensitive background potassium (K(2P)9.1/TASK3) channels.

Neurotrophins provide an effective tool for the rescue and regeneration of spiral ganglion neurons (SGNs) following sensorineural hearing loss. However, these nerve growth factors are also potent modulators of ion channel activity and expression, and in the peripheral auditory system brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) have previously been shown to alter the firing properties of auditory neurons and differentially regulate the expression of some potassium channels in vitro. In this study we examined the activity of the hyperpolarization-mediated mixed-cation current (I(h)) in early post-natal cultured rat SGNs following exposure to combined BDNF and NT3. Whole-cell patch-clamp recordings made after 1 or 2 days in vitro revealed no change in the firing adaptation of neurons in the presence of BDNF and NT3. Resting membrane potentials were also maintained, but spike latency and firing threshold was subject to regulation by both neurotrophins and time in vitro. Current clamp recordings revealed an activity profile consistent with activation of the hyperpolarization-activated current. Rapid membrane hyperpolarization was followed by a voltage- and time-dependent depolarizing voltage sag. In voltage clamp, membrane hyperpolarization evoked a slowly-activating inward current that was reversibly blocked with cesium and inhibited by ZD7288. The amplitude and current density of I(h) was significantly larger in BDNF and NT3 supplemented cultures, but this did not translate to a significant alteration in voltage sag magnitude. Neurotrophins provided at 50 ng/ml produced a hyperpolarizing shift in the voltage-dependence and slower time course of I(h) activation compared to SGNs in control groups or cultured with 10 ng/ml BDNF and NT3. Our results indicate that combined BDNF and NT3 increase the activity of hyperpolarization-activated currents and that the voltage-dependence and activation kinetics of I(h) in SGNs are sensitive to changes in neurotrophin concentration. In addition, BDNF and NT3 applied together induce a decrease in firing threshold, but does not generate a shift in firing adaptation.

Combined application of brain-derived neurotrophic factor and neurotrophin-3 and its impact on spiral ganglion neuron firing properties and hyperpolarization-activated currents.

In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.

An in vitro model of developmental synaptogenesis using cocultures of human neural progenitors and cochlear explants.

In the auditory system, a specialized subset of sensory neurons are responsible for correctly relaying precise pitch and temporal cues to the brain. In individuals with severe-to-profound sensorineural hearing impairment these sensory auditory neurons can be directly stimulated by a cochlear implant, which restores sound input to the brainstem after the loss of hair cells. This neural prosthesis therefore depends on a residual population of functional neurons in order to function effectively.

Article	Year
Fast inhibition underlies the transmission of auditory information between cochlear nuclei. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 12867521	2003
Fast inhibition alters first spike timing in auditory brainstem neurons. Journal of neurophysiology\| PubMed ID: 15140909	2004
Balanced inhibition and excitation underlies spike firing regularity in ventral cochlear nucleus chopper neurons. The European journal of neuroscience\| PubMed ID: 15813933	2005
Neural timing, inhibition and the nature of stellate cell interaction in the ventral cochlear nucleus. Hearing research\| PubMed ID: 16554129	2006
The commissural pathway and cochlear nucleus bushy neurons: an in vivo intracellular investigation. Brain research\| PubMed ID: 17174943	2007
Identification of endocrine cells of the stomach that express acid-sensitive background potassium (K(2P)9.1/TASK3) channels. Journal of molecular histology\| PubMed ID: 20607593	2010
Combined application of brain-derived neurotrophic factor and neurotrophin-3 and its impact on spiral ganglion neuron firing properties and hyperpolarization-activated currents. Hearing research\| PubMed ID: 22796476	2012
An in vitro model of developmental synaptogenesis using cocultures of human neural progenitors and cochlear explants. Stem cells and development\| PubMed ID: 23078657	2013
Challenges for stem cells to functionally repair the damaged auditory nerve. Expert opinion on biological therapy\| PubMed ID: 23094991	2013

Karina Needham

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