Search

Burst discharges in the immature brain may contribute to its enhanced seizure susceptibility. The cellular mechanisms underlying burst discharges in the CA1 area of the immature versus adult hippocampus were investigated with simultaneous wholecell and field-potential recordings. When GABAA receptors were blocked pharmacologically, bursts in CA1 were either graded or all-or-none (or mixed) as a function of electrical stimulation intensity. Most CA1 minislices from immature rats displayed all-or-none or mixed bursts, whereas the slices from adult rats predominantly elicited graded bursts. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were greater in CA1 pyramidal cells from the immature than the adult slices. The developmental differences in CA1 bursting were also detected in slices adjusted for maturational changes in brain volume (i.e., 350 mm thick for immature vs. 450 mm thick for adult rats). Neither N-methyl-D-aspartate (NMDA) nor group I metabotropic glutamate (mGlu1) receptor antagonists blocked the network-driven bursts in immature CA1, but an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker abolished them. Robust excitatory postsynaptic potentials (EPSPs) occurred after bursts in some immature CA1 slices (23%) but never in slices from the adult. The input-output (amount of current injected vs. number of action potentials generated) relationship was markedly greater in CA1 pyramidal cells in the immature compared with the adult hippocampus. These data suggest that the CA1 area of the immature brain is capable of generating network-driven bursts, which declines in adult rats. The increased propensity of burst generation in immature CA1 appears to involve a greater AMPA receptor-mediated synaptic network and an increased intrinsic spike-generating ability. [ABSTRACT FROM AUTHOR]

Neural circuits require balanced synaptic excitation and inhibition to ensure accurate neural computation. Our knowledge about the development and maturation of inhibitory synaptic inputs is less well developed than that concerning excitation. Here we describe the maturation of an inhibitory circuit within the mammalian auditory brainstem where counterintuitively, inhibition drives action potential firing of principal neurons. With the use of combined anatomical tracing and electrophysiological recordings from mice, neurons of the superior paraolivary nucleus (SPN) are shown to receive converging glycinergic input from at least four neurons of the medial nucleus of the trapezoid body (MNTB). These four axons formed 30.71 ± 2.72 (means ± SE) synaptic boutons onto each SPN neuronal soma, generating a total inhibitory conductance of 80 nS. Such strong inhibition drives the underlying postinhibitory rebound firing mechanism, which is a hallmark of SPN physiology. In contrast to inhibitory projections to the medial and lateral superior olives, the inhibitory projection to the SPN does not exhibit experience-dependent synaptic refinement following the onset of hearing. These findings emphasize that the development and function of neural circuits cannot be inferred from one synaptic target to another, even if both originate from the same neuron. NEW & NOTEWORTHY Neuronal activity regulates development and maturation of neural circuits. This activity can include spontaneous burst firing or firing elicited by sensory input during early development. For example, auditory brainstem circuits involved in sound localization require acoustically evoked activity to form properly. Here we show, that an inhibitory circuit, involved in processing sound offsets, gaps, and rhythmically modulated vocal communication signals, matures before the onset of acoustically evoked activity. [ABSTRACT FROM AUTHOR]

The endocannabinoid (eCB) signaling system has been functionally implicated in many brain regions. Our understanding of the role of cannabinoid receptor type 1 (CB1) in olfactory processing remains limited. Cannabinoid signaling is involved in regulating glomerular activity in the main olfactory bulb (MOB). However, the cannabinoid-related circuitry of inputs to mitral cells in the MOB has not been fully determined. Using anatomical and functional approaches we have explored this question. CB1 was present in periglomerular processes of a GAD65-positive subpopulation of interneurons but not in mitral cells. We detected eCBs in the mouse MOB as well as the expression of CB1 and other genes associated with cannabinoid signaling in the MOB. Patch-clamp electrophysiology demonstrated that CB1 agonists activated mitral cells and evoked an inward current, while CB1 antagonists reduced firing and evoked an outward current. CB1 effects on mitral cells were absent in subglomerular slices in which the olfactory nerve layer and glomerular layer were removed, suggesting the glomerular layer as the site of CB1 action. We previously observed that GABAergic periglomerular cells show the inverse response pattern to CB1 activation compared with mitral cells, suggesting that CB1 indirectly regulates mitral cell activity as a result of cellular activation of glomerular GABAergic processes. This hypothesis was supported by the finding that cannabinoids modulated synaptic transmission to mitral cells. We conclude that CB1 directly regulates GABAergic processes in the glomerular layer to control GABA release and, in turn, regulates mitral cell activity with potential effects on olfactory threshold and behavior. [ABSTRACT FROM AUTHOR]

Patch clamping is the gold standard measurement technique for cell-type characterization in vivo, but it has low throughput, is difficult to scale, and requires highly skilled operation. We developed an autonomous robot that can acquire multiple consecutive patch-clamp recordings in vivo. In practice, 40 pipettes loaded into a carousel are sequentially filled and inserted into the brain, localized to a cell, used for patch clamping, and disposed. Automated visual stimulation and electrophysiology software enables functional cell-type classification of whole cell-patched cells, as we show for 37 cells in the anesthetized mouse in visual cortex (V1) layer 5. We achieved 9% yield, with 5.3 min per attempt over hundreds of trials. The highly variable and low-yield nature of in vivo patch-clamp recordings will benefit from such a standardized, automated, quantitative approach, allowing development of optimal algorithms and enabling scaling required for large-scale studies and integration with complementary techniques. [ABSTRACT FROM AUTHOR]

Much innovation is currently aimed at improving the number, density, and geometry of electrodes on extracellular multielectrode arrays for in vivo recording of neural activity in the mammalian brain. To choose a multielectrode array configuration for a given neuroscience purpose, or to reveal design principles of future multielectrode arrays, it would be useful to have a systematic way of evaluating the spike recording capability of such arrays. We describe an automated system that performs robotic patch-clamp recording of a neuron being simultaneously recorded via an extracellular multielectrode array. By recording a patch-clamp data set from a neuron while acquiring extracellular recordings from the same neuron, we can evaluate how well the extracellular multielectrode array captures the spiking information from that neuron. To demonstrate the utility of our system, we show that it can provide data from the mammalian cortex to evaluate how the spike sorting performance of a close-packed extracellular multielectrode array is affected by bursting, which alters the shape and amplitude of spikes in a train. We also introduce an algorithmic framework to help evaluate how the number of electrodes in a multielectrode array affects spike sorting, examining how adding more electrodes yields data that can be spike sorted more easily. Our automated methodology may thus help with the evaluation of new electrode designs and configurations, providing empirical guidance on the kinds of electrodes that will be optimal for different brain regions, cell types, and species, for improving the accuracy of spike sorting. [ABSTRACT FROM AUTHOR]

The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, NaV1.6, and NaV1.7) and -resistant (TTX-R, NaV1.8, and NaV1.9) voltage-gated sodium (NaV) channels, but NaV1.9 current has not yet been demonstrated. Using a F--containing internal solution, we found a NaV current in muscle afferent neurons that activates at around -70 mV with slow activation and inactivation kinetics, as expected from NaV1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following NaV1.9 current rundown (internal F-), application of the NaV1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl-), which suggests that NaV1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were NaV1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also NaV1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of NaV1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR. [ABSTRACT FROM AUTHOR]

Noradrenergic signaling in the amygdala is important for processing threats and other emotionally salient stimuli, and β-adrenergic receptor activation is known to enhance neuronal spiking in the lateral amygdala (LA) of juvenile animals. Nevertheless, intracellular recordings have not yet been conducted to determine the effect of β-adrenergic receptor activation on spike properties in the adult LA, despite the potential significance of developmental changes between adolescence and adulthood. Here we demonstrate that the β-adrenergic agonist isoproterenol (15 μM) enhances spike frequency in dorsal LA principal neurons of juvenile male C57BL/6 mice and fails to do so in strain- and sex-matched adults. Furthermore, we find that the age-dependent effect of isoproterenol on spike frequency is occluded by the GABAA receptor blocker picrotoxin (75 μM), suggesting that β-adrenergic receptors downregulate tonic inhibition specifically in juvenile animals. These findings indicate a significant shift during adolescence in the cellular mechanisms of β-adrenergic modulation in the amygdala. [ABSTRACT FROM AUTHOR]

Patch-clamp electrophysiology is widely used to characterize neuronal electrical phenotypes. However, there are no standard experimental conditions for in vitro whole cell patch-clamp electrophysiology, complicating direct comparisons between data sets. In this study, we sought to understand how basic experimental conditions differ among laboratories and how these differences might impact measurements of electrophysiological parameters. We curated the compositions of external bath solutions (artificial cerebrospinal fluid), internal pipette solutions, and other methodological details such as animal strain and age from 509 published neurophysiology articles studying rodent neurons. We found that very few articles used the exact same experimental solutions as any other, and some solution differences stem from recipe inheritance from advisor to advisee as well as changing trends over the years. Next, we used statistical models to understand how the use of different experimental conditions impacts downstream electrophysiological measurements such as resting potential and action potential width. Although these experimental condition features could explain up to 43% of the studyto-study variance in electrophysiological parameters, the majority of the variability was left unexplained. Our results suggest that there are likely additional experimental factors that contribute to cross-laboratory electrophysiological variability, and identifying and addressing these will be important to future efforts to assemble consensus descriptions of neurophysiological phenotypes for mammalian cell types. [ABSTRACT FROM AUTHOR]

Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a whole mount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at the cost of severing the lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application to and removal of filter paper from the photo-receptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell end feet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a whole mount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and nonpharmacological removal of light input. NEW & NOTEWORTHY This study reports a method for removing photoreceptors from rodent whole mount retina while preserving the architecture of the inner retina. The method enables easier access to the inner retina for studies of neural processing, such as by patch clamp recording. [ABSTRACT FROM AUTHOR]

Chronic experimentally induced hyperglycemia augments subunit-specific γ-aminobutyric acid A (GABAA) receptor-mediated inhibition of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). However, the contribution of α1 or γ GABAA receptor subunits, which are ubiquitously expressed on central nervous system neurons, to this elevation in inhibitory tone have not been determined. This study investigated the effect of chronic hyperglycemia/hypoinsulinemia on α1- and γ-subunit-specific GABAA receptor-mediated inhibition using electrophysiological recordings in vitro and quantitative RT-PCR. DMV neurons from streptozotocin-treated mice demonstrated enhancement of both phasic and tonic inhibitory currents in response to application of the α1-subunit-selective GABAA receptor-positive allosteric modulator zolpidem. Responses to low concentrations of the GABAA receptor antagonist gabazine suggested an additional increased contribution of γ-subunit-containing receptors to tonic currents in DMV neurons. Consistent with the functional elevation in α1- and γ-subunit-dependent activity, transcription of both the α1- and γ2-subunits was increased in the dorsal vagal complex of streptozotocin-treated mice. Overall, these findings suggest an increased sensitivity to both zolpidem and gabazine after several days of hyperglycemia/hypoinsulinemia, which could contribute to altered parasympathetic output from DMV neurons in diabetes. NEW & NOTEWORTHY Glutamate and GABA signaling in the dorsal vagal complex is elevated after several days of chronic hyperglycemia in a mouse model of type 1 diabetes. We report persistently enhanced GABAA receptor-mediated responses to the somnolescent zolpidem in preganglionic vagal motor neurons. These results imply a broader impact of chronic hyperglycemia on central vagal function than previously appreciated and reinforce the hypothesis that diabetes effects in the brain can impact regulation of metabolic homeostasis. [ABSTRACT FROM AUTHOR]

In vivo studies of neurophysiology using the whole cell patch-clamp technique enable exquisite access to both intracellular dynamics and cytosol of cells in the living brain but are underrepresented in deep subcortical nuclei because of fouling of the sensitive electrode tip. We have developed an autonomous method to navigate electrodes around obstacles such as blood vessels after identifying them as a source of contamination during regional pipette localization (RPL) in vivo. In mice, robotic navigation prevented fouling of the electrode tip, increasing RPL success probability 3 mm below the pial surface to 82% (n = 72/88) over traditional, linear localization (25%, n = 24/95), and resulted in high-quality thalamic whole cell recordings with average access resistance (32.0 MΩ) and resting membrane potential (-62.9 mV) similar to cortical recordings in isoflurane-anesthetized mice. Whole cell yield improved from 1% (n = 1/95) to 10% (n = 9/88) when robotic navigation was used during RPL. This method opens the door to whole cell studies in deep subcortical nuclei, including multimodal cell typing and studies of long-range circuits. NEW & NOTEWORTHY This work represents an automated method for accessing subcortical neural tissue for intracellular electrophysiology in vivo. We have implemented a novel algorithm to detect obstructions during regional pipette localization and move around them while minimizing lateral displacement within brain tissue. This approach leverages computer control of pressure, manipulator position, and impedance measurements to create a closed-loop platform for pipette navigation in vivo. This technique enables whole cell patching studies to be performed throughout the living brain. [ABSTRACT FROM AUTHOR]

Neuronal activity is energy demanding and coupled to cellular metabolism. In this study, we investigated the effects of glycolytic inhibition with 2-deoxy-d-glucose (2-DG) on basal membrane properties, spontaneous neuronal firing, and epileptiform network bursts in hippocampal slices. The effect of glycolytic inhibition on basal membrane properties was examined in hippocampal CA1 neurons, which are not ordinarily active spontaneously. Intracellular application of 2-DG did not significantly alter the membrane input resistance, action-potential threshold, firing pattern, or input-output relationship of these neurons compared with simultaneously recorded neighboring neurons without intracellular 2-DG. The effect of glycolytic inhibition on neuronal firing was tested in spontaneously active hippocampal neurons (CA3) when synaptic transmission was left intact or blocked with AMPA, NMDA, and GABAA receptor antagonists (DNQX, APV, and bicuculline, respectively). Under both conditions (synaptic activity intact or blocked), bath application of 2-DG (2 mM) blocked spontaneous firing in ~2/3 (67 and 71%, respectively) of CA3 pyramidal neurons. In contrast, neuronal firing of CA3 neurons persisted when 2-DG was applied intracellularly, suggesting that glycolytic inhibition of individual neurons is not sufficient to stop neuronal firing. The effects of 2-DG on epileptiform network bursts in area CA3 were tested in Mg2+-free medium containing 50 µM 4-aminopyridine. Bath application of 2-DG abolished these epileptiform bursts in a dose-dependent and all-or-none manner. Taken together, these data suggest that altered glucose metabolism profoundly affects cellular and network hyperexcitability and that glycolytic inhibition by 2-DG can effectively abrogate epileptiform activity. NEW & NOTEWORTHY Neuronal activity is highly energy demanding and coupled to cellular metabolism. In this study, we demonstrate that glycolytic inhibition with 2-deoxy-d-glucose (2-DG) effectively suppresses spontaneous neuronal firing and epileptiform bursts in hippocampal slices. These data suggest that an altered metabolic state can profoundly affect cellular and network excitability, and that the glycolytic inhibitor 2-DG may hold promise as a novel treatment of drug-resistant epilepsy. [ABSTRACT FROM AUTHOR]

Whole cell patch clamp has provided much insight into the function of voltage-gated ion channels in central neurons. However, voltage errors caused by the resistance of the recording electrode [series resistance ( R s currents and the other directly recording the membrane potential. We hypothesized that Ohm's law-based correction would approximate the measured voltage error. We found that voltage errors averaged <5 mV for currents considered to be large for patch clamp (∼7-13 nA) and <10 mV for massive currents thought to be experimentally intractable (25-30 nA), each error falling within commonly accepted inclusion limits. In most cases Ohm's law-based correction overpredicted these measured voltage errors by roughly 2.5-fold. Consequently, the use of Ohm's law to correct for voltage errors led to erroneous current-voltage ( + currents and the other directly recording the membrane potential. We hypothesized that Ohm's law-based correction would approximate the measured voltage error. We found that voltage errors averaged <5 mV for currents considered to be large for patch clamp (∼7-13 nA) and <10 mV for massive currents thought to be experimentally intractable (25-30 nA), each error falling within commonly accepted inclusion limits. In most cases Ohm's law-based correction overpredicted these measured voltage errors by roughly 2.5-fold. Consequently, the use of Ohm's law to correct for voltage errors led to erroneous current-voltage ( I-V ) relationships, showing the greatest distortion for inactivating currents. Finally, recordings with low electrode R s compensated moderately by the amplifier circuitry appeared to have smaller voltage errors than those with larger R s and high compensation despite the same "effective R s " and current magnitude. Therefore, when R s is low, large currents may be studied with better-than-expected voltage control. These results suggest that patch-clamp may be used to study ionic currents often interpreted to be off limits because of size. NEW & NOTEWORTHY Voltage errors occur in whole cell voltage clamp. We make, to our knowledge, the first direct measurements of these errors and find that voltage errors are far smaller than standard calculations would predict. Since voltage errors were often minimal during the measurement of large ion channel currents, this technique may be applied to large neurons of adults to gain insight into ion channel function across the life span and progression of disease.

The input-output and discharge properties of neurons are shaped by both passive and active electrophysiological membrane properties. Whole cell patchclamp recordings in lamina I-III neurons in an isolated preparation of the whole spinal cord of juvenile rats with attached dorsal roots and dorsal root ganglia were used to further define which of these properties provides the most impactful classification strategy. A total of 95 neurons were recorded in segment L5 and were classified based on the responses to L4 dorsal root stimulation. The results showed that high-threshold and silent neurons had higher membrane resistance and more negative resting membrane potential than low-threshold or wide-dynamic-range neurons. Rheobase in low-threshold and wide-dynamic-range neurons was significantly lower than that of highthreshold or silent neurons. Four types of firing patterns were identified in response to depolarizing current injections. Low-threshold cells most frequently showed a phasic firing pattern characterized by a short initial burst of action potentials, single spiking or irregular firing bursts at the onset of a depolarizing pulse. High-threshold and wide-dynamic-range neurons were characterized by tonic firing with trains of spikes occurring at regular intervals throughout the current pulse. The majority of silent neurons displayed a delayed onset of firing in response to current injection. These results indicate that the passive membrane properties of spinal neurons are tuned to optimize the responses to particular subsets of afferent stimuli. [ABSTRACT FROM AUTHOR]

Olanzapine, an atypical antipsychotic, is widely prescribed for the treatment of schizophrenia and bipolar disorder despite causing undesirable metabolic side effects. A variety of mechanisms and brain sites have been proposed as contributors to the side effects; however, the role of the dorsal motor nucleus of the vagus nerve (DMV), which plays a crucial role in the regulation of subdiaphragmatic organs and thus governs energy and glucose homeostasis, is largely unknown. Identifying the effect of olanzapine on the excitability of DMV neurons in both sexes is thus crucial to understanding possible underlying mechanisms. Whole cell patch-clamp electrophysiological recordings were conducted in stomach- and liver-related DMV neurons identified with retrograde viral tracers and in random DMV neurons. The effect of olanzapine on the neuronal excitability of DMV neurons both in male and female mice was established. Our data demonstrate that olanzapine hyperpolarizes the DMV neurons in both sexes and this effect is reversible. The hyperpolarization is associated with decreased firing rate and input resistance. Olanzapine also decreases the excitability of a subset of stomach- and liver-related DMV neurons. Our study demonstrates that olanzapine has a powerful effect on DMV neurons in both sexes, indicating its ability to reduce vagal output to the subdiaphragmatic organs, which likely contributes to the metabolic side effects observed in both humans and experimental models. These findings suggest that the metabolic side effects of olanzapine may partially originate in the DMV. [ABSTRACT FROM AUTHOR]

Electrophysiological recordings from brain slices are typically performed in small recording chambers that allow for the superfusion of the tissue with artificial extracellular solution (ECS), while the chamber holding the tissue is mounted in the optical path of a microscope to image neurons in the tissue. ECS itself is inexpensive, and thus superfusion rates and volumes of ECS consumed during an experiment using standard ECS are not critical. However, some experiments require the addition of expensive pharmacological agents or other chemical compounds to the ECS, creating a need to build superfusion systems that operate on small volumes while still delivering appropriate amounts of oxygen and other nutrients to the tissue. We developed a closed circulation tissue chamber for slice recordings that operates with small volumes of bath solution in the range of 1.0 to 2.6 ml and a constant oxygen/carbon dioxide delivery to the solution in the bath. In our chamber, the ECS is oxygenated and recirculated directly in the recording chamber, eliminating the need for tubes and external bottles/containers to recirculate and bubble ECS and greatly reducing the total ECS volume required for superfusion. At the same time, the efficiency of tissue oxygenation and health of the section are comparable to standard superfusion methods. We also determined that the small volume of ECS contains a sufficient amount of nutrients to support the health of a standard brain slice for several hours without concern for either depletion of nutrients or accumulation of waste products. [ABSTRACT FROM AUTHOR]

Automation has been an important part of biomedical research for decades, and the use of automated and robotic systems is now standard for such tasks as DNA sequencing, microfluidics, and high-throughput screening. Recently, Kodandaramaiah and colleagues (Nat Methods 9: 585-587, 2012) demonstrated, using anesthetized animals, the feasibility of automating blind patch-clamp recordings in vivo. Blind patch is a good target for automation because it is a complex yet highly stereotyped process that revolves around analysis of a single signal (electrode impedance) and movement along a single axis. Here, we introduce an automated system for blind patch-clamp recordings from awake, head-fixed mice running on a wheel. In its design, we were guided by 3 requirements: easy-to-use and easy-to-modify software; seamless integration of behavioral equipment; and efficient use of time. The resulting system employs equipment that is standard for patch recording rigs, moderately priced, or simple to make. It is written entirely in MATLAB, a programming environment that has an enormous user base in the neuroscience community and many available resources for analysis and instrument control. Using this system, we obtained 19 whole cell patch recordings from neurons in the prefrontal cortex of awake mice, aged 8-9 wk. Successful recordings had series resistances that averaged 52 ± 4 MΩ and required 5.7 ± 0.6 attempts to obtain. These numbers are comparable with those of experienced electrophysiologists working manually, and this system, written in a simple and familiar language, will be useful to many cellular electrophysiologists who wish to study awake behaving mice. [ABSTRACT FROM AUTHOR]

Histamine receptors are densely expressed in the mesencephalic trigeminal nucleus (MesV) and trigeminal motor nucleus. However, little is known about the functional roles of neuronal histamine in controlling oral-motor activity. Thus, using the whole-cell recording technique in brainstem slice preparations from Wistar rats aged between postnatal days 7 and 13, we investigated the effects of histamine on the MesV neurons innervating the masseter muscle spindles and masseter motoneurons (MMNs) that form a reflex arc for the jaw-closing reflex. Bath application of histamine (100 µM) induced membrane depolarization in both MesV neurons and MMNs in the presence of tetrodotoxin, whereas histamine decreased and increased the input resistance in MesV neurons and MMNs, respectively. The effects of histamine on MesV neurons and MMNs were mimicked by an H1 receptor agonist, 2-pyridylethylamine (100 µM). The effects of an H2 receptor agonist, dimaprit (100 µM), on MesV neurons were inconsistent, whereas MMNs were depolarized without changes in the input resistance. An H3 receptor agonist, immethridine (100 µM), also depolarized both MesV neurons and MMNs without changing the input resistance. Histamine reduced the peak amplitude of postsynaptic currents (PSCs) in MMNs evoked by stimulation of the trigeminal motor nerve (5N), which was mimicked by 2-pyridylethylamine but not by dimaprit or immethridine. Moreover, 2-pyridylethylamine increased the failure rate of PSCs evoked by minimal stimulation and the pairedpulse ratio. These results suggest that histaminergic inputs to MesV neurons through H1 receptors are involved in the suppression of the jaw-closing reflex although histamine depolarizes MesV neurons and/or MMNs. [ABSTRACT FROM AUTHOR]

Early cognitive impairment in Alzheimer's disease (AD) correlates with medial temporal lobe dysfunction, including two areas essential for memory formation: the entorhinal cortex and dentate gyrus (DG). In the Tg2576 animal model for AD amyloidosis, activation of the peroxisome proliferator-activated receptor-gamma (PPARγ) with rosiglitazone (RSG) amelio-rates hippocampus-dependent cognitive impairment and restores ab-errant synaptic activity at the entorhinal cortex to DG granule neuron inputs. It is unknown, however, whether intrinsic firing properties of DG granule neurons in these animals are affected by amyloid-β pathology and if they are sensitive to RSG treatment. Here, we report that granule neurons from 9-mo-old wild-type and Tg2576 animals can be segregated into two cell types with distinct firing properties and input resistance that correlate with less mature type I and more mature type II neurons. The DG type I cell population was greater than type II in wild-type littermates. In the Tg2576 animals, the type I and type II cell populations were nearly equal but could be restored to wild-type levels through cognitive enhancement with RSG. Furthermore, Tg2576 cell firing frequency and spike after depolarization were decreased in type I and increased in type II cells, both of which could also be restored to wild-type levels upon RSG treatment. That these parameters were restored by PPARγ activation emphasizes the therapeutic value of RSG against early AD cognitive impairment. [ABSTRACT FROM AUTHOR]

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 µV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation. [ABSTRACT FROM AUTHOR]

The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4-) were hardly affected. GBP (100 µM) or PGB (10 µM) were less effective than 20 µM Mn2+ in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 µM Mn2+ in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca2+ channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca2+ imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa. [ABSTRACT FROM AUTHOR]

Burst discharges in the immature brain may contribute to its enhanced seizure susceptibility. The cellular mechanisms underlying burst discharges in the CA1 area of the immature versus adult hippocampus were investigated with simultaneous whole-cell and field-potential recordings. When GABA A receptors were blocked pharmacologically, bursts in CA1 were either graded or all-or-none (or mixed) as a function of electrical stimulation intensity. Most CA1 minislices from immature rats displayed all-or-none or mixed bursts, whereas the slices from adult rats predominantly elicited graded bursts. The frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were greater in CA1 pyramidal cells from the immature than the adult slices. The developmental differences in CA1 bursting were also detected in slices adjusted for maturational changes in brain volume (i.e., 350 µm thick for immature vs. 450 µm thick for adult rats). Neither N -methyl-d-aspartate (NMDA) nor group I metabotropic glutamate (mGlu1) receptor antagonists blocked the network-driven bursts in immature CA1, but an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker abolished them. Robust excitatory postsynaptic potentials (EPSPs) occurred after bursts in some immature CA1 slices (23%) but never in slices from the adult. The input-output (amount of current injected vs. number of action potentials generated) relationship was markedly greater in CA1 pyramidal cells in the immature compared with the adult hippocampus. These data suggest that the CA1 area of the immature brain is capable of generating network-driven bursts, which declines in adult rats. The increased propensity of burst generation in immature CA1 appears to involve a greater AMPA receptor-mediated synaptic network and an increased intrinsic spike-generating ability. NEW & NOTEWORTHY Burst discharges in the developing brain can provide valuable insights into epileptogenesis. We show that the immature hippocampal CA1 area is capable of generating all-or-none (i.e., network) bursts, which transitions to graded (i.e., nonnetwork) bursts in the mature brain via both synaptic and intrinsic mechanisms. Our results provide new clues to help understand possible mechanisms that may be shared in the immature and epileptic brain and how the normal brain becomes seizure prone (i.e., epileptogenesis).

Serotonin (5-HT), and its 5-HT1A receptor (5-HT1AR) subtype, is a powerful modulator of the cardiorespiratory system and its sensory reflexes. The nucleus tractus solitarii (nTS) serves as the first central station for visceral afferent integration and is critical for cardiorespiratory reflex responses. However, the physiological and synaptic role of 5-HT1ARs in the nTS is relatively unknown. In the present study, we examined the distribution and modulation of 5-HT1ARs on cardiorespiratory and synaptic parameters in the nTS. 5-HT1ARs were widely distributed to cell bodies within the nTS but not synaptic terminals. In anesthetized rats, activation of 5-HT1ARs by microinjection of the 5-HT1AR agonist 8-OH-DPAT into the caudal nTS decreased minute phrenic neural activity via a reduction in phrenic amplitude. In brain stem slices, 8-OH-DPAT decreased the amplitude of glutamatergic tractus solitarii- evoked excitatory postsynaptic currents, and reduced overall spontaneous excitatory nTS network activity. These effects persisted in the presence of GABAA receptor blockade and were antagonized by coapplication of 5-HT1AR blocker WAY-100135. 5-HT1AR blockade alone had no effect on tractus solitarii-evoked excitatory postsynaptic currents, but increased excitatory network activity. On the other hand, GABAergic nTS-evoked inhibitory postsynaptic currents did not change by activation of the 5-HT1ARs, but spontaneous inhibitory nTS network activity decreased. Blocking 5-HT1ARs tended to increase nTS-evoked inhibitory postsynaptic currents and inhibitory network activity. Taken together, 5-HT1ARs in the caudal nTS decrease breathing, likely via attenuation of afferent transmission, as well as overall nTS network activity. [ABSTRACT FROM AUTHOR]

The electrophysiological and morphological characteristics of premotor neurons in the supratrigeminal region (SupV) targeting the trigeminal motor nucleus (MoV) were examined in neonatal rat brain stem slice preparations with Ca2+ imaging, whole cell recordings, and intracellular biocytin labeling. First, we screened SupV neurons that showed a rapid rise in intracellular free Ca2+ concentration ([Ca2+]i) after single-pulse electrical stimulation of the ipsilateral MoV. Subsequent whole cell recordings were generated from the screened SupV neurons, and their antidromic responses to MoV stimulation were confirmed. We divided the antidromically activated premotor neurons into two groups according to their discharge patterns during the steady state in response to 1-s depolarizing current pulses: those firing at a frequency higher (HF neurons, n = 19) or lower (LF neurons, n = 17) than 33 Hz. In addition, HF neurons had a narrower action potential and a larger afterhyperpolarization than LF neurons. Intracellular labeling revealed that the axons of all HF neurons (6/6) and half of the LF neurons (4/9) entered the MoV from its dorsomedial aspect, whereas the axons of the remaining LF neurons (5/9) entered the MoV from its dorsolateral aspect. Furthermore, the dendrites of three HF neurons penetrated into the principal sensory trigeminal nucleus (Vp), whereas the dendrites of all LF neurons were confined within the SupV. These results suggest that the types of SupV premotor neurons targeting the MoV with different firing properties have different dendritic and axonal morphologies, and these SupV neuron classes may play unique roles in diverse oral motor behaviors, such as suckling and mastication. [ABSTRACT FROM AUTHOR]

Cellular mechanisms for antinociception produced by oxytocin in the spinal dorsal horn have not yet been investigated thoroughly. We examined how oxytocin affects synaptic transmission in substantia gelatinosa neurons, which play a pivotal role in regulating nociceptive transmission, by applying the whole-cell patch-clamp technique to the substantia gelatinosa neurons of adult rat spinal cord slices. Bathapplied oxytocin did not affect glutamatergic spontaneous, monosynaptically- evoked primary-afferent Aδ-fiber and C-fiber excitatory transmissions. On the other hand, oxytocin produced an inward current at -70 mV and enhanced GABAergic and glycinergic spontaneous inhibitory transmissions. These activities were repeated with a slow recovery from desensitization, concentration-dependent and mimicked by oxytocin-receptor agonist. The oxytocin current was inhibited by oxytocin-receptor antagonist, intracellular GDPβS, U-73122, 2-aminoethoxydiphenyl borate, but not dantrolene, chelerythrine, dibutyryl cyclic-AMP, CNQX, Ca2+-free and tetrodotoxin, while the spontaneous inhibitory transmission enhancements were depressed by tetrodotoxin. Current-voltage relation for the oxytocin current reversed at negative potentials more than the equilibrium potential for K+, or around 0 mV. The oxytocin current was depressed in high-K+, low-Na+ or Ba2+-containing solution. Vasopressin V1A- receptor antagonist inhibited the oxytocin current, but there was no correlation in amplitude between a vasopressin-receptor agonist [Arg8]vasopressin and oxytocin responses. It is concluded that oxytocin produces a membrane depolarization mediated by oxytocin but not vasopressin-V1A receptors, which increases neuronal activity, resulting in the enhancement of inhibitory transmission, a possible mechanism for antinociception. This depolarization is due to a change in membrane permeabilities to K+ and/or Na+, which is possibly mediated by phospholipase C and inositol 1,4,5-triphosphate-induced Ca2+-release. [ABSTRACT FROM AUTHOR]

The rostral ventrolateral medulla (RVLM) is a critical component of the sympathetic nervous system regulating homeostatic functions including arterial blood pressure. Using the transsynaptic retrograde viral tracer PRV-152, we identified kidney-related neurons in the RVLM. We found that PRV-152-labeled RVLM neurons displayed an unusually large persistent, tonic current to both glutamate, via N-methyl-d-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/kainate receptors, and to γ-aminobutyric acid (GABA), via GABAA receptors, in the absence of large-scale phasic neurotransmission with whole cell patch-clamp recordings. A cocktail of potent NMDA and AMPA/kainate ionotropic glutamate receptor antagonists AP-5 (50 μM) and CNQX (10 μM) revealed a two-component somatic tonic excitatory current with an overall amplitude of 42.6 ± 13.4 pA. Moreover, application of the GABAA receptor blockers gabazine (15 μM) and bicuculline (30 μM) revealed a robust somatic tonic inhibitory current with an average amplitude of 196.3 ± 39.3 pA. These findings suggest that the tonic current plays a role in determining the resting membrane potential, input resistance, and firing rate of RVLM neurons. The magnitude of the tonic inhibitory current demonstrates that GABAergic inhibition plays a critical role in regulation of kidney-related RVLM neurons. Our results indicate that the GABAergic tonic current may determine the basal tone of firing activity in kidney-related RVLM neurons. [ABSTRACT FROM AUTHOR]

Activity of presympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus is known to play an important role in the regulation of sympathetic outflow. Sympathetic overactivity is associated with many pathophysiological conditions such as diabetes mellitus and hypertension; however, the underlying synaptic mechanisms are poorly understood. In this study, we examined the GABAergic inhibitory synaptic control of kidney-related presympathetic PVN neurons in the streptozotocin-treated type 1 diabetic mouse model, using patch-clamp slice electrophysiology in combination with retrograde labeling. Type 1 diabetes resulted in decreased frequency of miniature inhibitory postsynaptic currents (mIPSCs). Our data also demonstrated a reduction of mIPSC amplitude and mean inhibitory current without alteration of input resistance. Furthermore, our data revealed decreased tonic GABAergic inhibition of kidney-related PVN neurons in diabetic conditions, which was consistent with the observed increased excitability of the presympathetic PVN neurons. In summary, our data demonstrated decreased phasic and tonic inhibitory control of kidney-related presympathetic PVN neurons that suggest altered sympathetic circuitry in type 1 diabetes. [ABSTRACT FROM AUTHOR]

Olfactory perception is influenced by behavioral states, presumably via efferent regulation. Using the whole cell version of patch-clamp recording technique, we discovered that acetylcholine, which is released from efferent fibers in the olfactory mucosa, can directly affect the signal encoding in newt olfactory receptor cells (ORCs). Under current-clamp conditions, application of carbachol, an acetylcholine receptor agonist, increased the spike frequency of ORCs and lowered their spike threshold. When a 3-pA current to induce near-threshold depolarization was injected into ORCs, 0.0 spikes/s were generated in control solution and 0.5 spikes/s in the presence of carbachol. By strong stimuli of injection of a 13-pA current into ORCs, 9.1 and 11.0 spikes/s were generated in control and carbachol solutions, respectively. A similar result was observed by bath application of 50 M acetylcholine. Under voltageclamp conditions, carbachol increased the peak amplitude of a voltage- gated sodium current by 32% and T-type calcium current by 39%. Atropine, the specific muscarinic receptor antagonist, blocked the enhancement by carbachol of the voltage-gated sodium current and T-type calcium current, suggesting that carbachol increases those currents via the muscarinic receptor rather than via the nicotinic receptor. In contrast, carbachol did not significantly change the amplitude of the L-type calcium current or the delayed rectifier potassium current in the ORCs. Because T-type calcium current is known to lower the threshold in ORCs, we suggest that acetylcholine enhance excitability by lowering the threshold of spike generation in ORCs via the muscarinic receptor. [ABSTRACT FROM AUTHOR]

The properties of neurons participating in masticatory rhythmogenesis are not clearly understood. Neurons within the dorsal trigeminal principal sensory nucleus (dPrV) are potential candidates as components of the masticatory central pattern generator (CPG). The present study examines in detail the ionic mechanisms controlling burst generation in dPrV neurons in rat (postnatal day 8–12) brain stem slices using whole cell and perforated patch-clamp methods. Nominal extracellular Ca2+ concentration transformed tonic discharge in response to a maintained step pulse of current into rhythmical bursting in 38% of nonbursting neurons. This change in discharge mode was suppressed by riluzole, a persistent Na+ current (INaP) antagonist. Veratridine, which suppresses the Na+ channel inactivation mechanism, induced rhythmical bursting in nonbursting neurons in normal artificial cerebrospinal fluid, suggesting that INaP contributes to burst generation. Nominal extracellular Ca2+ exposed a prominent afterdepolarizing potential (ADP) following a single spike induced by a 3-ms current pulse, which was suppressed, but not completely blocked, by riluzole. Application of BAPTA, a Ca2+ chelator, intracellularly, or flufenamic acid, a Ca2+-activated nonspecific cationic channel (ICAN) antagonist, extracellularly to the bath, suppressed rhythmical bursting and the postspike ADP. Application of drugs to alter Ca2+ release from endoplasmic reticulum also suppressed bursting. Finally, voltage-clamp methods demonstrated that nominal Ca2+ facilitated INaP and induced ICAN. These data demonstrate for the first time that the previously observed induction in dPrV neurons of rhythmical bursting in nominal Ca2+ is mediated by enhancement of INaP and onset of ICAN, which are dependent on intracellular Ca2+. [ABSTRACT FROM AUTHOR]

The rostral nucleus of the solitary tract (rNTS), the first-order relay of gustatory information, not only transmits sensory information to more rostral brain areas but also connects to various brain stem sites responsible for orofacial reflex activities. While much is known regarding ascending projections to the parabrachial nucleus, intramedullary projections to the reticular formation (which regulate oromotor reflexive behaviors) remain relatively unstudied. The present study examined the intrinsic firing properties of these neurons as well as their morphological properties and synaptic connectivity with primary sensory afferents. Using in vitro whole cell patch-clamp recording, we found that intramedullary projection neurons respond to depolarizing current injection with either tonic or bursting action potential trains and subsets of these groups of neurons express A-type potassium, H-like, and postinhibitory rebound currents. Approximately half of the intramedullary projection neurons tested received monosynaptic innervation from primary afferents, while the rest received polysynaptic innervation, indicating that at least a subpopulation of these neurons can be directly activated by incoming sensory information. Neuron morphological reconstructions revealed that many of these neurons possessed numerous dendritic spines and that neurons receiving monosynaptic primary afferent input have a greater spine density than those receiving polysynaptic primary afferent input. These results reveal that intramedullary projection neurons represent a heterogeneous class of rNTS neurons and, through both intrinsic voltage-gated ion channels and local circuit interactions, transform incoming gustatory information into signals governing oromotor reflexive behaviors. [ABSTRACT FROM AUTHOR]

The timing of neurotransmitter release from neurons can be modulated by many presynaptic mechanisms. The retina uses synaptic ribbons to mediate slow graded glutamate release from bipolar cells that carry photoreceptor inputs. However, many inhibitory amacrine cells, which modulate bipolar cell output, spike and do not have ribbons for graded release. Despite this, slow glutamate release from bipolar cells is modulated by slow GABAergic inputs that shorten the output of bipolar cells, changing the timing of visual signaling. The time course of light-evoked inhibition is slow due to a combination of receptor properties and prolonged neurotransmitter release. However, the light-evoked release of GABA requires activation of neurons upstream from the amacrine cells, so it is possible that prolonged release is due to slow amacrine cell activation, rather than slow inherent release properties of the amacrine cells. To test this idea, we directly activated primarily action potential-dependent amacrine cell inputs to bipolar cells with electrical stimulation. We found that the decay of GABAC receptor-mediated electrically evoked inhibitory currents was significantly longer than would be predicted by GABAC receptor kinetics, and GABA release, estimated by deconvolution analysis, was inherently slow. Release became more transient after increasing slow Ca2+ buffering or blocking prolonged L-type Ca2+ channels and Ca2+ release from intracellular stores. Our results suggest that GABAergic amacrine cells have a prolonged buildup of Ca2+ in their terminals that causes slow, asynchronous release. This could be a mechanism of matching the time course of amacrine cell inhibition to bipolar cell glutamate release. [ABSTRACT FROM AUTHOR]

TRP channels are thought to play a role in regulating nociceptive transmission to spinal substantia gelatinosa (SG) neurons. It remains to be unveiled whether the TRP channels in the CNS are different in property from those involved in receiving nociceptive stimuli in the PNS. We examined the effect of vanilloid compound zingerone, which activates TRPV1 channels in the cell body of primary-afferent neuron, on glutamatergic excitatory transmission in the SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. Bath-applied zingerone reversibly and concentration-dependently increased spontaneous excitatory postsynaptic current (EPSC) frequency. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate-receptor antagonists. These zingerone effects were repeated and persisted in Na+-channel blocker tetrodotoxin-, La3+-, IP3-induced Ca2+-release inhibitor 2-aminoethoxydiphenyl borate-containing or Ca2+-free Krebs solution. Zingerone activity was resistant to selective TRPV1 antagonist capsazepine, whereas sensitive to non-selective TRP antagonist ruthenium red, TRPA1 antagonist HC-030031 and Ca2+-induced Ca2+-release inhibitor dantrolene. TRPA1 agonist allyl isothiocyanate but not capsaicin inhibited the facilitatory effect of zingerone. On the other hand, zingerone reduced monosynaptically-evoked EPSC amplitudes, as TRPA1 agonists did. Like allyl isothiocyanate, zingerone enhanced GABAergic spontaneous inhibitory transmission in a manner sensitive to tetrodotoxin. We conclude that zingerone presynaptically facilitates spontaneous excitatory transmission, probably through Ca2+-induced Ca2+-release mechanisms, and produces a membrane depolarization in SG neurons by activating TRPA1 but not TRPV1 channels. [ABSTRACT FROM AUTHOR]

The retina responds to a wide range of light stimuli by adaptation of retinal signaling to background light intensity and the use of two different photoreceptors: rods that sense dim light and cones that sense bright light. Rods signal to rod bipolar cells that receive significant inhibition from amacrine cells in the dark, especially from a rod bipolar cell-activated GABAergic amacrine cell. This inhibition modulates the output of rod bipolar cells onto downstream neurons. However, it was not clear how the inhibition of rod bipolar cells changes when rod signaling is limited by an adapting background light and cone signaling becomes dominant. We found that both light-evoked and spontaneous rod bipolar cell inhibition significantly decrease with light adaptation. This suggests a global decrease in the activity of amacrine cells that provide input to rod bipolar cells with light adaptation. However, inhibition to rod bipolar cells is also limited by GABAergic connections between amacrine cells, which decrease GABAergic input to rod bipolar cells. When we removed this serial inhibition, the light-evoked inhibition to rod bipolar cells remained after light adaptation. These results suggest that decreased inhibition to rod bipolar cells after light adaptation is due to decreased rod pathway activity as well as an active increase in inhibition between amacrine cells. Together these serve to limit rod bipolar cell inhibition after light adaptation, when the rod pathway is inactive and modulation of the signal is not required. This suggests an efficiency mechanism in the retina to limit unnecessary signaling. [ABSTRACT FROM AUTHOR]

Sensory systems need to tease out stimulation-evoked activity against a noisy background. In the olfactory system, the odor response profile of an olfactory sensory neuron (OSN) is dependent on the type of odorant receptor it expresses. OSNs also exhibit spontaneous activity, which plays a role in establishing proper synaptic connections and may also increase the sensitivity of the cells. However, where the spontaneous activity originates and whether it informs sensory-evoked activity remain unclear. We addressed these questions by examining patch-clamp recordings of genetically labeled mouse OSNs with defined odorant receptors in intact olfactory epithelia. We show that OSNs expressing different odorant receptors had significantly different rates of basal activity. Additionally, OSNs expressing an inactive mutant I7 receptor completely lacked spontaneous activity, despite being able to fire action potentials in response to current injection. This finding strongly suggests that the spontaneous firing of an OSN originates from the spontaneous activation of its G protein-coupled odorant receptor. Moreover, OSNs expressing the same receptor displayed considerable variation in their spontaneous activity, and the variation was broadened upon odor stimulation. Interestingly, there is no significant correlation between the spontaneous and sensory-evoked activity in these neurons. This study reveals that the odorant receptor type determines the spontaneous firing rate of OSNs, but the basal activity does not correlate with the activity induced by near-saturated odor stimulation. The implications of these findings on olfactory information processing are discussed. [ABSTRACT FROM AUTHOR]

To account for benzodiazepine-induced spinal analgesia observed in association with an inflammation-induced shift in the influence of the GABAA receptor antagonist gabazine on nociceptive threshold, the present study was designed to determine whether persistent inflammation is associated with the upregulation of high-affinity GABAA receptors in primary afferents. The cell bodies of afferents innervating the glabrous skin of the rat hind paw were retrogradely labeled, acutely dissociated, and studied before and after the induction of persistent inflammation. A timedependent increase in GABAA current density was observed that was more than twofold by 72 h after the initiation of inflammation. This increase in current density included both high- and low-affinity currents and was restricted to neurons in which GABA increased intracellular Ca2+. No increases in GABAA receptor subunit mRNA or protein were detected in whole ganglia. In contrast, the increased current density was completely reversed by 20-min preincubation with the tyrosine kinase inhibitor genistein and partially reversed with the Src kinase inhibitor PP2. Genistein reversal was partially blocked by the dynamin inhibitor peptide P4. Changes in nociceptive threshold following spinal administration of genistein and muscimol to inflamed rats indicated that the pronociceptive actions of muscimol observed in the presence of inflammation were reversed by genistein. These results suggest that persistent changes in relative levels of tyrosine kinase activity following inflammation provide not only a sensitive way to dynamically regulate spinal nociceptive signaling but a viable target for the development of novel therapeutic interventions for the treatment of inflammatory pain. [ABSTRACT FROM AUTHOR]

Mammals rely on their acute olfactory sense for their survival. The most anterior olfactory subsystem in the nose, the Grueneberg ganglion (GG), plays a role in detecting alarm pheromone, cold, and urinary compounds. GG neurons respond homogeneously to these stimuli with increases in intracellular [Ca2+] or transcription of immediate-early genes. In this electrophysiological study, we used patch-clamp techniques to characterize the membrane properties of GG neurons. Our results offer evidence of functional heterogeneity in the GG. GG neurons fire spontaneously and independently in several stable patterns, including phasic and repetitive single-spike modes of discharge. Whole cell recordings demonstrated two distinct voltage-gated fast-inactivating Na+ currents with different steady-state voltage dependencies and different sensitivities to tetrodotoxin. Hodgkin-Huxley simulations showed that these Na+ currents confer dual mechanisms of action potential generation and contribute to different firing patterns. Additionally, GG neurons exhibited hyperpolarization-activated inward currents that modulated spontaneous firing in vitro. Thus, in GG neurons, the heterogeneity of firing patterns is linked to the unusual repertoire of ionic currents. The membrane properties described here will aid the interpretation of chemosensory function in the GG. [ABSTRACT FROM AUTHOR]

The effect of interleukin- 1β (IL-1β) on the electrical properties of sensory neurons was assessed at levels and exposure times comparable to those found in animal models of neuropathic pain. Experiments involved whole cell current-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined-medium, neuron-enriched cultures. Five- to sixday exposure to 100 pM IL-1β produced subpopulation-dependent effects on DRG neurons. These included an increase in the excitability of medium-diameter and small-diameter isolectin B4 (IB4)-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large-diameter neurons, while no effect was found in small-diameter IB4-negative neurons. Further characterization of changes in medium and small IB4-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Although the acute actions of IL-1β on sensory neurons have been well studied and related to acute and/or inflammatory pain, the present study shows how sensory neurons respond to long-term cytokine exposure. Such effects are relevant to understanding processes that contribute to the onset of neuropathic pain. [ABSTRACT FROM AUTHOR]

Whole cell patchclamp recordings were used to investigate the contribution of transient, low-threshold calcium currents (IT) to firing properties of hamster spinal dorsal horn neurons. IT was widely, though not uniformly, expressed by cells in Rexed's laminae I-IV and correlated with the pattern of action potential discharge evoked under current-clamp conditions: IT in neurons responding to constant membrane depolarization with one or two action potentials was nearly threefold larger than IT in cells responding to the same activation with continuous firing. IT was evoked by depolarizing voltage ramps exceeding 46 mV/s and increased with ramp slope (240 -2,400 mV/s). Bath application of 200 µM Ni2+ depressed ramp-activated IT. Phasic firing recorded in current clamp could only be activated by membrane depolarizations exceeding ~43-46 mV/s and was blocked by Ni2+ and mibefradil, suggesting IT as an underlying mechanism. Two components of IT, "fast" and "slow," were isolated based on a difference in time constant of inactivation (12 ms and 177 ms, respectively). The amplitude of the fast subtype depended on the slope of membrane depolarization and was twice as great in burst-firing cells than in cells having a tonic discharge. Post hoc single-cell RT-PCR analyses suggested that the fast component is associated with the CaV3.1 channel subtype. IT may enhance responses of phasic-firing dorsal horn neurons to rapid membrane depolarizations and contribute to an ability to discriminate between afferent sensory inputs that encode high- and low-frequency stimulus information. [ABSTRACT FROM AUTHOR]

Although intrathecally administrated galanin modulates nociceptive transmission in a biphasic manner, this has not been fully examined previously. In the present study, the action of galanin on synaptic transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices was examined, using the whole cell patch-clamp technique. Galanin concentration-dependently increased the frequency of spontaneous excitatory postsynaptic current (EPSC; EC50 = 2.0 nM) without changing the amplitude, indicating a presynaptic effect. This effect was reduced in a Ca2+-free, or voltage-gated Ca2+ channel blocker La3+-containing Krebs solution and was produced by a galanin type-2/3 receptor (GalR2/R3) agonist, galanin 2-11, but not by a galanin type-1 receptor (GalR1) agonist, M617. Galanin also concentration-dependently produced an outward current at -70 mV (EC50 = 44 nM), although this appeared to be contaminated by a small inward current. This outward current was mimicked by M617, but not by galanin 2-11. Moreover, galanin reduced monosynaptic Aδ-fiber- and C-fiber-evoked EPSC amplitude; the former reduction was larger than the latter. A similar action was produced by galanin 2-11, but not by M617. Spontaneous and focally evoked inhibitory (GABAergic and glycinergic) transmission was unaffected by galanin. These findings indicate that galanin at lower concentrations enhances the spontaneous release of l-glutamate from nerve terminals by Ca2+ entry from the external solution following GalR2/R3 activation, whereas galanin at higher concentrations also produces a membrane hyperpolarization by activating GalR1. Moreover, galanin reduces l-glutamate release onto SG neurons from primary afferent fibers by activating GalR2/R3. These effects could partially contribute to the behavioral effect of galanin. [ABSTRACT FROM AUTHOR]