Higher Resting Membrane Potential Research Articles

FRI274 Dopamine Transporter-Expressing Leptin-Sensitive Neurons In The Ventral Premammillary Nucleus Innervate Kiss1 Neurons

Abstract Disclosure: C. Sáenz de Miera: None. L. Heikkinen: None. C. Broberger: None. C.F. Elias: None. Pubertal development is tightly regulated by energy balance, and easily disrupted in states of excess or negative energy reserves. This crosstalk between metabolism and reproduction is orchestrated by complex neural networks coordinated by circulating hormones, notably, leptin action in the hypothalamus plays a critical role. The ventral premammillary nucleus (PMv) is rich in leptin receptor (LepRb) neurons, and acts as an essential relay for leptin action on reproduction. Here, we describe a novel subpopulation of leptin sensitive neurons within the PMv that express dopamine transporter (DATPMv). We observed that in both prepubertal and adult females in diestrous, virtually all DATPMv neurons express LepRb mRNA and respond to acute leptin treatment with phosphorylation of the intracellular marker signal transducer and activator of transcription 3 (pSTAT3). Electrophyisological recordings from DAT-CRE;R26tdTomato (DAT-tdTom) mice show that DATPMv cells in adult females have a higher resting membrane potential than those in prepubertal females. DAT mRNA expression in the PMv is higher in prepubertal than in adult females. However, DAT mRNA expression was similar in adult females in diestrous, ovariectomized (OVX), and OVX treated with estradiol, indicating that the age difference observed is not due to the increase in estradiol during pubertal maturation. To trace DATPMv projections, we injected an adenoassociated virus expressing Cre-dependent channelrhodopsin (ChR2-mCherry) in the PMv of adult female DAT-Cre;Kiss1-hrGFP mice, which express GFP driven by kiss1 regulatory elements. ChR2-mCherry projections were observed in the anteroventral periventricular nucleus (AVPV) surrounding Kiss1-hrGFP neurons, a population critical for sexual maturation and positive estrogen feedback in females. No projections were observed near arcuate nucleus Kiss1-hrGFP neurons, in the medial preoptic area (MPOA) or near GnRH neurons. This contrasts with previous observations of dense projections from the overall PMv LepRb population to the MPOA, surrounding GnRH cell bodies suggesting the DATPMv subpopulation has different target areas than the non-DAT LepRb neurons. Using DAT-tdTOM females we observed that overall tdTom projections to the AVPV are denser in adult diestrous than in prepubertal females. Overall, these findings suggest that the DAT expressing LepR subpopulation in the PMv play a role in leptin regulation of sexual maturation via actions on AVPV kisspeptin neurons. Presentation: Friday, June 16, 2023

Molecular mechanisms responsible for the sexual dimorphism in pancreatic β-cell insulin release

In humans, type 2 diabetes mellitus (T2DM) has a higher incidence in males compared to females, a phenotype recapitulated by many rodent models. While the sex difference in insulin sensitivity partially accounts for this phenomenon, hitherto uncharacterized differences in pancreatic β-cell insulin release strongly contribute. Here, we show that stepwise increase in extracellular glucose concentration (2, 5, 7.5, 10, 15, 20 mM) induced electrical activity in β cells of both sexes with similar glucose sensitivity (female, EC50 = 9.45 ± 0.15 mM; male, EC50 = 9.42 ± 0.16 mM). However, female β cells' resting membrane potential (RMP) and inter-spike potential (IP) were significantly higher compared to males (e.g., at 15 mM glucose: male RMP = -82.7 ± 6.3, IP = -74.3 ± 6.8 mV; female RMP = -50.0 ± 7.1, IP = -41.2 ± 7.3 mV). Females also showed higher frequency of trains of action potential (AP; at 10 mM glucose: male F = 1.13 ± 0.15 trains/min; female F = 1.78 ± 0.25 trains/min) and longer AP-burst duration (e.g., at 10 mM glucose: male, 241 ± 30.8 ms; female, 419 ± 60.2 ms). The higher RMP in females reduced the voltage-gated calcium channel (CaV) availability by ∼60%. This explains the paradoxical observation that, despite identical CaV expression levels and higher electrical activity, the islet Ca2+ transients were smaller in females compared to males. Interestingly, the different RMPs are not caused by altered KATP, TASK, or TALK K+ currents. However, stromatoxin-1-sensitive KV2.1 K+ current amplitude was almost double in males (IK = 130.93 ± 7.05 pA/pF) compared to females (IK = 75.85 ± 11.3 pA/pF) when measured at +80 mV. Our results are in agreement with previous findings showing that KV2.1 genetic deletion or pharmacological block leads to higher insulin release and β-cell survival. Therefore, we propose the sex-specific expression of KV2.1 to be the mechanism underlying the observed sexual dimorphism in insulin release and the incidence of T2DM.

Analysis of spontaneous depolarization-linked hyperpolarizations in mouse detrusor smooth muscle cells

Background: Urinary bladder detrusor smooth muscle cells exhibit spontaneous electrical activities comprising various signal types. Aims and Objectives: This article introduces and analyzes a rare category of signals observed in such activity, named spontaneous depolarization-linked hyperpolarization (sDLH). Materials and Methods: A mouse model was used in the study, where all the occurrences of sDLHs were pooled together from multiple intracellular recording sessions. Four features – (i) resting membrane potential (RMP) (R, in mV), (ii) depolarization amplitude (D, in mV), (iii) hyperpolarization amplitude (H, in mV), and (iv) time course of the hyperpolarization (T, in ms) – were evaluated from all sDLHs. Results: The analysis of results indicated that (a) the signals appear more frequently in cells with higher RMP, (b) the depolarization amplitudes seem to be distributed randomly and have no correlation with other features, (c) hyperpolarization amplitudes show two distinct clusters and exhibit strong correlation with the RMP, and (d) time course of hyperpolarization phase shows no distinct groups and is distributed in a window larger than that of any other signals seen in the intracellular recordings. With the help of the results obtained from the analysis, a hypothesis for the biophysical origin of these signals is proposed. Conclusions: This needs to be tested experimentally, and if proved right, would help extend the boundaries of our current understanding of the detrusor smooth muscle system.

Sex differences in the nicotinic excitation of principal neurons within the developing hippocampal formation.

The hippocampal formation (HF) plays an important role to facilitate higher order cognitive functions. Cholinergic activation of heteromeric nicotinic acetylcholine receptors (nAChRs) within the HF is critical for the normal development of principal neurons within this brain region. However, previous research investigating the expression and function of heteromeric nAChRs in principal neurons of the HF is limited to males or does not differentiate between the sexes. We used whole-cell electrophysiology to show that principal neurons in the CA1 region of the female mouse HF are excited by heteromeric nAChRs throughout postnatal development, with the greatest response occurring during the first two weeks of postnatal life. Excitability responses to heteromeric nAChR stimulation were also found in principal neurons in the CA3, dentate gyrus, subiculum, and entorhinal cortex layer VI (ECVI) of young postnatal female HF. A direct comparison between male and female mice found that principal neurons in ECVI display greater heteromeric nicotinic passive and active excitability responses in females. This sex difference is likely influenced by the generally more excitable nature of ECVI neurons from female mice, which display a higher resting membrane potential, greater input resistance, and smaller afterhyperpolarization potential of medium duration (mAHP). These findings demonstrate that heteromeric nicotinic excitation of ECVI neurons differs between male and female mice during a period of major circuitry development within the HF, which may have mechanistic implications for known sex differences in the development and function of this cognitive brain region.

Gender in the brain

Neurodevelopment The hippocampus of the brain is important for higher-order cognitive function, such as learning and memory. Derangements in this region are associated with autism spectrum, attention-deficit hyperactivity disorder, and epilepsy. While looking at the development of neurons during formation of the hippocampus in mice, Chung and Bailey found differences between males and females. In the early postnatal period, when hippocampal circuitry is being built, these neurons depend on signaling through nicotinic acetylcholine receptors. Whole-cell electrophysiology showed that these nascent hippocampal neurons in female mice showed greater excitability, higher resting membrane potential, and greater input resistance than neurons of male mice. These sex-dependent differences occurred during development of circuits that, at maturity, facilitate cognitive functions. Dev. Neurobiol. 10.1002/dneu.22646 (2018).

Reduced cooperativity of voltage-gated sodium channels in the hippocampal interneurons of an aged mouse model of Alzheimer's disease.

Beta amyloid (A[Formula: see text] ) associated with Alzheimer's disease (AD) leads to abnormal behavior in inhibitory neurons, resulting in hyperactive neuronal networks, epileptiform behavior, disrupted gamma rhythms, and aberrant synaptic plasticity. Previously, we used a dual modeling-experimental approach to explain several observations, including failure to reliably produce action potentials (APs), smaller AP amplitudes, higher resting membrane potential, and higher membrane depolarization in response to a range of stimuli in hippocampal inhibitory neurons from 12- to 16-month-old female AP Pswe/PSEN1DeltaE9 (APdE9) AD mice as compared to age-matched non-transgenic (NTG) mice. Our experimental results also showed that AP initiation in interneurons from APdE9 mice are significantly different from that of NTG mice. APs in interneurons from NTG mice are characterized by abrupt onset and an upstroke that is much steeper and occurs with larger variability as compared to cells from APdE9 mice. The phase plot (the rate of change of membrane potential versus the instantaneous membrane potential) of APs produced by interneurons from APdE9 mice shows a biphasic behavior, whereas that from NTG mice shows a monophasic behavior. Here we show that using the classic Hodgkin-Huxley (HH) formalism for the gating of voltage-gated sodium channels (VGSCs) in a single-compartment neuron, we cannot reproduce these features, and a model that takes into account a cooperative activation of VGSCs is needed. We also argue that considering a realistic multi-compartment neuron where the kinetics of VGSC is modeled by HH formalism, as done in the past, would not explain our observations when APs from both NTG and APdE9 mice are considered simultaneously. Wefurther show that VGSCs in interneurons from APdE9 mice exhibit significantly lower cooperativity in their activation as compared to those from NTG mice.

Developmental Changes in Electrophysiological Properties and a Transition from Electrical to Chemical Coupling between Excitatory Layer 4 Neurons in the Rat Barrel Cortex.

During development, sensory systems switch from an immature to an adult mode of function along with the emergence of the active cortical states. Here, we used patch-clamp recordings from neocortical slices in vitro to characterize the developmental changes in the basic electrophysiological properties of excitatory L4 neurons and their connectivity before and after the developmental switch, which occurs in the rat barrel cortex in vivo at postnatal day P8. Prior to the switch, L4 neurons had higher resting membrane potentials, higher input resistance, lower membrane capacity, as well as action potentials (APs) with smaller amplitudes, longer durations and higher AP thresholds compared to the neurons after the switch. A sustained firing pattern also emerged around the switch. Dual patch-clamp recordings from L4 neurons revealed that recurrent connections between L4 excitatory cells do not exist before and develop rapidly across the switch. In contrast, electrical coupling between these neurons waned around the switch. We suggest that maturation of electrophysiological features, particularly acquisition of a sustained firing pattern, and a transition from the immature electrical to mature chemical synaptic coupling between excitatory L4 neurons, contributes to the developmental switch in the cortical mode of function.

Postnatal Excitability Development and Innervation by Functional Transient Receptor Potential Vanilloid 1 (TRPV1) Terminals in Neurons of the Rat Spinal Sacral Dorsal Commissural Nucleus: an Electrophysiological Study.

The sacral dorsal commissural nucleus (SDCN) in the spinal cord receives both somatic and visceral primary afferents. Transient receptor potential vanilloid 1 (TRPV1) channels are preferentially expressed in certain fine primary afferents. However, knowledge of the SDCN neurons postnatal excitability development and their contacts with TRPV1 fibers remains elusive. Here, whole-cell recordings were conducted in spinal cord slices to evaluate the postnatal development of SDCN neurons and their possible contacts with functional TRPV1-expressing terminals. SDCN neurons in neonatal (postnatal day (P) 1-2), young (P8-10), and adult rats (P35-40) have different electrophysiological properties. SDCN neurons in neonatal rats have higher frequency of spontaneous firing, higher resting membrane potential, and lower presynaptic glutamate release probability. However, no difference in quantal release was found. At all developmental stages, TRPV1 activation with the selective agonist capsaicin increases glutamate release in the presence of tetrodotoxin, which blocks action potential-dependent and polysynaptic neurotransmission, indicating that functional TRPV1 fibers innervate SDCN neurons directly. Capsaicin-induced presynaptic glutamate release onto SDCN neurons depends on external Ca2+ influx through TRPV1 channels; voltage-dependent calcium channels had a slighter impact. In contrast, capsaicin blocked C fiber-evoked synaptic transmission, indicating that TRPV1 activation has opposite effects on spontaneous asynchronous and action potential-dependent synchronous glutamate release. These data indicate that excitability of SDCN neurons undergoes a developmental shift, and these neurons receive functional TRPV1 terminals from early postnatal stage. The opposite action of capsaicin on asynchronous and synchronous glutamate release should be taken into account when TRPV1 channels are considered as therapeutic targets.

Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba2+ 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K+, while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K+ typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba2+-sensitive inward rectifier K+ (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.

Bachmann’s Bundle

Bachmann’s bundle (BB), also known as the interatrial bundle, is well recognized as a muscular bundle comprising of parallel aligned myocardial strands connecting the right and left atrial walls and is considered to be the main pathway of interatrial conduction.1 Disruption of the bundle’s structure causes interatrial conduction block (IAB),2 which is associated with development of various atrial tachyarrhythmias3,4 and with electromechanical dysfunction of the left atrium.5 Technological progress providing sophisticated mapping and imaging techniques in the past decade has increased our knowledge of specific anatomic structures and their role in development of both atrial brady- and tachyarrhythmias. This review outlines the current knowledge of the relation between anatomic and electrophysiological properties of BB and its possible role in initiation and perpetuation of atrial fibrillation (AF). In 1963, Thomas N. James described 3 pathways connecting the sinus node to the atrioventricular node (AVN), namely the anterior, medial, and posterior internodal pathways.6 Whether these conduction pathways were because of the presence of specialized conduction tissue or because of the anisotropic orientation of the muscle fibers remains controversial. Nevertheless, James described the anterior pathway as leaving the sinus node in anterior direction and giving off a secondary branch at the level of the superior vena cava to form BB.1 BB stretches subepicardially across the interatrial groove (septal raphe). It is at the interatrial groove that the BB can be identified as a discrete bundle (Figures 1 and 2) separated by fatty tissues from the infolded right atrial wall that is the limbus of the oval fossa. Notably, the bundle is not surrounded by a fibrous tissue sheath. Instead, the bundle is comprised of strands of atrial myocardium that are similarly aligned in parallel fashion. Its rightward and leftward extensions bifurcate to pass to either …

A pair of identified giant visual projection neurons demonstrates rhythmic activities before eclosion

A small set of neurons acting as an internal clock in the Drosophila brain is critical for regulating circadian activities behavior and pre-adult development. However, the cell basis for the circadian rhythm in correlation with light sensitivity is not fully understood. Here we identified a pair of giant visual projection neurons located laterally to the calyx of the mushroom bodies, and investigated their electrophysiological, morphological characteristics, as well as the development pathways during eclosion. The typical morphology of these giant neurons showed the size of the soma (16.0±0.6 microns in diameter) and its processes. Interestingly during development, the three major branches shrunk significantly along with gradually decreased rhythmic spikes. Furthermore, the electrical activity of the giant visual projection neurons is circadian-regulated, shown with significantly higher resting membrane potential, increase in frequency of spontaneous action potential firing, and burst firing pattern during circadian day and night time. The similarities in the morphological characteristics with other visual projection neurons highly suggest that this neuron is a type of novel visual projection neurons in this area, which has special properties in light sensitivities and rhythmic activities. Our data provided supporting evidence for the visual projection neurons with light sensitivities, and pointed to the potential correlation of visual projection neurons and circadian rhythms during the eclosion period or an adaptive development for higher sensitivity of light in adult visual systems.

Computational modeling of bimodal resting membrane potential (RP) of arteriolar cells

The mechanism of bimodal RP in arterioles is not fully understood. We modeled this phenomenon by three steps. First, we mathematically modeled the whole‐cell I/V with a sum of five currents: (1) IKir=(Vm−EK)*GMAX‐Kir/(1+exp((Vm−V0.5Kir)/kKir)); (2) IKdr=(Vm−EK)*(GMAX‐Kdr − GMAX‐Kdr/(1+exp((x−V0.5Kdr)/kKdr))); (3) IKnon‐v=(Vm−EK)*GKnon‐v; (4) Icat= (Vm−ECat)*Gcat and (5) Ipump=(Vm−Vr‐pump)*Gpump. Using the sum of these equations and initial parameters estimated by vascular experiments, our composed program (in MATLAB) could fit to a ramp‐constructed I/V curve and yield parameter measurements of the individual currents of the recorded cell. Second, using GHK zero‐current equation: Vm (RP) = (EK*GKir + EK*GKdr + EKGKnon‐v + ECat*GCat + Epump*Gpump)/(GKir + GKdr + GKnon‐v+ GCat + Gpump) and the above five equations with approximate parameters, the program's iteration process could search the stable Vm, i.e. the RP, after inputting an initial Vm value to equations (1) and (2). When we applied normal distribution random values (n≥50000) of the yielded GmaxKir and one or more of other conductances, the program produced a bimodal distribution of RPs peaked at ∼−75 and ∼−40 mV. Third, using the program and data yielded, the program simulated the observed RP shifts such as those induced by Ba2+‐inhibition of Kir and NO‐activation of KATP. We conclude that the bimodal RP results from sum between optimal Kir and other membrane currents: generally the other currents set a ∼−40 mV RP background, and the Kir causes a high RP ∼−75 mV only when the background enables a Kir activation that triggers the Kir a positive‐feedback activation. Supported by NIH DC004716 & P30 CD005983

Morphological correlates of intrinsic electrical excitability in neurons of the deep cerebellar nuclei.

To what degree does neuronal morphology determine or correlate with intrinsic electrical properties within a particular class of neuron? This question has been examined using microelectrode recordings and subsequent neurobiotin filling and reconstruction of neurons in the deep cerebellar nuclei (DCN) of brain slices from young rats (P13-16). The neurons reconstructed from these recordings were mostly large and multipolar (17/21 cells) and were likely to represent glutamatergic projection neurons. Within this class, there was considerable variation in intrinsic electrical properties and cellular morphology. Remarkably, in a correlation matrix of 18 electrophysiological and 6 morphological measures, only one morphological characteristic was predictive of intrinsic excitability: neurons with more spines had a significantly slower basal firing rate. To address the possibility that neurons with fewer spines represented a slowly maturing subgroup, recordings and reconstructions were also made from neurons at a younger age (P6-9). While P6-9 neurons were morphologically indistinguishable from P13 to 16 neurons, they were considerably less excitable: P6-9 neurons had a lower spontaneous spiking rate, larger fast AHPs, higher resting membrane potentials, and smaller rebound depolarizations. Thus while the large projection neurons of the DCN are morphologically mature by P6-9, they continue to mature electrophysiologically through P13-16 in a way that renders them more responsive to the burst-and-pause pattern that characterizes Purkinje cell inhibitory synaptic drive.

Electrophysiological properties of mitogen-expanded adult rat spinal cord and subventricular zone neural precursor cells.

Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.

Potassium channels of myenteric neurons in guinea-pig small intestine

Patch-clamp recording was used to study rectifying K + currents in myenteric neurons in short-term culture. In conditions that suppressed Ca 2+-activated K + current, three kinds of voltage-activated K + currents were identified by their voltage range of activation, inactivation, kinetics and pharmacology. These were A-type current, delayed outwardly rectifying current (I K.dr) and inwardly rectifying current (I K.ir). I K.ir consisted of an instantaneous component followed by a time-dependent current that rapidly increased at potentials negative to −80 mV. Time-constant of activation was voltage-dependent with an e-fold decrease for a 31-mV hyperpolarization amounting to a decrease from 800 to 145 ms between −80 and −100 mV. I K.ir did not inactivate. I K.ir was abolished in K +-free solution. Increases in external K + increased I K.ir conductance in direct relation to the square root of external K + concentration. Activation kinetics were accelerated and the activation range shifted to more positive K + equilibrium potentials. I K.ir was suppressed by external Cs + and Ba 2+ in a concentration-dependent manner. Ca 2+ and Mg 2+ were less effective than Ba 2+ I K, ir was unaffected by tetraethylammonium ions. I K.dr was activated at membrane potentials positive to −30 mV with an e-fold decrease in time-constant of activation from 145 to 16 ms between −20 and 30 mV. It was half-activated at 5 mV and fully activated at 50 mV. Inactivation was indiscernible during 2.5 s test pulses. I K.dr was suppressed in a concentration-, but not voltage-dependent manner by either tetraethylammonium or 4-aminopyridine and was insensitive to Cs 2+. The results suggest that I K.ir may be important in maintaining the high resting membrane potentials found in afterhyperpolarization-type enteric neurons. They also suggest importance of I K.ir channels in augmentation of the large hyperpolarizing after-potentials in afterhyperpolarization-type neurons and the hyperpolarization associated with inhibitory postsynaptic potentials. I K.dr in afterhyperpolarization-type enteric neurons has overall kinetics and voltage behaviour like delayed rectifier currents in other excitable cells where the currents can also be distinguished from A-type and Ca 2+-activated K + current.

Membrane ultrastructure preservation and membrane potentials after isolation of rabbit retinal glial (Müller) cells by papain

Enzymatically isolated retinal glial (Müller) cells have been the subject of many electrophysiological studies. Local high membrane conductivities for potassium ions have been speculated to correspond with local occurrence of orthogonal arrays of intramembranous particles (OAP) observed in freeze-fracture replicas of retinal Müller cells in situ. We studied whether such OAP are preserved after enzymatic digestion of the retinal tissue which is necessary for isolation of living cells for electrophysiology. We found that strong papain digestion leads not only to disturbances in the cell's ultrastructure as seen in ultrathin sections but evokes both a redistribution of intramembranous particles and a disappearance of OAP as seen in the freeze-fracture replica. Furthermore, such isolated cells have low membrane potentials and lose their topographical specialization in K + conductance. If, however, the retinae were exposed to papain as short as possible to get just some isolated cells, their cytoplasmic and membranous ultrastructure was preserved very well, and high resting membrane potentials were recorded in cells with marked regional specialization of membrane conductivity. Our results show that indeed sites of high K + conductance may correspond with the occurrence of OAP, even in isolated cells.

Intracellular recordings from pancreatic ganglia of the cat.

1. The anatomy, morphology, and electrophysiology of parasympathetic ganglia of cat pancreas were studied in vitro. 2. Pancreatic ganglia existed as an interconnected plexus of small ganglia (ten to fifty cells) lying in the interlobular connective tissue. Occasionally smaller ganglia (four to ten cells) were observed lying on or within nerve trunks. 3. Electron micrographs revealed the presence of neurones and satellite cells as well as unmyelinated axons and nerve terminals. Nerve terminals contained small clear vesicles and/or large, dense-cored vesicles. 4. Intracellular recording of electrical activity revealed the presence of two types of ganglion cells. Type I ganglion cells exhibited resting membrane potentials that ranged from -40 to -63 mV and input resistances that ranged from 8 to 168 M omega. They responded to intracellular depolarizing current with action potentials, and received synaptic inputs which when activated caused fast and slow depolarizing responses. Type I cells were considered to be ganglionic neurones. Type II ganglion cells had higher resting membrane potentials that ranged from -61 to -83 mV, lower input resistances that ranged from 5 to 83 M omega and were electrically unexcitable. Repetitive stimulation of preganglionic nerves evoked a slow depolarization that was frequency dependent. Type II cells were considered to be satellite cells. 5. Stimulation of nerve trunks both central and peripheral to the ganglia evoked multiple, subthreshold, fast EPSPs in all type I cells tested. Fast EPSPs were blocked by the nicotinic antagonist hexamethonium. 6. Antidromic potentials were also observed following stimulation of either central or peripheral nerve trunks but never both. 7. In type I cells repetitive stimulation of both central and peripheral nerve trunks resulted in a slow, synaptically mediated depolarization which persisted during superfusion with nicotinic and muscarinic receptor antagonists. 8. Periods of low-frequency, spontaneous fast EPSPs and action potentials were observed in all type I cells tested. 9. It was concluded that parasympathetic neurones in cat pancreatic ganglia receive convergent fast and slow synaptic inputs from central and possibly peripheral sources and may function in vivo as sites of integration. The occurrence of spontaneous synaptic potentials in pancreatic ganglia suggests the possibility of intrinsic neural control of pancreatic function.

Separation of different types of afterpotentials following penicillin-induced paroxysmal depolarization shifts of neurons in the motor cortex of the rat.

Afterpotentials of penicillin-induced paroxysmal depolarization shifts (PDS) of neurones in the motor cortex of the rat in vivo were investigated with intracellular recordings. Following the PDS, 4 different types of afterpotentials were observed: fast afterhyperpolarizations with an average duration of 600 ms, fast afterdepolarizations with an average duration of 700 ms, slow afterhyperpolarizations with an average duration of 1.9 s and slow afterdepolarizations with an average duration of 1.8 s. The fast and slow afterpotentials could occur in various combinations with the exception of a fast afterhyperpolarization followed by a slow afterdepolarization. Neurones displaying afterdepolarizations had higher resting membrane potentials than those displaying afterhyperolarizations, i.e. the polarity of the afterpotential depended on membrane potential. Experiments with intracellular Cl- injection indicated that the fast but not the slow afterpotentials are associated with an increase in membrane Cl- conductance. The slow afterhyperpolarizations are suggested to result from a calcium-dependent or a synaptically generated potassium current.

The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine.

Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.

Stratum lacunosum-moleculare interneurons of hippocampal CA1 region. I. Intracellular response characteristics, synaptic responses, and morphology

Stable intracellular recordings were obtained from nonpyramidal cells (interneurons) in stratum lacunosum-moleculare (L-M) of the CA1 region of guinea pig hippocampal slices. The intracellular response characteristics of these interneurons were distinctly different from responses of pyramidal cells and of other interneurons (basket cells and oriens-alveus interneurons). L-M interneurons had a high resting membrane potential (-58 mV), a high input resistance (64 M omega), and a large amplitude (60 mV), relatively long duration (2 msec) action potential. A large afterhyperpolarization (11 mV, 34 msec) followed a single action potential. Most L-M interneurons did not display any spontaneous firing. Lucifer yellow (LY)-filled L-M interneurons showed nonpyramidal morphology. Cells were generally fusiform or multipolar, with aspinous, beaded dendritic processes ramifying in stratum lacunosum-moleculare, radiatum, and (sometimes) oriens. The varicose axon originated from a primary dendrite, projected along stratum lacunosum-moleculare, branched profusely in stratum radiatum, and coursed toward and into stratum pyramidale and occasionally into oriens. Processes of cells with somata in the L-M region of CA1 were not restricted to the CA1 region. The dendritic and axonal processes of some L-M interneurons were seen ascending in stratum lacunosum-moleculare, crossing the hippocampal fissure, and coursing in stratum moleculare of the dentate gyrus. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) were evoked in L-M interneurons from stimulation of major hippocampal afferents. EPSPs were most effectively elicited by stimulation of fiber pathways in transverse slices, whereas IPSPs were predominantly evoked when major pathways were stimulated in longitudinal slices. We have identified a population of interneurons with intracellular response characteristics and morphology distinctly different from previously described pyramidal and nonpyramidal neurons of CA1 region. The possible role of these interneurons in hippocampal circuitry is discussed.