Cell type-specific and frequency-dependent centrifugal modulation in olfactory bulb output neurons in vivo.

Mitral/tufted cells (M/TCs) form complex local circuits with interneurons in the olfactory bulb and are powerfully inhibited by these interneurons. The horizontal limb of the diagonal band of Broca (HDB), the only GABAergic/inhibitory source of centrifugal circuit with the olfactory bulb, is known to target olfactory bulb interneurons, and we have shown targeting also to olfactory bulb glutamatergic neurons in vitro. However, the net efficacy of these circuits under different patterns of activation in vivo and the relative balance between the various targeted intact local and centrifugal circuits was the focus of this study. Here channelrhodopsin-2 (ChR2) was expressed in HDB GABAergic neurons to investigate the short-term plasticity of HDB-activated disinhibitory rebound excitation of M/TCs. Optical activation of HDB interneurons increased spontaneous M/TC firing without odor presentation and increased odor-evoked M/TC firing. HDB activation induced disinhibitory rebound excitation (burst or cluster of spiking) in all classes of M/TCs. This excitation was frequency dependent, with short-term facilitation only at higher HDB stimulation frequency (5 Hz and above). However, frequency-dependent HDB regulation was more potent in the deeper layer M/TCs compared with more superficial layer M/TCs. In all neural circuits the balance between inhibition and excitation in local and centrifugal circuits plays a critical functional role, and this patterned input-dependent regulation of inhibitory centrifugal inputs to the olfactory bulb may help maintain the precise balance across the populations of output neurons in different environmental odors, putatively to sharpen the enhancement of tuning specificity of individual or classes of M/TCs to odors.NEW & NOTEWORTHY Neuronal local circuits in the olfactory bulb are modulated by centrifugal long circuits. In vivo study here shows that inhibitory horizontal limb of the diagonal band of Broca (HDB) modulates all five types of mitral/tufted cells (M/TCs), by direct inhibitory circuits HDB → M/TCs and indirect disinhibitory long circuits HDB → interneurons → M/TCs. The HDB net effect exerts excitation in all types of M/TCs but more powerful in deeper layer output neurons as HDB activation frequency increases, which may sharpen the tuning specificity of classes of M/TCs to odors during sensory processing.

Frequency-dependent centrifugal modulation of the activity of different classes of mitral and tufted cells in olfactory bulb.

Mitral/tufted cells (M/TCs), the principal output neuron classes form complex circuits with bulbar neurons and long-range centrifugal circuits with higher processing areas such as the horizontal limb of the diagonal band of Broca (HDB). The precise excitability of output neurons is sculpted by local inhibitory circuits. Here, light-gated cation channel channelrhodopsin-2 (ChR2) was expressed in HDB GABAergic neurons to investigate the short-term plasticity of evoked postsynaptic currents/potentials of HDB input to all classes of M/TCs and effects on firing in the acute slice preparation. Activation of the HDB directly inhibited all classes of output neurons exhibiting frequency-dependent short-term depression of evoked inhibitory postsynaptic current (eIPSC)/potential (eIPSP), resulting in decreased inhibition of responses to olfactory nerve input as a function of input frequency. In contrast, activation of an indirect circuit of HDB→interneurons→M/TCs induced frequency-dependent disinhibition, resulting in short-term facilitation of evoked excitatory postsynaptic current (eEPSC) eliciting a burst or cluster of spiking in M/TCs. The facilitatory effects of elevated HDB input frequency were strongest on deeper output neurons (deep tufted and mitral cells) and negligible on peripheral output neurons (external and superficial tufted cells). Taken together, GABAergic HDB activation generates frequency-dependent regulation that differentially affects the excitability and responses across the five classes of M/TCs. This regulation may help maintain the precise balance between inhibition and excitation of neuronal circuits across the populations of output neurons in the face of changes in an animal sniffing rate, putatively to enhance and sharpen the tuning specificity of individual or classes of M/TCs to odors.NEW & NOTEWORTHY Neuronal circuits in the olfactory bulb closely modulate olfactory bulb output activity. Activation of GABAergic circuits from the HDB to the olfactory bulb has both direct and indirect action differentially across the five classes of M/TC bulbar output neurons. The net effect enhances the excitability of deeper output neurons as HDB frequency increases, altering the relative inhibition-excitation balance of output circuits. We hypothesize that this sharpens the tuning specificity of classes of M/TCs to odors during sensory processing.

Short-Term Plasticity in Cortical GABAergic Synapses on Olfactory Bulb Granule Cells Is Modulated by Endocannabinoids.

Olfactory bulb and higher processing areas are synaptically interconnected, providing rapid regulation of olfactory bulb circuit dynamics and sensory processing. Short-term plasticity changes at any of these synapses could modulate sensory processing and potentially short-term sensory memory. A key olfactory bulb circuit for mediating cortical feedback modulation is granule cells, which are targeted by multiple cortical regions including both glutamatergic excitatory inputs and GABAergic inhibitory inputs. There is robust endocannabinoid modulation of excitatory inputs to granule cells and here we explored whether there was also endocannabinoid modulation of the inhibitory cortical inputs to granule cells. We expressed light-gated cation channel channelrhodopsin-2 (ChR2) in GABAergic neurons in the horizontal limb of the diagonal band of Broca (HDB) and their projections to granule cells in olfactory bulb. Selective optical activation of ChR2 positive axons/terminals generated strong, frequency-dependent short-term depression of GABA A -mediated-IPSC in granule cells. As cannabinoid type 1 (CB1) receptor is heavily expressed in olfactory bulb granule cell layer (GCL) and there is endogenous endocannabinoid release in GCL, we investigated whether activation of CB1 receptor modulated the HDB IPSC and short-term depression at the HDB→granule cell synapse. Activation of the CB1 receptor by the exogenous agonist Win 55,212-2 significantly decreased the peak amplitude of individual IPSC and decreased short-term depression, while blockade of the CB1 receptor by AM 251 slightly increased individual IPSCs and increased short-term depression. Thus, we conclude that there is tonic endocannabinoid activation of the GABAergic projections of the HDB to granule cells, similar to the modulation observed with glutamatergic projections to granule cells. Modulation of inhibitory synaptic currents and frequency-dependent short-term depression could regulate the precise balance of cortical feedback excitation and inhibition of granule cells leading to changes in granule cell mediated inhibition of olfactory bulb output to higher processing areas.

Short-term plasticity in glomerular inhibitory circuits shapes olfactory bulb output.

Short-term plasticity is a fundamental synaptic property thought to underlie memory and neural processing. The glomerular microcircuit comprises complex excitatory and inhibitory interactions and transmits olfactory nerve signals to the excitatory output neurons, mitral/tufted cells (M/TCs). The major glomerular inhibitory interneurons, short axon cells (SACs) and periglomerular cells (PGCs), both provide feedforward and feedback inhibition to M/TCs and have reciprocal inhibitory synapses between each other. Olfactory input is episodically driven by sniffing. We hypothesized that frequency-dependent short-term plasticity within these inhibitory circuits could influence signals sent to higher-order olfactory networks. To assess short-term plasticity in glomerular circuits and MC outputs, we virally delivered channelrhodopsin-2 (ChR2) in glutamic acid decarboxylase-65 promotor (GAD2-cre) or tyrosine hydroxylase promoter (TH-cre) mice and selectively activated one of these two populations while recording from cells of the other population or from MCs. Selective activation of TH-ChR2-expressing SACs inhibited all recorded GAD2-green fluorescent protein(GFP)-expressing presumptive PGC cells, and activation of GAD2-ChR2 cells inhibited TH-GFP-expressing SACs, indicating reciprocal inhibitory connections. SAC synaptic inhibition of GAD2-expressing cells was significantly facilitated at 5-10 Hz activation frequencies. In contrast, GAD2-ChR2 cell inhibition of TH-expressing cells was activation-frequency independent. Both SAC and PGC inhibition of MCs also exhibited short-term plasticity, pronounced in the 5-20 Hz range corresponding to investigative sniffing frequency ranges. In paired SAC and olfactory nerve electrical stimulations, the SAC to MC synapse was able to markedly suppress MC spiking. These data suggest that short-term plasticity across investigative sniffing ranges may differentially regulate intra- and interglomerular inhibitory circuits to dynamically shape glomerular output signals to downstream targets.NEW & NOTEWORTHY Short-term plasticity is a fundamental synaptic property that modulates synaptic strength based on preceding activity of the synapse. In rodent olfaction, sensory input arrives episodically driven by sniffing rates ranging from quiescent respiration (1-2 Hz) through to investigative sniffing (5-10 Hz). Here we show that glomerular inhibitory networks are exquisitely sensitive to input frequencies and exhibit plasticity proportional to investigative sniffing frequencies. This indicates that olfactory glomerular circuits are dynamically modulated by episodic sniffing input.

Short-Term Plasticity at Olfactory Cortex to Granule Cell Synapses Requires CaV2.1 Activation.

Output projections of the olfactory bulb (OB) to the olfactory cortex (OCX) and reciprocal feedback projections from OCX provide rapid regulation of OB circuit dynamics and odor processing. Short-term synaptic plasticity (STP), a feature of many synaptic connections in the brain, can modulate the strength of feedback based on preceding network activity. We used light-gated cation channel channelrhodopsin-2 (ChR2) to investigate plasticity of excitatory synaptic currents (EPSCs) evoked at the OCX to granule cell (GC) synapse in the OB. Selective activation of OCX glutamatergic axons/terminals in OB generates strong, frequency-dependent STP in GCs. This plasticity was critically dependent on activation of CaV2.1 channels. As acetylcholine (ACh) modulates CaV2.1 channels in other brain regions and as cholinergic projections from the basal forebrain heavily target the GC layer (GCL) in OB, we investigated whether ACh modulates STP at the OCX→GC synapse. ACh decreases OCX→GC evoked EPSCs, it had no effect on STP. Thus, ACh impact on cortical feedback is independent of CaV2.1-mediated STP. Modulation of OCX feedback to the bulb by modulatory transmitters, such as ACh, or by frequency-dependent STP could regulate the precise balance of excitation and inhibition of GCs. As GCs are a major inhibitory source for OB output neurons, plasticity at the cortical feedback synapse can differentially impact OB output to higher-order networks in situations where ACh inputs are activated or by active sniff sampling of odors.

Activation of ß-noradrenergic receptors enhances rhythmic bursting in mouse olfactory bulb external tufted cells.

The main olfactory bulb (MOB) receives a rich noradrenergic innervation from the nucleus locus coeruleus. Despite the well-documented role of norepinephrine and ß-adrenergic receptors in neonatal odor preference learning, identified cellular physiological actions of ß-receptors in the MOB have remained elusive. ß-Receptors are expressed at relatively high levels in the MOB glomeruli, the location of external tufted (ET) cells that exert an excitatory drive on mitral and other cell types. The present study investigated the effects of ß-receptor activation on the excitability of ET cells with patch-clamp electrophysiology in mature mouse MOB slices. Isoproterenol and selective ß2-, but not ß1-, receptor agonists were found to enhance two key intrinsic currents involved in ET burst initiation: persistent sodium (INaP) and hyperpolarization-activated inward (Ih) currents. Together, the positive modulation of these currents increased the frequency and strength of ET cell rhythmic bursting. Rodent sniff frequency and locus coeruleus neuronal firing increase in response to novel stimuli or environments. The increase in ET excitability by ß-receptor activation may better enable ET cell rhythmic bursting, and hence glomerular network activity, to pace faster sniff rates during heightened norepinephrine release associated with arousal.

Functional integration of human neural precursor cells in mouse cortex.

This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation, 1.21% of transplanted hNPCs survived. In these hNPCs, parvalbumin (PV)-, calretinin (CR)-, somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-, CR-, and SS-positive cells among GFP+ cells were 35.5%, 15.7%, and 17.1%, respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude, frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion, GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy.

Reduced chemical and electrical connections of fast-spiking interneurons in experimental cortical dysplasia.

Aberrant neural connections are regarded as a principal factor contributing to epileptogenesis. This study examined chemical and electrical connections between fast-spiking (FS), parvalbumin (PV)-immunoreactive (FS-PV) interneurons and regular-spiking (RS) neurons (pyramidal neurons or spiny stellate neurons) in a rat model of prenatal irradiation-induced cortical dysplasia. Presynaptic action potentials were evoked by current injection and the elicited unitary inhibitory or excitatory postsynaptic potentials (uIPSPs or uEPSPs) were recorded in the postsynaptic cell. In dysplastic cortex, connection rates between presynaptic FS-PV interneurons and postsynaptic RS neurons and FS-PV interneurons, and uIPSP amplitudes were significantly smaller than controls, but both failure rates and coefficient of variation of uIPSP amplitudes were larger than controls. In contrast, connection rates from RS neurons to FS-PV interneurons and uEPSPs amplitude were similar in the two groups. Assessment of the paired pulse ratio showed a significant decrease in synaptic release probability at FS-PV interneuronal terminals, and the density of terminal boutons on axons of biocytin-filled FS-PV interneurons was also decreased, suggesting presynaptic dysfunction in chemical synapses formed by FS-PV interneurons. Electrical connections were observed between FS-PV interneurons, and the connection rates and coupling coefficients were smaller in dysplastic cortex than controls. In dysplastic cortex, we found a reduced synaptic efficiency for uIPSPs originating from FS-PV interneurons regardless of the type of target cell, and impaired electrical connections between FS-PV interneurons. This expands our understanding of the fundamental impairment of inhibition in this model and may have relevance for certain types of human cortical dysplasia.

TRPC3 mediates hyperexcitability and epileptiform activity in immature cortex and experimental cortical dysplasia.

Neuronal hyperexcitability plays an important role in epileptogenesis. Conditions of low extracellular calcium (Ca) or magnesium (Mg) can induce hyperexcitability and epileptiform activity with unclear mechanisms. Transient receptor potential canonical type 3 (TRPC3) channels play a pivotal role in neuronal excitability and are activated in low-Ca and/or low-Mg conditions to depolarize neurons. TRPC3 staining was highly enriched in immature, but very weak in mature, control cortex, whereas it was strong in dysplastic cortex at all ages. Depolarization and susceptibility to epileptiform activity increased with decreasing Ca and Mg. Combinations of low Ca and low Mg induced larger depolarization in pyramidal neurons and greater susceptibility to epileptiform activity in immature and dysplastic cortex than in mature and control cortex, respectively. Intracellular application of anti-TRPC3 antibody to block TRPC3 channels and bath application of the selective TRPC3 inhibitor Pyr3 greatly diminished depolarization in immature control and both immature and mature dysplastic cortex with strong TRPC3 expression. Epileptiform activity was initiated in low Ca and low Mg when synaptic activity was blocked, and Pyr3 completely suppressed this activity. In conclusion, TRPC3 primarily mediates low Ca- and low Mg-induced depolarization and epileptiform activity, and the enhanced expression of TRPC3 could make dysplastic and immature cortex more hyperexcitable and more susceptible to epileptiform activity.

Impaired hippocampal memory function and synaptic plasticity in experimental cortical dysplasia.

PURPOSE: Memory impairment is a common comorbidity in people with epilepsy-associated malformations of cortical development. We studied spatial memory performance and hippocampal synaptic plasticity in an animal model of cortical dysplasia. METHODS: Embryonic day 17 rats were exposed to 2.25 Gy external radiation. One-month-old rats were tested for spatial recognition memory. After behavioral testing, short-term and long-term synaptic plasticity in the hippocampal CA1 region was studied in an in vitro slice preparation. KEY FINDINGS: Behavioral assessments showed impaired hippocampal CA1-dependent spatial recognition memory in irradiated rats. Neurophysiologic assessments showed that baseline synaptic transmission was significantly enhanced, whereas paired-pulse facilitation, long-term potentiation, and long-term depression of the field excitatory postsynaptic potential (fEPSP) slope at Schaffer collateral/commissural fiber-CA1 synapses were significantly reduced in the irradiated rats. Histologic observations showed dysplastic cortex and dispersed hippocampal pyramidal neurons. SIGNIFICANCE: This study has shown that prenatally irradiated rats with cortical dysplasia exhibit a severe impairment of spatial recognition memory accompanied by disrupted short-term and long-term synaptic plasticity and may help to guide development of potential therapeutic interventions for this important problem.

Altered behavior in experimental cortical dysplasia.

PURPOSE: Developmental delay and cognitive impairment are common comorbidities in people with epilepsy associated with malformations of cortical development (MCDs). We studied cognition and behavior in an animal model of diffuse cortical dysplasia (CD), in utero irradiation, using a battery of behavioral tests for neuromuscular and cognitive function. METHODS: Fetal rats were exposed to 2.25 Gy external radiation on embryonic day 17 (E17). At 1 month of age they were tested using an open field task, a grip strength task, a grid walk task, inhibitory avoidance, an object recognition task, and the Morris water maze task. KEY FINDINGS: Rats with CD showed reduced nonlocomotor activity in the open field task and impaired motor coordination for grid walking but normal grip strength. They showed a reduced tendency to recognize novel objects and reduced retention in an inhibitory avoidance task. Water maze testing showed that learning and memory were impaired in irradiated rats for both cue discrimination and spatially oriented tasks. These results demonstrate significant deficits in cortex- and hippocampus-dependent cognitive functions associated with the diffuse abnormalities of cortical and hippocampal development that have been documented in this model. SIGNIFICANCE: This study documents multimodal cognitive deficits associated with CD and can serve as the foundation for future investigations into the mechanisms of and possible therapeutic interventions for this problem.

Altered firing rates and patterns in interneurons in experimental cortical dysplasia.

Cortical dysplasia (CD) is associated with severe epilepsy in humans, and the in utero irradiation of fetal rats provides a model of this disorder. These animals show a selective loss of inhibitory interneurons, and the surviving interneurons have a reduced excitatory synaptic drive. The current study was undertaken to see how alterations in synaptic input would affect spontaneous firing of interneurons in dysplastic cortex. We recorded spontaneous action potentials and excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) from somatostatin (SST)-, parvalbumin (PV)-, and calretinin (CR)-immunoreactive (ir) interneurons. We found that SST- and PV-ir interneurons fired less frequently and with less regularity than controls. This corresponded to a relative imbalance in the ratio of EPSCs to IPSCs that favored inhibition. In contrast, CR-ir interneurons from CD showed no differences from controls in spontaneous firing or ratio of EPSCs to IPSCs. Additional studies demonstrated that synaptic input had a powerful effect on spontaneous firing in all interneurons. These findings demonstrate that a relative reduction in excitatory drive results in less active SST- and PV-ir interneurons in irradiated rats. This would further impair cortical inhibition in these animals and may be an important mechanism of epileptogenesis.

Densities of glutamatergic and GABAergic presynaptic terminals are altered in experimental cortical dysplasia.

PURPOSE: Cortical dysplasia (CD) is a major cause of epilepsy in children and adults, but underlying mechanisms of epileptogenesis in this disorder are poorly understood. We have utilized the irradiated rat model to study an injury-based form of diffuse CD in rats. Prior studies in this model have shown reduced numbers of γ-aminobutyric acid (GABA)ergic interneurons and reduced inhibitory synaptic currents in pyramidal cells in CD. We analyzed the number of excitatory and inhibitory presynaptic terminals in the neocortex of irradiated rats to better characterize altered connectivity in experimental CD. METHODS: Antibodies to vesicular glutamate transporter 1 (VGLUT1), vesicular glutamate transporter 2 (VGLUT2), vesicular GABA transporter (VGAT), and parvalbumin (PV) were used to quantify glutamatergic and GABAergic presynaptic terminals in control and dysplastic cortex. RESULTS: We found that the density of VGLUT1 terminals was increased in CD in comparison to layers IV, V, and VI in control cortex. VGLUT2 terminals were increased in CD compared to layers IV and VI. VGAT terminals were reduced in CD compared to layers II/III, IV, and V in controls as were PV-immunoreactive somata and terminals. DISCUSSION: These findings suggest an overall increase in excitatory synaptic connectivity and decrease in inhibitory synaptic connectivity in CD in irradiated rat. We propose that these changes contribute to hyperexcitability in these animals and may contribute to epileptogenicity in some forms of human CD.

Multiple therapeutic effects of adjunctive baicalin therapy in experimental bacterial meningitis.

This study aimed to examine effects of adjunctive baicalin therapy to ampicillin for experimental bacterial meningitis in rabbits. After Escherichia Coli inoculation, mean leukocyte counts, concentrations of protein, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and lactate in cerebrospinal fluid (CSF), brain water content and mean arterial and intracranial pressures substantially increased in the meningitis group. Ampicillin alone for 5 h markedly exacerbated the enhanced leukocyte counts and protein concentration, and showed no significant effect on the elevated CSF TNF-alpha, IL-1 and lactate concentration, mean arterial and intracranial pressures, and brain water content. Baicalin (7-D-glucuronic acid-5,6-dihydroxyflavone, C(21)H(18)O(11)) completely counteracted ampicillin-induced exacerbation, and further alleviated the enhanced mean leukocyte counts and protein concentration when combined with ampicillin. Adjunctive baicalin also significantly ameliorated the elevated CSF TNF-alpha, IL-1 and lactate concentration, mean arterial and intracranial pressures, and brain water content. Baicillin, as an adjunctive treatment exerted multiple therapeutic effects in experimental bacterial meningitis.

Balance of inhibitory and excitatory synaptic activity is altered in fast-spiking interneurons in experimental cortical dysplasia.

Cortical dysplasia (CD) is a common cause of intractable epilepsy in children and adults. We have studied rats irradiated in utero as a model of CD to better understand mechanisms that underlie dysplasia-associated epilepsy. Prior studies have shown a reduction in the number of cortical interneurons and in the frequency of inhibitory postsynaptic currents (IPSCs) in pyramidal cells in this model. They have also shown a reduced frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) in the surviving cortical interneurons. However, the inhibitory synaptic contacts were not examined in that study. The current experiments were performed to assess inhibitory synaptic activity in fast-spiking (FS) interneurons in irradiated rats and controls and the balance of excitatory and inhibitory synaptic activity in these cells. Whole cell recordings were obtained from layer IV FS cells in controls and comparable FS cells in irradiated rats. The frequency of spontaneous and miniature IPSCs was reduced in dysplastic cortex, but the amplitude of these currents was unchanged. Stimulus-evoked IPSCs showed short-term depression in control and short-term facilitation in dysplastic cortex. Simultaneous recording of spontaneous EPSCs and IPSCs showed a shift in the ratio of excitation-to-inhibition in favor of inhibition in FS cells from dysplastic cortex. The same shift toward inhibition was seen when miniature EPSCs and IPSCs were examined. These results show that FS cells in dysplastic cortex have a relative lack of excitatory drive. This may result in an important class of inhibitory cells that are less able to perform their normal function especially in periods of increased excitatory activity.

An ultra-short dopamine pathway regulates basal ganglia output.

Substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus critical for movement control. Its GABA-containing projection neurons intermingle with nigral dopamine (DA) neuron dendrites. Here we show that SNr GABA neurons coexpress dopamine D(1) and D(5) receptor mRNAs and also mRNA for TRPC3 channels. Dopamine induced an inward current in these neurons and increased their firing frequency. These effects were mimicked by D(1)-like agonists, blocked by a D(1)-like antagonist. D(1)-like receptor blockade reduced SNr GABA neuron firing frequency and increased their firing irregularity. These D(1)-like effects were absent in D(1) or D(5) receptor knock-out mice and inhibited by intracellularly applied D(1) or D(5) receptor antibody. These D(1)-like effects were also inhibited when the tonically active TRPC3 channels were inhibited by intracellularly applied TRPC3 channel antibody. Furthermore, stimulation of DA neurons induced a direct inward current in SNr GABA neurons that was sensitive to D(1)-like blockade. Manipulation of DA neuron activity and DA release and inhibition of dopamine reuptake affected SNr GABA neuron activity in a D(1)-like receptor-dependent manner. Together, our findings indicate that dendritically released dopamine tonically excites SNr GABA neurons via D(1)-D(5) receptor coactivation that enhances constitutively active TRPC3 channels, forming an ultra-short substantia nigra pars compacta --> SNr dopamine pathway that regulates the firing intensity and pattern of these basal ganglia output neurons.

Increasing extracellular potassium results in subthalamic neuron activity resembling that seen in a 6-hydroxydopamine lesion.

Abnormal neuronal activity in the subthalamic nucleus (STN) plays a crucial role in the pathophysiology of Parkinson's disease (PD). Although altered extracellular potassium concentration ([K+]o) and sensitivity to [K+]o modulates neuronal activity, little is known about the potassium balance in the healthy and diseased STN. In vivo measurements of [K+]o using ion-selective electrodes demonstrated a twofold increase in the decay time constant of lesion-induced [K+]o transients in the STN of adult Wistar rats with a unilateral 6-hydroxydopamine (6-OHDA) median forebrain bundle lesion, employed as a model of PD, compared with nonlesioned rats. Various [K+]o concentrations (1.5-12.5 mM) were applied to in vitro slice preparations of three experimental groups of STN slices from nonlesioned control rats, ipsilateral hemispheres, and contralateral hemispheres of lesioned rats. The majority of STN neurons of nonlesioned rats and in slices contralateral to the lesion fired spontaneously, predominantly in a regular pattern, whereas those in slices ipsilateral to the lesion fired more irregularly or even in bursts. Experimentally increased [K+]o led to an increase in the number of spontaneously firing neurons and action potential firing rates in all groups. This was accompanied by a decrease in the amplitude of post spike afterhyperpolarization (AHP) and the amplitude and duration of the posttrain AHP. Lesion effects in ipsilateral neurons at physiological [K+]o resembled the effects of elevated [K+]o in nonlesioned rats. Our data suggest that changed potassium sensitivity due to conductivity alterations and delayed clearance may be critical for shaping STN activity in parkinsonian states.

Constitutively active TRPC3 channels regulate basal ganglia output neurons.

A hallmark of the GABA projection neurons of the substantia nigra pars reticulata (SNr), a key basal ganglia output nucleus, is its depolarized membrane potential and rapid spontaneous spikes that encode the basal ganglia output. Parkinsonian movement disorders are often associated with abnormalities in SNr GABA neuron firing intensity and/or pattern. A fundamental question remains regarding the molecular identity of the ion channels that drive these neurons to a depolarized membrane potential. We show here that SNr GABA projection neurons selectively express type 3 canonical transient receptor potential (TRPC3) channels. These channels are tonically active and mediate an inward, Na+-dependent current, leading to a substantial depolarization in these neurons. Inhibition of TRPC3 channels induces hyperpolarization, decreases firing frequency, and increases firing irregularity. These data demonstrate that TRPC3 channels play important roles in ensuring the appropriate firing intensity and pattern in SNr GABA projection neurons that are crucial to movement control.

Opposite functions of histamine H1 and H2 receptors and H3 receptor in substantia nigra pars reticulata.

The substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus. Inhibitory outputs from SNr are encoded in spike frequency and pattern of the inhibitory SNr projection neurons. SNr output intensity and pattern are often abnormal in movement disorders of basal ganglia origin. In Parkinson's disease, histamine innervation and histamine H3 receptor expression in SNr may be increased. However, the functional consequences of these alterations are not known. In this study, whole cell patch-clamp recordings were used to elucidate the function of different histamine receptors in SNr. Histamine increased SNr inhibitory projection neuron firing frequency and thus inhibitory output. This effect was mediated by activation of histamine H1 and H2 receptors that induced inward currents and depolarization. In contrast, histamine H3 receptor activation hyperpolarized and inhibited SNr inhibitory projection neurons, thus decreasing the intensity of basal ganglia output. By the hyperpolarization, H3 receptor activation also increased the irregularity of the interspike intervals or changed the pattern of SNr inhibitory neuron firing. H3 receptor-mediated effects were normally dominated by those mediated by H1 and H2 receptors. Furthermore, endogenously released histamine provided a tonic, H1 and H2 receptor-mediated excitation that helped keep SNr inhibitory projection neurons sufficiently depolarized and spiking regularly. These results suggest that H1 and H2 receptors and H3 receptor exert opposite effects on SNr inhibitory projection neurons. Functional balance of these different histamine receptors may contribute to the proper intensity and pattern of basal ganglia output and, as a consequence, exert important effects on motor control.

Effect of vasoactive intestinal peptide on pulmonary surfactants phospholipid synthesis in lung explants.

AIM: To investigate the effect of vasoactive intestinal peptide (VIP) on pulmonary surfactants (PS) phospholipid synthesis in cultured lung explants. METHODS: Lung explants were cultured with serum-free medium, [methyl-3H]choline incorporation, total phospholipid, phosphatidylcholine, activity of choline-phosphate cytidylyltransferase (CCT) and CCTalpha mRNA level in lung explants were determined. RESULTS: (1) VIP (10(-10)-10(-7) mol/L) for 16 h promoted [methyl-3H]choline incorporation in dose dependence and VIP (10(-8) mol/L) for 2 h-16 h promoted [methyl-3H]choline incorporation in time dependence. (2) VIP (10(-8) mol/L) enhanced the contents of total phospholipids and phosphatidylcholine in lung explants. (3) VIP (10(-10)-10(-7) mol/L) elevated microsomal CCT activity of lung explants in dose dependence. (4) VIP (10(-8) mol/L) increased expression of CCTalpha mRNA in lung explants and alveolar type II cells (ATII). (5) [D-P-Cl-Phe(6)-Leu(17)]-VIP (10(-6) mol/L), a VIP receptors antagonist, abolished the increase of [3H]choline incorporation, microsomal CCT activity and CCTalpha mRNA level induced by VIP (10(-8) mol/L) in lung explants. CONCLUSION: VIP could enhance synthesis of phosphatidylcholine, the major component of pulmonary surfactants by enhancing microsomal CCT activity and CCTalpha mRNA level via VIP receptor-mediated pathway.