Neurons In Brainstem Slices Research Articles

Chronic intermittent hypoxia and hypercapnia inhibit the hypothalamic paraventricular nucleus neurotransmission to parasympathetic cardiac neurons in the brain stem.

Obstructive sleep apnea is associated with chronic intermittent hypoxia/hypercapnia (CIHH) episodes during sleep that heighten sympathetic and diminish parasympathetic activity to the heart. Although one population of neurons in the paraventricular nucleus of the hypothalamus strongly influences sympathetic tone and has increased activity after CIHH, little is known about the role of this pathway to parasympathetic neurons and how this network is altered in CIHH. We hypothesized that CIHH inhibits the excitatory pathway from the paraventricular nucleus of the hypothalamus to parasympathetic cardiac vagal neurons in the brain stem. To test this hypothesis, channelrhodopsin was selectively expressed, using viral vectors, in neurons in the paraventricular nucleus of the hypothalamus and channelrhodopsin-expressing fibers were photoactivated to evoke postsynaptic currents in cardiac vagal neurons in brain stem slices. Excitatory postsynaptic currents were diminished in animals exposed to CIHH. The paired-pulse and prolonged facilitation of the postsynaptic current amplitudes and frequencies evoked by paired and bursts of photoactivation of channelrhodopsin fibers, respectively, occurred in unexposed rats but were blunted in CIHH animals. In response to an acute challenge of hypoxia/hypercapnia, the amplitude of postsynaptic events was unchanged during, but increased after hypoxia/hypercapnia in unexposed animals. In contrast, postsynaptic currents were inhibited during hypoxia/hypercapnia in rats exposed to CIHH. In conclusion, the excitatory pathway to cardiac vagal neurons is diminished in response to both acute and chronic exposures to hypoxia/hypercapnia. This could elicit a reduced cardioprotective parasympathetic activity and an enhanced risk of adverse cardiovascular events in episodes of apnea and chronic obstructive sleep apnea.

Intrinsic properties of rostral ventrolateral medulla presympathetic and bulbospinal respiratory neurons of juvenile rats are not affected by chronic intermittent hypoxia.

The presympathetic neurons in the rostral ventrolateral medulla (RVLM) are considered to be the source of the sympathetic activity, and there is experimental evidence that these cells present intrinsic autodepolarization. There is also evidence that an important respiratory neuronal population located in the RVLM/Bötzinger complex (BötC) corresponds to augmenting expiratory neurons (aug-E), which send projections to the phrenic nucleus in the spinal cord. However, the pacemaker activity of presympathetic neurons and the intrinsic properties of aug-E neurons had not been evaluated in brainstem slices of juvenile rats (postnatal day35). Chronic intermittent hypoxia (CIH) is a sympathetic-mediated hypertension model, which seems to produce an associated increase in the activity of aug-E neurons. In this study, we evaluated the effects of CIH on the intrinsic properties of RVLM/BötC presympathetic and phrenic nucleus-projecting neurons (aug-E) in brainstem slices of juvenile rats (postnatal day35). We observed that all presympathetic neurons presented spontaneous action potential firing (n=18), which was not abolished by ionotropic receptor antagonism. In addition, exposure to 10days of CIH produced no changes in their intrinsic passive properties, firing pattern or excitability. Most aug-E neurons presented spontaneous firing in control conditions (13 of 15 neurons), and this characteristic was preserved after blocking fast synaptic transmission (12 of 15 neurons), clearly demonstrating their intrinsic pacemaker activity. Chronic intermittent hypoxia also produced no changes in intrinsic passive properties, frequency and pattern of discharge or excitability of the aug-E neurons. The present study shows that: (i)it is possible to record the electrophysiological properties of RVLM/BötC presympathetic and aug-E neurons in brainstem slices from juvenile rats; (ii)these neurons present characteristics of intrinsic pacemakers; and (iii)their intrinsic properties were not altered by chronic intermittent hypoxia.

Reduced GABAergic synaptic transmission in the nucleus tractus solitarius in Mecp2‐null mice (1130.20)

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused by loss‐of‐function mutations in the X‐linked methyl‐CpG binding protein 2 (Mecp2) gene. Respiratory dysfunction is one of the major clinical features, presumably due to defective GABAergic function because enhancing GABAergic transmission improved the respiratory phenotype and prolonged survival in RTT mice. We test the hypothesis that deficiency in Mecp2 gene reduces GABAergic signal transmission in the NTS where respiratory information is first received and integrated. Spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded from NTS neurons in brainstem slices obtained from age‐matched male Mecp2‐null mice and wild‐type littermates. Compared to those from wild‐type mice (n = 11 neurons), NTS neurons from Mecp2‐null mice (n = 12 neurons) had a left‐ward shift in cumulative probability of sIPSC amplitudes (p < 0.05) without significant changes in inter‐event intervals (p > 0.05). Similarly, there was a significant left‐ward shift in mIPSC cumulative probability of amplitudes (p < 0.05) but not inter‐event intervals (p > 0.05). The data are consistent with the hypothesis and suggest that reduced GABAergic signaling via post‐synaptic mechanism(s) in the NTS contributes to respiratory dysfunction in RTT.Grant Funding Source: supported by: 1R01 HD064817, International Rett Syndrome Foundation

Temperature influences neuronal activity and CO2/pH sensitivity of locus coeruleus neurons in the bullfrog,Lithobates catesbeianus

The locus coeruleus (LC) is a chemoreceptive brain stem region in anuran amphibians and contains neurons sensitive to physiological changes in CO2/pH. The ventilatory and central sensitivity to CO2/pH is proportional to the temperature in amphibians, i.e., sensitivity increases with increasing temperature. We hypothesized that LC neurons from bullfrogs, Lithobates catesbeianus, would increase CO2/pH sensitivity with increasing temperature and decrease CO2/pH sensitivity with decreasing temperature. Further, we hypothesized that cooling would decrease, while warming would increase, normocapnic firing rates of LC neurons. To test these hypotheses, we used whole cell patch-clamp electrophysiology to measure firing rate, membrane potential (V(m)), and input resistance (R(in)) in LC neurons in brain stem slices from adult bullfrogs over a physiological range of temperatures during normocapnia and hypercapnia. We found that cooling reduced chemosensitive responses of LC neurons as temperature decreased until elimination of CO2/pH sensitivity at 10°C. Chemosensitive responses increased at elevated temperatures. Surprisingly, chemosensitive LC neurons increased normocapnic firing rate and underwent membrane depolarization when cooled and decreased normocapnic firing rate and underwent membrane hyperpolarization when warmed. These responses to temperature were not observed in nonchemosensitive LC neurons or neurons in a brain stem slice 500 μm rostral to the LC. Our results indicate that modulation of cellular chemosensitivity within the LC during temperature changes may influence temperature-dependent respiratory drive during acid-base disturbances in amphibians. Additionally, cold-activated/warm-inhibited LC neurons introduce paradoxical temperature sensitivity in respiratory control neurons of amphibians.

Regulation of ventral surface chemoreceptors by purinergic signaling

A brainstem region called the retrotrapezoid nucleus (RTN) contains a population of CO2/H+‐sensitive neurons that appear to function as important central chemoreceptors (i.e. CO2/H+ sensors). Evidence also indicates that CO2/H+‐evoked ATP release contributes to RTN chemoreception. The goals of this study are to determine the extent to which ATP contributes to RTN chemoreception both in vivo and in vitro, and determine whether purinergic drive to chemoreceptors relies on extracellular Ca2+ or gap junction hemichannels. In vivo, phrenic nerve recordings of respiratory activity in adult rats show that bilateral injection of P2‐ receptor antagonists decreased the ventilatory response to CO2 by 30%. Likewise, in vitro, recordings from functionally identified RTN chemoreceptor neurons in brainstem slices from rat pups (p7–p12), we show P2‐receptor antagonists decreased the firing rate response of RTN neurons to CO2 also by 30%. In the slice, this purinergic drive to breathe did not increase with temperature and was retained in low [Ca2+]o. Conversely, gap junction blockers carbenoxolone (100 μM) and cobalt (500 μM) decreased neuronal CO2/H+‐sensitivity by an amount similar to P2‐receptor antagonists. These results support the possibility that astrocytes but not neurons mediate CO2/H+‐evoked ATP release possibly by a mechanism involving gap junction hemichannels.

Roux‐en‐Y Gastric Bypass Surgery Increases The Response Of Central Vagal Neurons To Satiety Neuropeptides

Roux‐en‐Y gastric bypass surgery (GBS) is the most effective treatment for morbid obesity although the effects on brainstem neurons responsible for vagal control of the viscera are unknown. The aim of the present study was to investigate the effects of obesity and GBS on the pharmacological responses of rat dorsal motor nucleus of the vagus (DMV) neurons.4‐week old Sprague‐Dawley rats were fed a high fat diet (60% kcal from fat) for 44–59 weeks prior to GBS surgery. Sham rats received the same surgical protocol except intestinal manipulation was performed instead. Three weeks post‐surgery, whole‐cell patch clamp recordings were made from DMV neurons in brainstem slices (300μm) and the effects of perfusion with either GLP‐1 or CCK8s (both 100nM) on neuronal excitability were measured.In GBS rats, 5 of 5 neurons tested responded to GLP‐1 with an increase in action potential firing rate (219±35%) whereas only 2 of 9 Sham neurons were similarly responsive (P<0.05). In contrast, while 3 of 6 GBS neurons responded to CCK8s with an increase in firing rate (190±21%) this was not significantly different from the response of Sham neurons (2 of 12 responsive neurons, 230±70%, P>0.05).These results suggest that the effects of GBS to reset obesity‐related alterations in ingestive behavior also involves modulation of the responsiveness of central vagal neurons to gastrointestinal satiety neuropeptides.Supported by NIH DK080899

Signaling Cascades for δ-Opioid Receptor-Mediated Inhibition of GABA Synaptic Transmission and Behavioral Antinociception

Membrane trafficking of the δ-opioid receptor (DOR) from intracellular compartments to plasma membrane in central neurons, induced by various pathological conditions such as long-term opioid exposure, represents unique receptor plasticity involved in the mechanisms of long-term opioid effects in opioid addiction and opioid treatment of chronic pain. However, the signaling pathways coupled to the newly emerged functional DOR in central neurons are largely unknown at present. In this study, we investigated the signaling cascades of long-term morphine-induced DOR for its cellular and behavioral effects in neurons of the rat brainstem nucleus raphe magnus (NRM), a key supraspinal site for opioid analgesia. We found that, among the three phospholipase A(2) (PLA(2))-regulated arachidonic acid (AA) metabolic pathways of lipoxygenase, cyclooxygenase, and epoxygenase, 12-lipoxygenase of the lipoxygenase pathway primarily mediated DOR inhibition of GABA synaptic transmission, because inhibitors of 12-lipoxygenase as well as lipoxygenases and PLA(2) largely blocked the DOR- or AA-induced GABA inhibition in NRM neurons in brainstem slices in vitro. Blockade of the epoxygenase pathway was ineffective, whereas blocking either 5-lipoxygenase of the lipoxygenase pathway or the cyclooxygenase pathway enhanced the DOR-mediated GABA inhibition. Behaviorally in rats in vivo, NRM infusion of 12-lipoxygenase inhibitors significantly reduced DOR-induced antinociceptive effect whereas inhibitors of 5-lipoxygenase and cyclooxygenase augmented the DOR antinociception. These findings suggest the PLA(2)-AA-12-lipoxygenase pathway as a primary signaling cascade for DOR-mediated analgesia through inhibition of GABA neurotransmission and indicate potential therapeutic benefits of combining 5-lipoxygenase and cyclooxygenase inhibitors for maximal pain inhibition.

Distinct tachykinin NK(1) receptor function in primate nucleus tractus solitarius neurons is dysregulated after second-hand tobacco smoke exposure.

Second-hand tobacco smoke (SHS) exposure in children increases the risk of asthma and sudden infant death syndrome. Epidemiological and experimental data have suggested SHS can alter neuroplasticity in the CNS, associated with substance P. We hypothesized that exposure to SHS in young primates changed the effect of substance P on the plasticity of neurons in the nucleus tractus solitarius (NTS), where airway sensory information is first processed in the CNS. Thirteen-month-old rhesus monkeys were exposed to filtered air (FA, n= 5) or SHS (n= 5) for >6 months from 50 days of their fetal age. Whole-cell patch-clamp recordings were performed on NTS neurons in brainstem slices from these animals to record the intrinsic cell excitability in the absence or presence of the NK(1) receptor antagonist, SR140333 (3 µM). Neurons were electrophysiologically classified based on their spiking onset from a hyperpolarized membrane potential into two phenotypes: rapid-onset spiking (RS) and delayed-onset spiking (DS) types. In RS neurons, SR140333 reduced the spiking response, similarly in both FA- and SHS-exposed animals. In DS neurons, SR140333 almost abolished the spiking response in FA-exposed animals, but had no effect in SHS-exposed animals. The contribution of NK(1) receptors to cell excitability depended on firing phenotype of primate NTS neurons and was disrupted by SHS exposure, specifically in DS neurons. Our findings reveal a novel NK(1) receptor function in the primate brainstem and support the hypothesis that chronic exposure to SHS in children causes tachykinin-related neuroplastic changes in the CNS.

Activation of adenosine A2A receptor on GABAergic inputs to 2nd‐order neurons of peripheral chemoreceptors in the NTS

Hypoxia elevates extracellular adenosine level in the NTS. Both adenosine A1 and A2A receptors exist in the NTS. Unlike inhibitory A1 receptor, activation of adenosine A2A receptor usually has a facilitation effect on neural function. In this project, we studied the effect of A2A receptor activation on GABAergic inputs to second‐order NTS neurons. Whole‐cell recordings were performed in NTS neurons in brainstem slices. Second‐order neurons were identified following DiA labeling of carotid bodies. Recordings were performed with CNQX but no TTX to study action potential‐dependent spontaneous pre‐synaptic GABA release (sIPSCs). In 3/7 NTS neurons, application of A2A receptor agonist CGS 21680 at 100 nM induced a significant increase in the frequency of sIPSCs (155±12% of control, p<0.05) without a significant change of the amplitude (95±3% of control, p>0.05). In the other 4/7 NTS neurons, CGS 21680 did not induce a significant change in sIPSCs frequency (93±4% of control, p>0.05). In 3 of these 4 neurons, CGS 21680 induced a significant decrease in sIPSCs amplitude (86±2% of control, p<0.05). The results indicate a heterogeneous and complex function of adenosine A2A receptor in the NTS. The lack of response in some neurons may be due to a lack of receptor on certain GABAergic neurons or indirect mechanisms involving other neurochemicals and ion channels. This work is supported by AHA BGIA 0865269F and NIH HL88052.

Chronic sustained hypoxia reduces neuronal excitability in 2nd‐order neurons of peripheral chemoreceptors in the NTS

Chronic sustained hypoxia (CSH) induces alterations in neural control of cardio‐respiratory function. Here we studied CSH effect on the excitability of NTS neurons receiving direct inputs from peripheral chemoreceptors. Whole‐cell recordings were performed in NTS neurons in brainstem slices. Second‐order neurons were identified following DiA labeling of carotid bodies. After exposure to 10% O2 for 7 d, current injection evoked fewer action potentials (APs) in CSH than normoxic (NORM) cells. Currents of 50, 100, 150 pA elicited 5±1, 10±1, 16±1 APs in CSH cells (n=13), 7±1, 16±1, 22±1 APs in NORM cells (n=14, p<0.001). There was no significant difference in intrinsic membrane properties between groups, including input resistance (NORM 1.0±0.1 GΩ vs CSH 1.1±0.1 GΩ, p>0.05). A‐type K+ currents potently regulate neuronal excitability. Although there was no difference in activation and inactivation of A‐currents in NTS neurons, peak steady‐state currents were greater in CSH (n=6) than NORM cells (n=5, p<0.05). An increased A‐current may contribute to decreased excitability. This could be one adaptive mechanism in response to we previously reported CSH‐induced enhancement of glutamatergic synaptic transmission in the NTS. Current results further reveal the complexity of CSH‐induced alteration of neural function in peripheral chemoreflex pathways. This work is supported by AHA BGIA 0865269F and NIH HL88052.

Effects of 17β-estradiol on glutamate synaptic transmission and neuronal excitability in the rat medial vestibular nuclei

We investigated the effects of the neurosteroid 17β-estradiol (E2) on the evoked and spontaneous activity of rat medial vestibular nucleus (MVN) neurons in brainstem slices. E2 enhances the synaptic response to vestibular nerve stimulation in type B neurons and depresses the spontaneous discharge in both type A and B neurons. The amplitude of the field potential, as well as the excitatory post-synaptic potential (EPSP) and current (EPSC), in type B neurons, are enhanced by E2. Both effects are long-term phenomena since they outlast the drug washout. The enhancement of synaptic response is mainly due to facilitation of glutamate release mediated by pre-synaptic n-methyl-d-aspartate receptors (NMDARs), since the reduction of paired pulse ratio (PPR) and the increase of miniature EPSC frequency after E2 are abolished under D-(−)-2-amino-5-phosphonopentanoic acid (AP-5). E2 also facilitates post-synaptic NMDARs, but it does not affect directly α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and group I-metabotropic glutamate receptors (mGluRs-I). In contrast, the depression of the spontaneous discharge of type A and type B neurons appears to depend on E2 modulation of intrinsic ion conductances, as the effect remains after blockade of glutamate, GABA and glycine receptors (GlyRs). The net effect of E2 is to enhance the signal-to-noise ratio of the synaptic response in type B neurons, relative to resting activity of all MVN neurons. These findings provide evidence for a novel potential mechanism to modulate the responsiveness of vestibular neurons to afferent inputs, and so regulate vestibular function in vivo.

Activation of TRPV1 Mediates Calcitonin Gene-Related Peptide Release, Which Excites Trigeminal Sensory Neurons and Is Attenuated by a Retargeted Botulinum Toxin with Anti-Nociceptive Potential

Excessive release of inflammatory/pain mediators from peripheral sensory afferents renders nerve endings hyper-responsive, causing central sensitization and chronic pain. Herein, the basal release of proinflammatory calcitonin gene-related peptide (CGRP) was shown to increase the excitability of trigeminal sensory neurons in brainstem slices via CGRP1 receptors because the effect was negated by an antagonist, CGRP8-37. This excitatory action could be prevented by cleaving synaptosomal-associated protein of M(r) 25,000 (SNAP-25) with botulinum neurotoxin (BoNT) type A, a potent inhibitor of exocytosis. Strikingly, BoNT/A proved unable to abolish the CGRP1 receptor-mediated effect of capsaicin, a nociceptive TRPV1 stimulant, or its elevation of CGRP release from trigeminal ganglionic neurons (TGNs) in culture. Although the latter was also not susceptible to BoNT/E, apparently attributable to a paucity of its acceptors (glycosylated synaptic vesicle protein 2 A/B), this was overcome by using a recombinant chimera (EA) of BoNT/A and BoNT/E. It bound effectively to the C isoform of SV2 abundantly expressed in TGNs and cleaved SNAP-25, indicating that its /A binding domain (H(C)) mediated uptake of the active /E protease. The efficacy of /EA is attributable to removal of 26 C-terminal residues from SNAP-25, precluding formation of SDS-resistant SNARE complexes. In contrast, exocytosis could be evoked after deleting nine of the SNAP-25 residues with /A but only on prolonged elevation of [Ca(2+)](i) with capsaicin. This successful targeting of /EA to nociceptive neurons and inhibition of CGRP release in vitro and in situ highlight its potential as a new therapy for sensory dysmodulation and chronic pain.

Pain relief by gabapentin and pregabalin via supraspinal mechanisms after peripheral nerve injury

The antihypersensitivity actions of gabapentin and pregabalin have been well characterized in a large number of studies, although the underlying mechanisms have yet to be defined. We have been focusing on the supraspinal structure as a possible site for their action and have demonstrated that intracerebroventricular (i.c.v.) administration of gabapentin and pregabalin indeed decreases thermal and mechanical hypersensitivity in a murine chronic pain model involving partial ligation of the sciatic nerve. This novel supraspinally mediated analgesic effect was markedly suppressed by either depletion of central noradrenaline (NA) or blockade of spinal alpha(2)-adrenergic receptors. Moreover, i.c.v. injection of gabapentin and pregabalin increased spinal NA turnover in mice only after peripheral nerve injury. In locus coeruleus (LC) neurons in brainstem slices prepared from mice after peripheral nerve injury, gabapentin reduced the gamma-aminobutyric acid (GABA) type A receptor-mediated inhibitory postsynaptic currents (IPSCs). Glutamate-mediated excitatory synaptic transmission was hardly affected. Moreover, gabapentin did not reduce IPSCs in slices taken from mice given a sham operation. Although gabapentin altered neither the amplitude nor the frequency of miniature IPSCs, it reduced IPSCs together with an increase in the paired-pulse ratio, suggesting that gabapentin acts on the presynaptic GABAergic nerve terminals in the LC. Together, the data suggest that gabapentin presynaptically reduces GABAergic synaptic transmission, thereby removing the inhibitory influence on LC neurons only in neuropathic pain states, leading to activation of the descending noradrenergic system.

Gabapentin produces PKA‐dependent pre‐synaptic inhibition of GABAergic synaptic transmission in LC neurons following partial nerve injury in mice

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to ameliorate pain hypersensitivity in mice with partial nerve ligation. To clarify the supraspinal mechanism of action of gabapentin, whole-cell patch-clamp recordings were performed on locus coeruleus (LC) neurons in brainstem slices prepared from mice after peripheral nerve injury or mice subjected to a sham-operation, and the effects of gabapentin in the modulation of synaptic transmission were studied. Bath application of gabapentin (10, 30 and 100 muM) in a concentration-dependent manner reduced the GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs) in slices prepared from partially nerve-ligated mice, whereas glutamate-mediated excitatory post-synaptic currents were hardly affected. By contrast, gabapentin did not reduce IPSCs in slices taken from mice given a sham operation. Although gabapentin altered neither the amplitude nor the frequency of miniature IPSCs, it reduced IPSCs together with an increase in the paired-pulse ratio, suggesting that gabapentin acts on the pre-synaptic GABAergic nerve terminals in the LC. As the protein kinase A (PKA) inhibitor H-89 but not the protein kinase C inhibitor chelerythrine abolished the inhibitory action of gabapentin on IPSCs, PKA-mediated phosphorylation seems to be important for supraspinal gabapentin responsiveness in neuropathic conditions. Together, gabapentin generates PKA-dependent pre-synaptic inhibition of GABAergic synaptic transmission, and thereby removes the inhibitory influence on LC neurons only under neuropathic pain states. These findings provide crucial evidence of how supraspinally acting gabapentin recruits the descending noradrenergic system.

Development of inhibitory synaptic transmission to the superior salivatory nucleus in rats

The primary parasympathetic center of the submandibular and sublingual salivary glands is the superior salivatory (SS) nucleus, neurons of which receive excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) synaptic transmissions in rats. In the present study, to examine postnatal neural development, we focused on inhibitory transmission to the SS neurons in neonatal rats from postnatal day 2 (P2) to P14. Conventional and gramicidin-perforated whole-cell patch-clamp techniques were applied to the neurons in brainstem slices. The decay time constants of GABAergic and glycinergic postsynaptic currents (PSCs) consisted of fast ( τ fast) and slow ( τ slow) components. Both τ fast and τ slow of PSC components tended to become faster with development. The equilibrium potential of Cl − ( E Cl−) was estimated from the reversal potentials of total PSCs (GABAergic plus glycinergic). The E Cl− in the P8–P14 group was significantly more negative than E Cl− in the P2–P7 group. Exogenous GABA application at the resting potentials produced depolarization in 83% of SS neurons at P2–P7 and accompanied the action potential in some neurons. In contrast, at P8–P14, GABA evoked hyperpolarization in 78% of SS neurons; therefore, SS neurons did not acquire mature inhibitory systems until P14. The development of SS neurons is discussed as compared with the development of peripheral salivary gland tissue and brainstem neurons that participate in oral motor and sensory functions.

Imaging of respiratory network topology in living brainstem slices

Topology of neuronal networks contributes to their functioning but the structure–function relationships are not yet understood. In order to reveal the spatial organisation of the respiratory network, we expressed enhanced green fluorescent proteins in neurons in brainstem slices containing the respiratory kernel (pre-Bötzinger complex). The expression was neuron specific due to use of adeno-associated viral vector driving transgene expression from synapsin 1 promoter. Both neuronal cell bodies and their dendrites were labelled with high efficacy. This labelling allowed for enhanced spatial resolution as compared to conventional calcium-sensitive dyes. Neurons occupied about 10% of tissue volume and formed an interconnected network. Using custom-developed software, we quantified the network structure that had a modular structure consisting of clusters having transverse (dorso-ventral) orientation. They contained in average seven neurons and connections between the cells in different clusters were less frequent. This novel in situ imaging technique is promising to gain new knowledge about the fine structure and function of neuronal networks in living slice preparations.

Seizures and Reduced Life Span in Mice Lacking the Potassium Channel Subunit Kv1.2, but Hypoexcitability and Enlarged Kv1 Currents in Auditory Neurons

Genes Kcna1 and Kcna2 code for the voltage-dependent potassium channel subunits Kv1.1 and Kv1.2, which are coexpressed in large axons and commonly present within the same tetramers. Both contribute to the low-voltage-activated potassium current I Kv1, which powerfully limits excitability and facilitates temporally precise transmission of information, e.g., in auditory neurons of the medial nucleus of the trapezoid body (MNTB). Kcna1-null mice lacking Kv1.1 exhibited seizure susceptibility and hyperexcitability in axons and MNTB neurons, which also had reduced I Kv1. To explore whether a lack of Kv1.2 would cause a similar phenotype, we created and characterized Kcna2-null mice (-/-). The -/- mice exhibited increased seizure susceptibility compared with their +/+ and +/- littermates, as early as P14. The mRNA for Kv1.1 and Kv1.2 increased strongly in +/+ brain stems between P7 and P14, suggesting the increasing importance of these subunits for limiting excitability. Surprisingly, MNTB neurons in brain stem slices from -/- and +/- mice were hypoexcitable despite their Kcna2 deficit, and voltage-clamped -/- MNTB neurons had enlarged I Kv1. This contrasts strikingly with the Kcna1-null MNTB phenotype. Toxin block experiments on MNTB neurons suggested Kv1.2 was present in every +/+ Kv1 channel, about 60% of +/- Kv1 channels, and no -/- Kv1 channels. Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, which may explain why MNTB neurons with larger proportions of such channels had larger I Kv1. If channel voltage dependence is determined by how many Kv1.2 subunits each contains, neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density.

Endomorphin-1 Modulates Intrinsic Inhibition in the Dorsal Vagal Complex

Mu-opioid receptor (MOR) agonists profoundly influence digestive and other autonomic functions by modulating neurons in nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). Whole cell recordings were made from NTS and DMV neurons in brain stem slices from rats and transgenic mice that expressed enhanced green fluorescent protein (EGFP) under the control of a GAD67 promoter (EGFP-GABA neurons) to identify opioid-mediated effects on GABAergic circuitry. Synaptic and membrane properties of EGFP-GABA neurons were assessed. The endogenous selective MOR agonist endomorphin-1 (EM-1) reduced spontaneous and evoked excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) in both rat and mouse DMV neurons. Electrical stimulation of the solitary tract evoked constant-latency EPSCs in approximately 50% of EGFP-GABA neurons, and the responses were reduced by EM-1 application. EM-1 reduced action potential firing, the frequency and amplitude of synaptic inputs in EGFP-GABA neurons and responses to direct glutamate stimulation. A subset of EGFP-GABA neurons colocalized mRFP1 after retrograde, transneuronal infection after gastric inoculation with PRV-614, indicating that they synapsed with gastric-projecting DMV neurons. Glutamate photolysis stimulation of intact NTS projections evoked IPSCs in DMV neurons, and EM-1 reduced the evoked response, most likely by activation of MOR on the soma of premotor GABA neurons in NTS. Naltrexone or H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), MOR antagonists, blocked the effects of EM-1. Our results show that GABA neurons in the NTS receive direct vagal afferent input and project to gastric-related DMV neurons. Furthermore, modulation by EM-1 of specific components of the vagal complex differentially suppresses excitatory and inhibitory synaptic input to the DMV by acting at different receptor locations.

Prostaglandin E2 depresses solitary tract-mediated synaptic transmission in the nucleus tractus solitarius

Prostaglandin E2 (PGE2) is a prototypical inflammatory mediator that excites and sensitizes cell bodies [Kwong K, Lee LY (2002) PGE2 sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli. J Appl Physiol 93:1419–1428; Kwong K, Lee LY (2005) Prostaglandin E2 potentiates a tetrodotoxin (TTX)-resistant sodium current in rat capsaicin-sensitive vagal pulmonary sensory neurons. J Physiol 56:437–450] and peripheral nerve terminals [Ho CY, Gu Q, Hong JL, Lee LY (2000) Prostaglandin E (2) enhances chemical and mechanical sensitivities of pulmonary C fibers in the rat. Am J Respir Crit Care Med 162:528–533] of primary vagal sensory neurons. Nearly all central nerve terminals of vagal afferents are in the nucleus tractus solitarius (NTS), where they operate with a high probability of release [Doyle MW, Andresen MC (2001) Reliability of monosynaptic sensory transmission in brain stem neurons in vitro. J Neurophysiol 85:2213–2223]. We studied the effect of PGE2 on synaptic transmission between tractus solitarius afferent nerve terminals and the second-order NTS neurons in brain stem slices of Sprague–Dawley rats. Whole-cell patch recording in voltage clamp mode was used to study evoked excitatory postsynaptic glutamatergic currents (evEPSCs) from NTS neurons elicited by electrical stimulation of the solitary tract (ST). In 34 neurons, bath-applied PGE2 (200 nM) decreased the evEPSC amplitude by 49±5%. In 22 neurons, however, PGE2 had no effect. We also tested 15 NTS neurons for capsaicin sensitivity. Seven neurons generated evEPSCs that were equally unaffected by PGE2 and capsaicin. Conversely, evEPSCs of the other eight neurons, which were PGE2-responsive, were abolished by 200 nM capsaicin. Furthermore, the PGE2-induced depression of evEPSCs was associated with an increase in the paired pulse ratio and a decrease in both the frequency and amplitude of the spontaneous excitatory postsynaptic currents (sEPSCs) and TTX-independent spontaneous miniature excitatory postsynaptic currents (mEPSCs). These results suggest that PGE2 acts both presynaptically on nerve terminals and postsynaptically on NTS neurons to reduce glutamatergic responses.

Effects of enkephaline on K + currents of oculomotor neurons in brainstem slices from rats

Effects of enkephaline on K + currents of oculomotor neurons in brainstem slices from rats