Rat Brainstem Slices Research Articles

Disturbed inspiratory rhythm in rat brainstem slices by seizure‐like bursting due to theophylline‐evoked GABAA receptor block

Methylxantines theophylline and caffeine can evoke seizures at clinical doses. Here we studied if this can disturb inspiratory activity and whether blockade of γ‐aminobutyric acid‐A (GABAA) receptors is involved.In newborn rat brainstem‐spinal cords, inspiratory‐related cervical nerve bursting driven by the pre‐Bötzinger Complex (preBötC) was perturbed by >0.1mM caffeine or theophylline. At millimolar levels, seizure‐like discharges occurred that resembled non‐respiratory activity due to bicuculline. In brainstem slices, seizure‐like bursting in inspiratory active hypoglossal nucleus or preBötC area was less pronounced than in the en bloc model for the same concentration. GABAA receptor activation with muscimol hyperpolarized preBötC neurons with a decrease of input resistance and blocked inspiratory rhythm. Rhythm restoration by 5–10mM theophylline in muscimol was accompanied by recovery of membrane potential and resistance.Findings suggest that methylxanthines at millimolar doses perturb preBötC bursting in the slice model via blockade of GABAA receptors, whereas disturbance of rhythm at submillimolar levels in the en bloc model may be due to another mechanism.Supported by AHFMR, CIHR, MFN‐Health

Multiphoton calcium imaging of methylxanthine‐reversal of opioid depression of inspiratory‐related pre‐Bötzinger complex rhythm in newborn rat brainstem slices

Breathing depression by opioids is reversed by methylxanthines (e.g. theophylline). Opioids block Ca2+ channels while methylxanthines cause Ca2+ release from cellular stores at high doses. We studied in newborn rat brainstem slices with 2photon imaging whether opioid block of inspiratory center pre‐Bötzinger Complex (preBötC) and its recovery by theophylline involved cytosolic Ca2+ change.DAMGO blocked bursting and related Ca2+ rises in most preBötC neurons. Ca2+ baseline fell by ~10% in these cells, but not in presumed astrocytes. Theophylline and phosphodiesterase‐4 (PDE4) blocker Rolipram restored rhythm with no effect on Ca2+ baseline. Ca2+ store blocker cyclopiazonic acid did not raise the baseline in preBötC neurons, but in astrocytes. Ca2+ rises became irregular upon block of L‐type Ca2+ channels with nifedipine or Ni2+(0.1mM) without effect on Ca2+ baseline or rhythm. At 2.5mM, Ni2+ blocked rhythm and Ca2+ baseline fell by 10%.SummaryOpioid depression is associated with a Ca2+ fall in preBötC cells that is not due to block of L‐type Ca2+ channels whereas actions of methylxanthines or PDE4 blockers do not involve a major change in Ca2+.Supported by CIHR, AHFMR, CFI‐ISRIP, MFN Health

Transient oxidative stress evokes early changes in the functional properties of neonatal rat hypoglossal motoneuronsin vitro

Oxidative stress of motoneurons is believed to be an important contributor to neurodegeneration underlying the familial (and perhaps even the sporadic) form of amyotrophic lateral sclerosis (ALS). This concept has generated numerous rodent genetic models with inborn oxidative stress to mimic the clinical condition. ALS is, however, a predominantly sporadic disorder probably triggered by environmental causes. Thus, it is interesting to understand how wild-type motoneurons react to strong oxidative stress as this response might cast light on the presymptomatic disease stage. The present study used, as a model, hypoglossal motoneurons from the rat brainstem slice to investigate how hydrogen peroxide could affect synaptic transmission and intrinsic motoneuron excitability in relation to their survival. Hydrogen peroxide (1 mm; 30 min) induced inward current or membrane depolarization accompanied by an increase in input resistance, enhanced firing and depressed spontaneous synaptic events. Despite enhanced intracellular oxidative processes, there was no death of motoneurons, although most cells were immunopositive for activating transcription factor 3, a stress-related transcription factor. Voltage-clamp experiments indicated increased frequency of excitatory or inhibitory miniature events, and reduced voltage-gated persistent currents of motoneurons. The global effect of this transient oxidative challenge was to depress the input flow from the premotor interneurons to motoneurons that became more excitable due to a combination of enhanced input resistance and impaired spike afterhyperpolarization. Our data show previously unreported changes in motoneuron activity associated with cell distress caused by a transient oxidative insult.

Involvement of Ca2+Channel Synprint Site in Synaptic Vesicle Endocytosis

The synaptic protein interaction (synprint) site of the voltage-gated Ca(2+) channel (VGCC) alpha1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the mu subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2-synprint interaction occurred over a wide range of Ca(2+) concentrations but was inhibited at high Ca(2+) concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca(2+)-dependent interactions with AP-2 and Syt.

Astrocytes Control Breathing Through pH-Dependent Vesicular Release of Atp

Extracellular signalling mediated by ATP has been associated with central chemosensory function. It has been shown that increase in pCO2 in the arterial blood triggers an immediate release of ATP from the chemosensitive regions located on the ventral surface of the medulla oblongata (VMS).Using in vitro and in vivo preparations and novel genetically encoded Ca2+ indicator based on cyclically permutated GFP - Case 12, we show that astrocytes in the ventral regions of the medulla oblongata are highly sensitive to changes in pH. Decrease in extracellular pH from 7.4 to 7.2 induced transient increases in [Ca2+]i in astrocytes from dissociated neuro-glial cultures prepared from the VMS, in ventral astrocytes of organotypic brainstem slice cultures and in acute horizontal brainstem slices of adult rats, as well as on the VMS in anaesthetized and artificially ventilated rats. ATP receptor antagonists such as MRS2179, PPADS and TNP-ATP effectively blocked [Ca2+]i responses evoked by lowering pH in VMS astrocytes. ATP hydrolyzing enzyme apyrase completely prevented propagation of [Ca2+]i excitation in VMS astrocytes evoked by lowering external pH. These data suggest that Ca2+ responses induced by lowering external pH are mediated by ATP release and subsequent activation of P2 receptors. Inhibitors of vesicular transport brefeldine A and bafilomycin A both effectively abolished [Ca2+]i excitation of VMS astrocytes evoked by lowering external pH. Incubation of astrocytes with FM 1-46 dye (vesicular marker) leads to selective labelling of intracellular vesicles. Acidification of external medium induced decrease in the fluorescence intensity of vesicles labelled with FM 1-43. These data suggest that VMS astrocytes respond to a decrease in pH by releasing ATP via vesicular exocytosis.

Large Conductance Calcium-Activated Potassium Channels (BKCa) Modulate Trigeminovascular Nociceptive Transmission

Migraine is a common, disabling, neurological problem whose acute management would benefit from the development of purely neurally acting therapies. The trigeminocervical complex is pivotal in nociceptive signaling in migraine, and is an accepted target for putative antimigraine agents. Whole-cell patch-clamp or extracellular recordings were made of trigeminal neurons identified in rat brainstem slices. Bath application of the large conductance calcium-activated potassium (BKCa) channel opener NS1619 caused a dramatic decrease of cell firing that could be reversed by the co-application of iberiotoxin. NS1619 hyperpolarized the resting membrane potential and reduced the frequency of spontaneous action potentials in these neurons. These data suggest the presence of BKCa channels in the trigeminocervical complex. In vivo in cat L-glutamate-evoked firing was facilitated in nociceptive neurons, also responding to stimulation of the superior sagittal sinus, in the trigeminal nucleus caudalis by the BKCa peptide antagonists, iberiotoxin and slotoxin. Of units tested, 70% responded to microiontophoretic application of the blockers, identifying a subpopulation of trigeminal neurons expressing toxin-sensitive BKCa channels. NS1619 inhibited 74% of cells tested, and this was reversed by slotoxin, suggesting that the action of NS1619 in these cells was mediated through BKCa channels. These data are consistent with the presence of BKCa channels in the trigeminal nucleus caudalis that are potential targets for the development of antimigraine treatments, and may also offer insights into receptor mechanisms involved in sensitization and thus allodynia, in migraine.

Progesterone and allopregnanolone enhance the miniature synaptic release of glycine in the rat hypoglossal nucleus

It is well known that progesterone is synthesised and metabolised within the nervous system, and that one of its metabolites, allopregnanolone, potentiates the activity of GABA receptor anionic channels and modulates GABAergic neurotransmission. Progesterone is now under clinical trial for its neuroprotective properties, but its possible effects on neurotransmission have not yet been fully explored. The present study investigated acute effects of progesterone on the other major type of synaptic inhibition, glycinergic neurotransmission. Spontaneous glycinergic miniature currents were recorded in hypoglossal motoneurons, using the whole-cell patch-clamp technique in rat brainstem slices. A 20-min superfusion with progesterone (1 mum) triggered an increase in the frequency of glycinergic miniatures, whereas no effect of progesterone was observed after block with finasteride (5 mum) of 5alpha -reductase, the first enzymatic step leading from progesterone to allopregnanolone. The effect of progesterone could be mimicked by superfusion with allopregnanolone (0.3 mum), whereas no effect was induced by epiallopregnanolone. Thus, progesterone can increase the synaptic miniature release of glycine and this effect appears to be indirect, resulting from its metabolism into 5alpha-reduced derivatives, in particular into allopregnanolone. A low concentration of an exogenous GABA(A) agonist can also increase the frequency of inhibitory miniature currents in hypoglossal motoneurons. Thus, the effects of progesterone and allopregnanolone on glycine release can be at least partly explained by the potentiation of the activity of depolarizing presynaptic GABA receptor channels. The increase in the tonic synaptic release of a major inhibitory neurotransmitter should reduce the excitability of the neurons and contribute to their protection against excitotoxicity.

Oxytocin and vasopressin enhance synaptic transmission in the hypoglossal motor nucleus of young rats by acting on distinct receptor types

Hypoglossal (XII) motoneurons innervate extrinsic and intrinsic muscles of the tongue and control behaviors such as suckling, swallowing, breathing or chewing. In young rats, XII motoneurons express V1a vasopressin and oxytocin receptors. Previous studies have shown that activation of these receptors induces direct powerful excitation in XII motoneurons. In addition, by activating V1a receptors vasopressin can also enhance inhibitory synaptic transmission in the XII nucleus. In the present work, we have further characterized the effect of these neuropeptides on synaptic transmission in the XII nucleus. We have used brainstem slices of young rats and whole-cell patch clamp recordings. Oxytocin enhanced the frequency of spontaneous inhibitory postsynaptic currents by a factor of two and a half. GABAergic and glycinergic events were both affected. The oxytocin effect was mediated by uterine-type oxytocin receptors. Vasopressin and oxytocin also increased the frequency of excitatory synaptic currents, the enhancement being sixfold for the former and twofold for the latter compound. These effects were mediated by V1a and oxytocin receptors, respectively. Miniature synaptic events were unaffected by either vasopressin or oxytocin. This indicates that the peptide-dependent facilitation of synaptic currents was mediated by receptors located on the somatodendritic membrane of interneurons or premotor neurons, and not by receptors sited on axon terminals contacting XII motoneurons. Accordingly, recordings obtained from non-motoneurons located near the border of the XII nucleus showed that part of these cells possess functional V1a and oxytocin receptors whose activation leads to excitation. Some of these neurons could be antidromically activated following electrical stimulation of the XII nucleus, suggesting that they may act as premotor neurons. We propose that in young rats, oxytocin and vasopressin may function as neuromodulators in brainstem motor circuits responsible of tongue movements.

Long-Term Potentiation in the Rat Medial Vestibular Nuclei Depends on Locally Synthesized 17β-Estradiol

In male rat brainstem slices, we investigated the involvement of locally synthesized 17beta-estradiol (E(2)) in the induction in the medial vestibular nucleus (MVN) of long-term potentiation (LTP) by high-frequency stimulation (HFS) of the primary vestibular afferents. We demonstrated that the blockade of aromatase by letrozole or of E(2) receptors (ERalpha and ERbeta) by ICI 182,780 prevented the HFS-induced LTP of the N1 wave of the evoked field potential (FP) without affecting baseline responses. Only prolonged afferent activation could induce low LTP. In contrast, HFS applied under a combined blockade of GABA(A) receptors and aromatase or ERs was still able to induce LTP, but it was significantly lower and slower. These findings demonstrate that E(2) does not have a tonic influence on the activity of the MVN neurons and provide the first evidence of the crucial role played by local synthesis of E(2) in inducing LTP. We suggest that the synthesis of E(2) occurs after aromatase activation during HFS and facilitates the development of vestibular synaptic plasticity by influencing glutamate and GABA transmission.

Electrophysiological effects of ghrelin on laterodorsal tegmental neurons in rats: An in vitro study

Ghrelin, a gut and brain peptide, is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep–wakefulness. Laterodorsal tegmental nucleus (LDT), that regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on LDT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes LDT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF (TTX ACSF), and is partially blocked by the application of [ d-Lys 3]-GHRP-6, a selective antagonist for GHS-Rs. Membrane resistance during the ghrelin-induced depolarization increased by about 18% than that before the depolarization. In addition, the ghrelin-induced depolarization was drastically reduced in high-K + TTX ACSF with a K + concentration of 13.25 mM. Reversal potentials obtained from I– V curves before and during the depolarization were about −83 mV, close to the equilibrium potential of the K + channel. Most of the LDT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca 2+ spike, and they were predominantly cholinergic. These results indicate that ghrelin depolarizes LDT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K + conductance. The results also suggest that LDT neurons are implicated in the cellular processes through which ghrelin participates in the regulation of sleep–wakefulness.

Potentiating Effect of Eszopiclone on GABAA Receptor-Mediated Responses in Pedunculopontine Neurons

The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and REM sleep. GABAergic modulation of this area appears to regulate sleep-wake cycles. Eszopiclone (ESZ), a nonbenzodiazepine hypnotic agent, appears to modulate GABAergic receptors. However, the action site of ESZ in the brain is still unresolved. We tested the hypothesis that ESZ acts by potentiating GABA(A) receptors on PPN neurons. Wholecell voltage clamp recordings were performed on PPN neurons in 7-15 day rat brainstem slices, and the potentiating effects of ESZ on the responses to the GABA(A) receptor agonist isoguvacine (IGV), and on GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), were determined. In the presence of tetrodotoxin, ESZ (1) increased the amplitude of the outward current induced by IGV, (2) increased its duration, and (3) enhanced the IGV-induced decrease in input resistance (Rin). The GABA(A) receptor antagonist gabazine (GBZ) blocked these effects. ESZ alone did not induce detectable currents or change Rin at a holding potential of -60 mV, but when held at 0 mV, ESZ induced an outward current in 13/21 PPN cells, an effect blocked by GBZ. ESZ also increased the amplitude (n = 18/21), duration (n = 17/21), and frequency (n = 13/15) of IPSCs. ESZ may potentiate GABA(A) inhibition in the PPN via pre- and post-synaptic modulation, which may underlie the hypnotic effects of ESZ. The differential effects of ESZ on both pre- and post-synaptic sites may partially explain why it has less significant side effects compared to other hypnotic agents.

Presynaptic modulation of tonic and respiratory inputs to cardiovagal motoneurons by substance P

Substance P (SP) has been implicated in vagal control of heart rate and cardiac functions, but the mechanisms of SP actions on cardiac vagal activity remain obscure. The present study has investigated the effects of SP on the synaptic inputs of preganglionic cardiovagal motoneurons (CVNs) in brainstem slices of neonatal rat. Whole-cell voltage-clamp recordings were performed on retrogradely labeled CVNs in the nucleus ambiguus. The results show that in thin slices (400 μm thickness) without respiratory-like rhythm, globally applied SP (1 μM) significantly enhanced both the GABAergic and the glycinergic inputs, but had no effect on the glutamatergic inputs, of CVNs. Since inspiratory-related augmentation of the inhibitory inputs of CVNs in individual respiratory cycles is known to play an important role in the genesis of respiratory sinus arrhythmia, the effects of SP on the inhibitory inputs of CVNs were further examined in thick slices (500–800 μm thickness) with respiratory-like rhythm, and SP (1 μM) was focally applied to the CVNs under patch-clamp recording. Focally applied SP caused frequency increases of the GABAergic and the glycinergic inputs both during inspiratory bursts and during inspiratory intervals. However, the inspiratory-related augmentation of the GABAergic and the glycinergic inputs of CVNs, measured by the frequency increases during inspiratory bursts in percentage of the frequency during inspiratory intervals, was significantly decreased by SP. These results suggest that SP inhibits CVNs via enhancement of their inhibitory synaptic inputs, and SP diminishes the respiratory-related fluctuation of cardiac vagal activity in individual respiratory cycles. These results also indicate that SP may play a role in altering the vagal control of the heart in some cardiovascular diseases such as myocardial ischemia and hypertension, since these diseases are characterized by weakened cardiac vagal tone and heart rate variability, and have been found to have increased central release and receptor binding of SP.

Akira Takeshita, MD, PhD

Akira Takeshita, MD, PhD, 69, Professor Emeritus of Cardiovascular Medicine and Director Emeritus of the Institute of Angiocardiology, Kyushu University Graduate School of Medicine, Japan, died on March 15, 2009 after a long illness. Dr Takeshita was an internationally recognized leader in cardiovascular medicine. His research focused on neural regulation and vascular biology. Dr Takeshita was born on January 24, 1940. He earned his MD degree from Kyushu University School of Medicine in 1965. His unique and international career started immediately after his graduation when, unlike many other Japanese physicians, especially at that time, he moved to the Tachikawa U.S. Air Force Hospital near Tokyo, where he was an intern and resident. He met Makiko, who was performing her externship in anesthesiology at the Hospital, and they married in 1968. They helped each other as the best partners in life and also as medical professionals, for 41 years until his death. In 1967, Dr Takeshita returned to Kyushu University where he trained in the Department of Cardiovascular Medicine. He then moved to Mount Sinai Hospital, Cleveland, in …

Structure–function analysis of rhythmogenic inspiratory pre-Bötzinger complex networks in “calibrated” newborn rat brainstem slices

Inspiratory pre-Bötzinger complex (preBötC) networks remain active in perinatal rodent brainstem slices. Our analysis of (crescendo-like) inspiratory-related population and cellular bursting in novel histologically identified transversal preBötC slices in physiological (3 mM) superfusate [K(+)] revealed: (i) the preBötC extent sufficient for rhythm in thin slices is at most 175 microm. (ii) In 700 microm thick slices with unilaterally exposed preBötC, a <100 microm kernel generates a eupnea-like inspiratory pattern under predominant influence of caudally adjacent structures or thyrotropin-releasing hormone-like transmitters, but a mixed eupnea-sigh-like pattern when influence of rostral structures or substance-P-like transmitters dominates. (iii) Primarily presynaptic processes may underlie inhibition of rhythm by opioids or raising superfusate [Ca(2+)] from lower to upper physiological limits (1-1.5 mM). (iv) High K(+) reverses depression of rhythm by raised Ca(2+), opioids and anoxia. In summary, distinct activity patterns of spatiochemically organized isolated inspiratory networks depend on both an extracellular "Ca(2+)-K(+) antagonism" and slice dimensions. This explains some discrepant findings between studies and suggests use of "calibrated" slices and more uniform experimental conditions.

Presynaptic inhibition of pre‐Bötzinger complex neurons in high calcium and disturbed seizure‐like inspiratory bursting in low calcium in newborn rat brainstem slices

Inspiratory pre‐Bötzinger complex (preBötC) rhythm in brainstem slices in physiological (3mM) K+ is depressed by raising superfusate Ca2+ from 1mM to 1.5mM. Here, we studied cellular mechanisms of such Ca2+ block of preBötC rhythm and quantified extracellular preBötC interneuron and preBötC‐driven hypoglossal (XII) motoneuron responses to varied Ca2+ and Mg2+. In 400µm thick newborn rat brainstem slices, preBötC/XII rhythm stopped after &lt;2h in 3K+‐1.2Ca2+, but was restored in 7mM K+. Stable preBötC/XII rhythm in 7K+‐1.2Ca2+ was blocked by 2‐3mM Ca2+ without effect on resting potential or input resistance of preBötC and non‐preBötC inspiratory neurons or tonic neurons. preBötC/XII burst rates increased while amplitudes decreased in &lt;1.2Ca2+ until rhythms stopped in &lt;0.25Ca2+. =0.8mM Ca2+evoked seizure‐like activity, partially masking inspiratory XII bursts. Lowering Mg2+ from 1mM to 0.25mM in 7K+‐1.2Ca2+ did not affect preBötC/XII rhythms, whereas 3mM Mg2+ depressed their rate and amplitude to 43% and 82% of control, respectively. Findings show that inspiratory interneuron and XII motoneuron networks in brainstem slices are relatively insensitive to extracellular Mg2+, but generate stable rhythm only in 0.8‐1.2mM Ca2+. Lack of postsynaptic membrane effects suggests that Ca2+ block of isolated preBötC networks involves primarily presynaptic mechanisms. Supported by CIHR‐MFN, CIHR, AHFMR.

Silencing by raised extracellular Ca 2+ of pre-Bötzinger complex neurons in newborn rat brainstem slices without change of membrane potential or input resistance

Breathing is controlled by inspiratory pre-Bötzinger complex (preBötC) networks that remain active in transversal brainstem slices from perinatal rodents. In 600 μm thick preBötC slices, inspiratory-related bursting in physiological (3 mM) [K +] is depressed by <1 mM elevation of superfusate [Ca 2+]. Here, we studied underlying cellular mechanisms in whole-cell-recorded neurons of 400 μm thin newborn rat slices with the <200 μm thin preBötC in the middle (“m-preBötC[400]” slices). Extracellular activity in the ventrolateral slice area in 3 mM K + and a most common physiological Ca 2+ range (1–1.2 mM) stopped spontaneously within 2 h (“in vitro apnea”). Contrary, rhythm was stable for >3 h at 6–8 bursts/min in 7 mM K + and 1.2 mM Ca 2+ solution. In non-pacemaker preBötC inspiratory cells and neighboring inspiratory or tonically active neurons, block or frequency depression by >90% of rhythm in the latter solution by 2–3 mM Ca 2+ changed neither resting potential nor input resistance. High Ca 2+ silenced inspiratory neurons and depressed tonic discharge of non-respiratory neurons. However, in both cell types current injection evoked normal action potentials with unchanged threshold potential. The findings show that m-preBötC[400] slices represent a good compromise between long term viability of rhythmogenic preBötC neurons and minimal modulation of these cells by adjacent tissue, but need to be studied in elevated K +. The lack of postsynaptic K + channel-mediated hyperpolarization suggests that saturation of surface charges, presynaptic block of transmission and/or inhibition of postsynaptic burst-promoting conductances such as Ca 2+ activated non-selective cation channels are involved in inspiratory depression by high Ca 2+.

Infant Brain Stem Is Prone to the Generation of Spreading Depression During Severe Hypoxia

Spreading depression (SD) resembles a concerted, massive neuronal/glial depolarization propagating within the gray matter. Being associated with cerebropathology, such as cerebral ischemia or hemorrhage, epileptic seizures, and migraine, it is well studied in cortex and hippocampus. We have now analyzed the susceptibility of rat brain stem to hypoxia-induced spreading depression-like depolarization (HSD), which could critically interfere with cardiorespiratory control. In rat brain stem slices, severe hypoxia (oxygen withdrawal) triggered HSD within minutes. The sudden extracellular DC potential shift of approximately -20 mV showed the typical profile known from other brain regions and was accompanied by an intrinsic optical signal (IOS). Spatiotemporal IOS analysis revealed that in infant brain stem, HSD was preferably ignited within the spinal trigeminal nucleus and then mostly spread out medially, invading the hypoglossal nucleus, the nucleus of the solitary tract (NTS), and the ventral respiratory group (VRG). The neuronal hypoxic depolarizations underlying the generation of HSD were massive, but incomplete. The propagation velocity of HSD and the associated extracellular K(+) rise were also less marked than in other brain regions. In adult brain stem, HSD was mostly confined to the NTS and its occurrence was facilitated by hypotonic solutions, but not by glial poisoning or block of GABAergic and glycinergic synapses. In conclusion, brain stem tissue reliably generates propagating HSD episodes, which may be of interest for basilar-type migraine and brain stem infarcts. The preferred occurrence of HSD in the infant brain stem and its propagation into the VRG may be of importance for neonatal brain stem pathology such as sudden infant death syndrome.

Fluorescent indication that nitric oxide formation in NTS neurons is modulated by glutamate and GABA

Nitric oxide (NO) in NTS plays an important role in regulating autonomic function to the cardiovascular system. Using the fluorescent dye DAF-2 DA, we evaluated the NO concentration in NTS. Brainstem slices of rats were loaded with DAF-2 DA, washed, fixed in paraformaldehyde and examined under fluorescent light. In different experimental groups, NTS slices were pre-incubated with 1 mM l-NAME (a non-selective NOS inhibitor), 1 mM d-NAME (an inactive enantiomere of l-NAME), 1 mM kynurenic acid (a non-selective ionotropic receptors antagonist) or 20 μM bicuculline (a selective GABA A receptors antagonist) before and during DAF-2 DA loading. Images were acquired using a confocal microscope and the intensity of fluorescence was quantified in three antero-posterior NTS regions. In addition, slices previously loaded with DAF-2 DA were incubated with NeuN or GFAP antibody. A semi-quantitative analysis of the fluorescence intensity showed that the basal NO concentration was similar in all antero-posterior aspects of the NTS (rostral intermediate, 15.5 ± 0.8 AU; caudal intermediate, 13.2 ± 1.4 AU; caudal commissural, 13.8 ± 1.4 AU, n = 10). In addition, the inhibition of NOS and the antagonism of glutamatergic receptors decreased the NO fluorescence in the NTS. On the other hand, d-NAME did not affect the NO fluorescence and the antagonism of GABA A receptors increased the NO fluorescence in the NTS. It is important to note that the fluorescence for NO was detected mainly in neurons. These data show that the fluorescence observed after NTS loading with DAF-2 DA is a result of NO present in the NTS and support the concept that NTS neurons have basal NO production which is modulated by l-glutamate and GABA.

Participation of Kv1 Channels in Control of Membrane Excitability and Burst Generation in Mesencephalic V Neurons

The function and biophysical properties of low threshold Kv1 current in control of membrane resonance, subthreshold oscillations, and bursting in mesencephalic V neurons (Mes V) were examined in rat brain stem slices (P8-P12) using whole cell current and voltage patch-clamp methods. alpha-dendrotoxin application, a toxin with high specificity for Kv1.1, 1.2, and 1.6 channels, showed the presence of a low-threshold K(+) current that activated rapidly around -50 mV and was relatively noninactivating over a 1-s period and had a V(1/2)max of -36.2 mV. Other toxins, specific for individual channels containing either Kv 1.1, 1.2, or 1.3 alpha-subunits, were applied individually, or in combination, and showed that Kv1 channels are heteromeric, composed of combinations of subunits. In current-clamp mode, toxin application transformed the high-frequency resonant properties of the membrane into a low-pass filter and concomitantly reduced the frequency of the subthreshold membrane oscillations. During this period, rhythmical bursting was transformed into low-frequency tonic discharge. Interestingly, in a subset of neurons that did not show bursting, low doses of alpha-dendrotoxin (alpha-DTX) sufficient to block 50% of the low threshold Kv1 channels induced bursting and increased the resonant peak impedance and subthreshold oscillations, which was replicated with computer simulation. This suggests that a critical balance between inward and outward currents is necessary for bursting. This was replicated with computer simulation. Single cell RT-PCR and immunohistochemical methods confirmed the presence of Kv1.1, 1.2, and 1.6 alpha-subunits in Mes V neurons. These data indicate that low threshold Kv1 channels are responsible for membrane resonance, contribute to subthreshold oscillations, and are critical for burst generation.

Postnatal development of inhibitory synaptic transmission to superior salivatory neurons in rats

The primary parasympathetic center of the submandibular and sublingual salivary glands is the superior salivatory (SS) nucleus, and its neurons receive excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) synaptic transmissions in rats. In the present study, we focused on the postnatal development of inhibitory transmission to SS neurons. Gramicidin-perforated whole-cell patch-clamp recordings were performed in rat brainstem slices on postnatal day 2 (P2)-P14. Developmental changes in the intracellular Cl(-) concentration ([Cl(-)](in)) were examined based on the reversal potentials of total inhibitory postsynaptic currents (GABAergic plus glycinergic), which were evoked by electrical stimulation near the recording neuron. The [Cl(-)](in) in the P8-P14 group was significantly lower than in the P2-P7 group. The effect of GABA application at the resting potentials changed from depolarization to hyperpolarization around P8, suggesting that SS neurons acquired mature inhibitory systems around P8. The period at which GABA responses change from excitatory to inhibitory in SS neurons was discussed compared with those of the forebrain, brainstem, and spinal neurons.