Kölliker-Fuse Research Articles

Desensitization of mu opioid receptors on Kölliker‐Fuse neurons

Tolerance to the respiratory depressant effects of opioids develops less readily than tolerance to analgesia, creating a dosing limit in chronic pain patients. Desensitization of mu opioid receptors is considered an initial step in the development of tolerance. Currently, most information about desensitization has been obtained in locus coeruleus (LC) neurons. Given the lack of respiratory tolerance, this study examined desensitization of mu opioid receptors on respiratory‐controlling Kölliker‐Fuse (KF) neurons. In voltage‐clamp recordings from KF neurons in rat brain slice, the opioid receptor agonist [Met5]enkephalin (ME) caused an outward current in a population of KF neurons due to activation of GIRK conductance. Compared to ME, the partial opioid agonist morphine produced a current that was proportionally larger than the morphine‐mediated current in LC brain slice preparations, indicating that KF neurons may have a large receptor reserve and thus be resistant to desensitization. Desensitization was induced by prolonged (10 min) perfusion of a high concentration of ME (30 µM). Desensitization of the ME‐mediated GIRK current was significantly less in KF neurons than in LC neurons. In contrast to LC, desensitization was not enhanced by activation of PKC or in slices from morphine‐treated rats. Thus, a lack of desensitization of mu opioid receptors on KF neurons may contribute to the relative lack of tolerance to the respiratory depressant effect of opioids. This will be tested using in situ and in vivo preparations of morphine‐treated rats. Supported by DA038069 (ESL) and DA08163 (JTW).

Respiratory-related outputs of glutamatergic, hypercapnia-responsive parabrachial neurons in mice.

In patients with obstructive sleep apnea, airway obstruction during sleep produces hypercapnia, which in turn activates respiratory muscles that pump air into the lungs (e.g., the diaphragm) and that dilate and stabilize the upper airway (e.g., the genioglossus). We hypothesized that these responses are facilitated by glutamatergic neurons in the parabrachial complex (PB) that respond to hypercapnia and project to premotor and motor neurons that innervate the diaphragm and genioglossus muscles. To test this hypothesis, we combined c-Fos immunohistochemistry with in situ hybridization for vGluT2 or GAD67 or with retrograde tracing from the ventrolateral medullary region that contains phrenic premotor neurons, the phrenic motor nucleus in the C3-C5 spinal ventral horn, or the hypoglossal motor nucleus. We found that hypercapnia (10% CO2 for 2 hours) activated c-Fos expression in neurons in the external lateral, lateral crescent (PBcr), and Kölliker-Fuse (KF) PB subnuclei and that most of these neurons were glutamatergic and virtually none γ-aminobutyric acidergic. Numerous CO2 -responsive neurons in the KF and PBcr were labeled after retrograde tracer injection into the ventrolateral medulla or hypoglossal motor nuclei, and in the KF after injections into the spinal cord, making them candidates for mediating respiratory-facilitatory and upper-airway-stabilizing effects of hypercapnia.

Respiratory and sympathetic chemoreflex regulation by Kölliker-Fuse neurons in rats

Chemoreceptor activation increases phrenic nerve activity (PNA) and sympathetic nerve activity (SNA). The dorsolateral pontine neurons, including the parabrachial nucleus and the Kölliker-Fuse (KF) region project to several brainstem areas involved in autonomic and respiratory regulation. Here the objective was to further test the hypothesis that the KF region could contribute to central and peripheral sympathetic chemoreflex activation. In urethane-anesthetized sino-aortic denervated or intact and vagotomized male Wistar rats (N = 7-8/group), hypercapnia (end-expiratory CO2 from 5 to 10 %) or KCN increased mean arterial pressure (MAP), splanchnic SNA, and PNA frequency and amplitude. Bilateral injection of muscimol (GABA-A agonist; 2 mM-50 nl) into the KF region increased resting PNA amplitude and reduced resting PNA frequency, without significant changes in resting MAP and SNA. Bilateral blockade of the KF region reduced the rise in MAP, sSNA, and PNA frequency and amplitude produced by hypercapnia or hypoxia. Our data suggest that the KF neurons could integrate and modulate breathing and sympathetic outflow during chemoreceptor activation.

Mu opioid receptors hyperpolarize respiratory‐controlling Kölliker‐Fuse neurons (712.1)

Respiratory depression is the primary cause of death from opioid overdose, yet little is known about the cellular mechanisms of opioids on respiratory‐controlling neurons. The pontine Kölliker‐Fuse (KF) regulates respiration, especially the inspiratory/expiratory phase transition. In the in situ arterially perfused working heart‐brainstem preparation of rat, injection of the mu opioid agonist DAMGO into the KF caused robust apneusis that was similar to complete silencing of the KF. The apneusis was rapidly reversed by perfusion of the opioid antagonist naloxone. In whole‐cell recordings from KF neurons contained in rat brain slices, activation of mu opioid receptors hyperpolarized a majority (75%) of KF neurons. The hyperpolarizing current produced by [Met5]enkephalin (ME) was concentration‐dependent, reversed at the potassium equilibrium potential and was blocked by BaCl2, characteristics of a GIRK conductance. Unexpectedly, the partial agonist morphine produced the same amplitude current as full agonists DAMGO or ME, indicating a large receptor reserve. The presence of many spare receptors could reduce the degree of desensitization induced by prolonged application of ME. Indeed, only minimal acute desensitization of the ME‐mediated GIRK current was observed. This lack of desensitization correlates with the lack of tolerance to the respiratory depressant effects of opioids.Grant Funding Source: Supported by DA08163 (JTW) and DA33036 (ESL)

Characterization of tauopathy in the ponto‐medullary brainstem nuclei of aged Tau‐P301L mice (728.7)

Neurodegenerative diseases, such as Alzheimer’s disease (AD) and dementia, are often linked to swallowing disorders such that aspiration pneumonia resulting from accidental inhalation of ingested material is a common phenomenon. Growing evidence suggests that malfunction in the coordination of swallowing and breathing leads to increased risk of aspiration. We investigated the underlying pathology linking dementia and swallowing dysfunction in Tau‐P301L transgenic mice, an established mouse model of neurodegeneration. Tauopathy and neurofibrillary tangle‐related neuropathological morphology in the brainstem of 10‐month old Tau‐P301L mice was identified by immunohistochemistry using antisera against: 1) pre‐neurofibrillary tangles (Tau phosphoThreonin 231); and 2) extra‐neuronal neurofibrillary tangles (Tau phosphoSerine 199/202). Cell bodies containing diffuse phospho‐tau‐immunoreactivity within the cytoplasm, together with preserved, collapsed or absent dendrites were identified in the Kölliker‐Fuse (KF) nucleus, ventral swallowing group (VSG) dorsomedial to the nucleus ambiguus, and nucleus of the solitary tract (NTS), regions known to co‐ordinate breathing and swallowing. Our results suggest that neurons in the KF, VSG and NTS are susceptible to neurodegeneration, and provide a platform for examining the effect of tauopathy in functionally defined and experimentally controllable brainstem circuits (for swallowing and breathing). This may facilitate a framework for the development of potential drug therapies for neuroprotection and prevention of aspiration and dementia.Grant Funding Source: ARC

Pontine respiratory group neuronal discharge is modulated by powerful GABAergic tonic inhibition (712.7)

Neurons within the parabrachial (PB) complex appear to play a key role in controlling breathing frequency. Densely labeled GABA‐immunoreactive varicosities have been observed throughout the PB and the Kölliker‐Fuse (KF) nuclei of rats, however, the role of GABAergic control of PRG neurons is inadequately understood. To characterize GABAergic inhibition in the control of PRG discharge frequency (Fn) patterns, we analyzed the effects of microejected GABA‐A receptor antagonists bicuculline and picrotoxin on PRG neuronal activity in a decerebrate, vagotomized, ventilated and paralyzed canine model. Histograms (CTHs) triggered from the phrenic neurogram were used to quantify neuronal unit activity. Both antagonists produced marked increases in Fn (>80%) either alone or combined (sequentially ejected) in 80‐90% of PRG neurons. Activity throughout the cycle of phase spanning PRG neurons was increased and phasic portions of Fn showed gain modulation. Effects of both antagonists appear to be qualitatively and quantitatively similar. The results show that most PRG neurons are under strong endogenous GABAergic inhibition that attenuates their activity and is expected to markedly impact phase‐timing.Grant Funding Source: Supported by VA Medical Research Funds I01 BX000721‐01

GLUTAMATE INJECTION INTO THE CUNEIFORM NUCLEUS IN RAT, PRODUCES CORRELATED SINGLE UNIT ACTIVITIES IN THE KOLLIKER-FUSE NUCLEUS AND CARDIOVASCULAR RESPONSES

The cuneiform (CnF) and Kolliker-Fuse (KF) nuclei are implicated in several functions including regulation of cardiovascular system and pain modulation. The KF also is a potential candidate for relaying the CnF cardiovascular responses to the rostral ventrolateral medulla (RVLM). In a previous study we showed that blockade of the KF strongly attenuated the short responses and moderately attenuated the long responses to glutamate microinjection into the CnF, suggesting that the cardiovascular effects of the CnF, especially the short responses, were mediated by the KF. In the present study the cellular basis of the cardiovascular responses of the CnF and possible role of the KF in relaying them to the RVLM were explored. In one group, l-glutamate was microinjected in the CnF and the cardiovascular responses were recorded. In another group the single unit responses of the KF to l-glutamate injection into the CnF were recorded. Our results showed that chemical stimulation of the CnF with glutamate produced mainly excitatory cardiovascular and single unit responses and a minority of mixed (excitatory and inhibitory) responses. In about one fourth of the cases there were no responses to stimulation. Various patterns of each group were presented and compared between cardiovascular and single unit responses. Similarities were found between cardiovascular and single unit response patterns, suggesting a significant role of KF neurons in mediating the CnF cardiovascular responses to the RVLM.

Isoflurane anesthesia precipitates tauopathy and upper airways dysfunction in pre-symptomatic Tau.P301L mice: Possible implication for neurodegenerative diseases

The postoperative cognitive decline resulting from volatile anesthesia is gaining acceptance as a major health problem. The common anesthetic isoflurane is suspected to precipitate neurodegeneration in Alzheimer's disease by unknown mechanisms. We previously validated that 8month old Tau.P301L mice suffer upper airways defects related to tauopathy within the Kolliker–Fuse nucleus that controls upper airways function. We now report that isoflurane anesthesia in young, pre-symptomatic Tau.P301L mice triggered precocious upper airways defects and tauopathy in several brainstem nuclei, including the nucleus ambiguus that contains upper airways motor neurons and the Kolliker–Fuse. The prescription drug memantine, identified as an NMDA receptor antagonist, prevented the post-anesthesia upper airways dysfunction and alleviated tauopathy in the nucleus ambiguus and Kolliker–Fuse. We further identified protocols of anesthesia in young Tau.P301L mice that mitigated adverse effects of isoflurane anesthesia. Thus, our experimental findings in a validated mouse model for tauopathy demonstrate the link between isoflurane anesthesia, earlier onset of tauopathy and earlier onset of functional deficits, highlight the implication of NMDA-receptors in the mechanisms mediating the adverse effects of isoflurane, and potentially identify safer protocols for anesthesia in patients with tauopathy.

Inactivation effect of Kolliker—Fuse nucleus on long cardiovascular response of L- glutamate in cuneiform nucleus in rats

Inactivation effect of Kolliker—Fuse nucleus on long cardiovascular response of L- glutamate in cuneiform nucleus in rats

Effect of glutamate stimulation of the cuneiform nucleus on cardiovascular regulation in anesthetized rats: Role of the pontine Kolliker–Fuse nucleus

Cuneiform nucleus (CnF) is a reticular nucleus of the midbrain involved in cardiovascular function and stress. There is no report on the cardiovascular effects of the glutamatergic system in the CnF. In the present study, we investigated the cardiovascular effects of glutamate and its NMDA and AMPA/kainate receptors in the CnF. In addition, the possible mediation of Kolliker–Fuse (KF) nucleus in the cardiovascular effects of the CnF was explored. l-glutamate, AP5 (an NMDA receptor antagonist), and CNQX (an AMPA/kainate receptor antagonist) (50–100nl) were microinjected into the CnF of anesthetized rats. Also, the KF was blocked by cobalt chloride (CoCl2) then l-glutamate was microinjected into the CnF. The maximum changes of blood pressure and heart rate were compared with the pre-injection (paired t-test) and control (independent t-test) values. Microinjection of glutamate (25nmol/100nl) into the CnF produced either a short pressor and bradycardic or a long pressor and tachycardic responses. Microinjection of AP5 or CNQX alone did not affect the basal arterial pressure and heart rate. However, co-injection of glutamate with AP5 strongly attenuated the short and moderately attenuated the long cardiovascular responses elicited by glutamate. Co-injection of glutamate with CNQX did not attenuate the short and weakly attenuated the long cardiovascular responses elicited by glutamate. These data suggest that the responses are mediated mainly through NMDA receptors. Blockade of the KF nucleus strongly attenuated the short response and weakly attenuated the long response to glutamate microinjection, suggesting that the cardiovascular effects of glutamate in the CnF, especially the short responses, were mediated by the KF nucleus.

GABAergic neurons in the ventrolateral subnucleus of the nucleus tractus solitarius are in contact with Kölliker-Fuse nucleus neurons projecting to the rostral ventral respiratory group and phrenic nucleus in the rat

After ipsilateral injections of biotinylated dextran amine (BDA) into the ventrolateral subnucleus of the nucleus tractus solitarius (vlNTS) and Fluoro-gold (FG) into the rostral ventral respiratory group (rVRG) region or into the phrenic nucleus (PhN) region in the rat, an overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the Kölliker-Fuse (KF) nucleus ipsilateral to the injection sites. Using retrograde tracing combined with immunohistochemistry for glutamic acid decarboxylase isoform 67 (GAD67), we indicated that as many as 40% of the vlNTS neurons projecting to the KF were immunoreactive for GAD67. Using a combination of anterograde and retrograde tracing techniques, and immunohistochemistry for GAD67, we further demonstrated that the vlNTS axon terminals with GAD67 immunoreactivity established close contact to the rVRG- or PhN-projecting KF neurons. The present results suggest that GABAergic vlNTS fibers may exert inhibitory influences on the rVRG- as well as PhN-projecting KF neurons and these circuits may be involved in the respiratory reflexes such as the Hering–Breuer reflex.

Expression of Fos protein in brainstem after application of l-menthol to the rat nasal mucosa

There are two functional pathways for the nasotrigeminal reflex: the spinal nucleus of trigeminal nerve (SPV) to the Kölliker-Fuse (KF) nucleus and the nucleus of solitary tract (NTS) to the lateral parabrachial nucleus (PBl). Although stimulation of the nasal mucosa by cool temperature induces respiratory depression, it is still unknown whether these nuclei are activated. In the present study, we examined the expression of Fos protein in rat brainstem neurons after nasal application of l-menthol, which is known to activate cold-sensitive nasal receptors. Application of l-menthol, but not paraffin oil, decreased the respiratory rate from 99.7+/-15.6 to 78.5+/-7.3 min(-1). Furthermore, a significantly higher density of Fos-immunoreactive cells was observed in the SPV and KF in the l-menthol rats than in the controls. In the SPV, the density of Fos-immunoreactive cells was highest at approximately 0.5mm rostral to the obex in both the l-menthol (48.5+/-11.5 cells/section) and paraffin oil (26.0+/-9.6 cells/section) groups. In the KF, the mean density of Fos-immunoreactive cells was highest at approximately 5.0mm rostral to the obex in both groups (l-menthol: 67.8+/-14.0 cells/section, control: 41.0+/-12.7 cells/section). The present study suggests that the SPV-KF pathway is important for the cold-induced respiratory depression.

Habituation, desensitization and sensitization of the Hering–Breuer reflex in normal and Mecp2−/y knockout mice

Habituation, desensitization and sensitization of the Hering–Breuer reflex in normal and <i>Mecp2^−/y</i> knockout mice

Expression and colocalization of NADPH-diaphorase and Fos in the subnuclei of the parabrachial nucleus in rats following visceral noxious stimulation

To investigate whether neural nitric oxide synthase (nNOS) in the parabrachial nucleus (PB) is involved in processing visceral noxious stimulation, we mapped the distribution of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nNOS, and immunohistochemical staining for Fos, a neuronal activity marker, in the subnuclei of the PB following 2% formalin injection into the stomach of rats. NADPH-d and noxious-stimuli induced Fos staining were also examined in tissue containing PB cells labeled by the retrograde transport of fluogold (FG) injected into the central nucleus of the amygdala (CeA). We found that the number of Fos immunoreactive (Fos-IR) neurons was significantly increased in the dorsal lateral (dl), external lateral (el) and Kölliker–Fuse (KF) subnuclei of the PB. We observed that intensely labeled (type 1) NADPH-d positive neurons were mainly located in the rostral part of the PB; they extended long processes adjacent Fos-IR neurons, but no Fos/type 1 NADPH-d double-labeled neurons were seen. In contrast, lightly labeled (type 2) NADPH-d positive neurons were principally localized in the dl of the PB, in which a few Fos/type 2 NADPH-d double-labeled neurons were detected. Additionally, a large number of FG/Fos double-labeled neurons were observed to be surrounded closely by the intensive NADPH-d staining in the el of the PB. These results suggest that neurons in the el of the PB that project to the CeA are activated by visceral noxious stimulation and could be indirectly influenced by nitric oxide in the PB.

A novel, more complex rhythmic slice preparation containing the preBötzinger (PBC) and Kölliker‐Fuse (KF) region

Rhythmic slice preparations for the study of neuronal control of breathing usually contain the PBC and associated medullary respiratory centers. The activity of XII rootlets or PBC elements indicates ongoing respiratory-like activity. Recent studies suggest that respiratory rhythm generation also involves more rostral aspects like the parafacial region (PFR). Also a potential role of the pontine KF is assumed. We developed a 700 μm rat brainstem slice that includes the PBC, PFR and KF. In this slice we imaged Ca2+ (see abstract Funke et al.) to analyze population activity (PA) in the PBC correlated with electrical PBC mass activity. The novel slice preparation is characterized by a low frequency PBC mass activity (3 burst/min, in 8 mM K+) compared to 6–12 bursts/minute in the “classic” PBC slice. Blocking inhibitory synapses with strychnine and bicuculline caused an initial, substantial augmentation of the PBC rhythm that stabilized at frequencies of 4–6 bursts/min; burst duration was increased. So far, imaging did not reveal a reconfiguration or recruitment of additional neuronal populations. Nevertheless, the response pattern reflects the situation after pontine ablation in en-bloc preparations. Therefore, our new slice preparation may prove substantial in the study of pontine modulation of PBC activity under a variety of physio- and pathophysiological conditions. Funded by the DFG (CMPB)

Projections from the parabrachial nucleus to the vestibular nuclei: potential substrates for autonomic and limbic influences on vestibular responses

Previous anatomical studies in rabbits and rats have shown that the superior vestibular nucleus (SVN), medial vestibular nucleus (MVN) and inferior vestibular nucleus (IVN) project to the parabrachial nucleus (PBN) and Kölliker–Fuse (KF) nucleus. Adult male albino rabbits and Long–Evans rats received iontophoretic injections of biotinylated dextran amine, Phaseolus vulgaris leucoagglutinin, Fluoro-Gold or tetramethylrhodamine dextran amine into either the vestibular nuclei or the PBN and KF nuclei. The results were similar in both rats and rabbits. Injections of retrograde tracers into the vestibular nuclei produced retrogradely labeled neurons bilaterally in caudal third of the medial, external medial, and external lateral PBN in both species, with more variable labeling in KF. Rats also had consistent bilateral (predominantly contralateral) labeling in the ventrolateral PBN. The most prominent labeling was produced from injections that included the SVN, with fewer labeled neurons observed from injections in the caudal MVN and the IVN. Anterograde transport of BDA from injections into the PBN and KF nuclei of rabbits revealed prominent projections to the SVN, dorsal aspect of the rostral MVN, caudal MVN, pars beta of the LVN and IVN. These connections appear to contain a component that is reciprocal to the vestibulo-parabrachial pathway and a non-reciprocal component to regions connected with the vestibulocerebellum and vestibulo-motor reflex pathways. These connections support the concept that a synthesis of autonomic, vestibular and limbic information is an integral property of pathways related to balance control in both the brain stem and forebrain. It is suggested that these projections may contribute broadly to both performance tradeoffs in vestibular-related pathways during variations in the behavioral context and affective state and the close association between anxiety and balance function.

Glutamatergic pathways from the Kölliker–Fuse nucleus to the phrenic nucleus in the rat

After ipsilateral injections of biotinylated dextran amine (BDA) into the Kölliker–Fuse (KF) nucleus and cholera toxin B subunit (CTb) into the ventral horn in C4 to C5 segments of the spinal cord, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the rostral ventral respiratory group (rVRG) region ipsilateral to the injection sites. After ipsilateral injections of BDA into the KF and Fluoro-Gold (FG) into the ventral horn in C4 to C5 segments of the spinal cord, BDA-labeled axons were found to make asymmetrical synapses with the somata and dendrites of FG-labeled neurons within the neuropil of the rVRG region. Using retrograde tracing combined with immunohistochemistry for phosphate-activated glutaminase (PAG), we observed that as many as 72% of the rVRG neurons projecting to the PhN were immunoreactive for PAG and that approximately 62% and 75% of the KF neurons projecting respectively to the rVRG region and PhN contain PAG immunoreactivity. Using anterograde tracing combined with immunohistochemistry for vesicular glutamate transporter 2 (VGluT2), we further demonstrated that the KF axon terminals in the rVRG and PhN regions as well as the rVRG axon terminals in the PhN region contain VGluT2 immunoreactivity. The present results suggest that the glutamatergic pathways from the KF to the PhN directly and indirectly via the rVRG region may exist and underlie the inspiratory responses that are elicited by activation of the KF neurons.

Responses of primate caudal parabrachial nucleus and Kölliker-fuse nucleus neurons to whole body rotation.

The caudal aspect of the parabrachial (PBN) and Kölliker-Fuse (KF) nuclei receive vestibular nuclear and visceral afferent information and are connected reciprocally with the spinal cord, hypothalamus, amygdala, and limbic cortex. Hence, they may be important sites of vestibulo-visceral integration, particularly for the development of affective responses to gravitoinertial challenges. Extracellular recordings were made from caudal PBN cells in three alert, adult female Macaca nemestrina through an implanted chamber. Sinusoidal and position trapezoid angular whole body rotation was delivered in yaw, roll, pitch, and vertical semicircular canal planes. Sites were confirmed histologically. Units that responded during rotation were located in lateral and medial PBN and KF caudal to the trochlear nerve at sites that were confirmed anatomically to receive superior vestibular nucleus afferents. Responses to whole-body angular rotation were modeled as a sum of three signals: angular velocity, a leaky integration of angular velocity, and vertical position. All neurons displayed angular velocity and integrated angular velocity sensitivity, but only 60% of the neurons were position-sensitive. These responses to vertical rotation could display symmetric, asymmetric, or fully rectified cosinusoidal spatial tuning about a best orientation in different cells. The spatial properties of velocity and integrated velocity and position responses were independent for all position-sensitive neurons; the angular velocity and integrated angular velocity signals showed independent spatial tuning in the position-insensitive neurons. Individual units showed one of three different orientations of their excitatory axis of velocity rotation sensitivity: vertical-plane-only responses, positive elevation responses (vertical plane plus ipsilateral yaw), and negative elevation axis responses (vertical plane plus negative yaw). The interactions between the velocity and integrated velocity components also produced variations in the temporal pattern of responses as a function of rotation direction. These findings are consistent with the hypothesis that a vestibulorecipient region of the PBN and KF integrates signals from the vestibular nuclei and relay information about changes in whole-body orientation to pathways that produce homeostatic and affective responses.

DIFFERENCES BETWEEN SHR AND WKY FOLLOWING THE AIRPUFF STARTLE STIMULUS IN THE NUMBER OF FOS EXPRESSING, RVLM PROJECTING NEURONS

The neurocircuitry responsible for excessive stress-induced cardiovascular responses in genetic hypertensive rats remains elusive. Prior studies detailed a differential cardiovascular response profile to airpuff startle stimuli between Spontaneously Hypertensive (SHR) and Wistar Kyoto (WKY) rats. We recently identified strain differential Fos expression in the rostroventrolateral medulla (RVLM) and several RVLM projecting sites following airpuff startle. The current study sought to define RVLM projecting neurons that also express Fos following placement in the test chamber and administration of the airpuff startle stimulus. Unilateral iontophoretic micro-injections of fluorogold were made into the RVLM of 9–10 week old SHR and WKY rats. Two to three weeks later, animals were subjected to a series of 60 airpuff startle stimuli. Brains were double labeled for Fos and fluorogold. Single fluorogold and single Fos cells, and double labeled cells were found in the nucleus tractus solitarius (NTS), caudal ventral lateral medulla (CVLM), Kölliker fuse (KF), ventral lateral, lateral, and dorsal central gray, lateral hypothalamus (LH), and paraventricular nucleus of the hypothalamus (PVN). These data are consistent with the notion that the RVLM receives differential excitatory and/or inhibitory input from higher brain centers, perhaps contributing to differential Fos expression in the RVLM, differential autonomic responding, or both.

Integral-differential calculus computations by short-term potentiation and depression in NTS-pontine pathways

Neural integrators are a class of neural networks that afford extended temporal summation of neural activity or, equivalently, low-pass frequency filtering of neurotransmission [1]. In the respiratory system, a biphasic neural integrator in the carotid chemoreflex response is demonstrated by the classic short-term potentiation (STP) and afterdischarge of phrenic activity [2]. Recently, a new class of neural networks called neural differentiators, which afford high-pass frequency filtering of afferent inputs through short-term depression (STD) of neurotransmission, has been identified in the vagal-carotid control of the expiratory off-switch [3]. The differentiator response is characterized by: 1) a habituation-like adaptation to the vagal Hering-Breuer (HB) reflex lengthening or carotid chemore-flex shortening of expiratory duration; 2) secondary adaptive effects in the form of pontine-mediated and NMDA receptor-dependent post-stimulus rebound and recovery of these vagal or carotid [4](or hypoxia [5]) mediated responses. To elucidate the mechanism of the habituation of the HB reflex, we obtained extracellular field potentials (FPs) in the medial and commissural regions of the nucleus tractus solitarius (NTS) evoked by electrical vagal stimulation (1 min) in urethane-anesthetized, paralyzed and mechanically ventilated rats (n =17) in vivo. FPs from vagal A-fibers (stimulus current, 20–70 μA, response latency, 4–5 ms) revealed a stimulus frequency-dependent (20–80 Hz) STD similar to the synaptic accommodation observed in vitro [6] and the habituation of HB reflex in vivo [4]. FPs from C-fibers (150–350 μA, 20–45 ms) also demonstrated a frequency-dependent (5–20 Hz) STD during afferent activation but their amplitude remained depressed for >50 s post-stimulation. Microinjection of the NMDA receptor antagonist D-APV abolished the short-term memory following C-fiber activation but not the frequency-dependent STD with A- or C-fibers. The characteristics of the vagal A-and C-fiber mediated responses are reminiscent of the type I and type II NTS neurons found in vitro [7]. To elucidate the mechanism of the pontine-mediated adaptation of the HB reflex, we obtained extracellular recordings of pontine neuronal activity simultaneously with phrenic nerve recording during electrical vagal stimulation (1.5×T, 80 Hz, 1 min). Neurons with tonic and respiratory modulated activity were found within or near the parabrachialis (PB) and Kolliker-Fuse (KF) complex. Most tonic neurons in KF (n = 11) were depressed during and/or shortly after vagal stimulation that elicited the characteristic biphasic adaptation of the HB reflex in the phrenic activity, whereas most tonic neurons in medial PB (n = 22) were not affected. Activity of respiratory modulated neurons (n = 4) recorded in KF remained in phase with phrenic activity during vagal stimulation. Results showed that vagal input induced STD of tonic neurotransmission in KF. These findings support a dual-process nonassociative learning model of integral-differential calculus computations in the brain [8] through activity-dependent STP and STD of neurotransmission in primary and secondary afferent pathways.