Horst Herbert

Cholinergic neurons in the pedunculopontine tegmental nucleus are involved in the mediation of prepulse inhibition of the acoustic startle response in the rat

The amplitude of the acoustic startle response (ASR) is markedly reduced when the startle eliciting pulse is preceded by a weak, non-startling stimulus at an appropriate lead time, usually about 100 ms. This phenomenon is termed prepulse inhibition (PPI) and has received considerable attention in recent years as a model of sensorimotor gating. We report here on experiments which were undertaken in order to investigate some of the neural mechanisms of PPI. We focused on the characterization of the cholinergic innervation of the pontine reticular nucleus, caudal part (PnC), an obligatory relay station in the primary startle pathway. The combination of retrograde tracing with choline acetyltransferase-immunocytochemistry revealed a cholinergic projection from the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) to the PnC. Extracellular recording from single PnC units, combined with microiontophoretic application of the acetylcholine (ACh) agonists acetyl-β-methylcholine (AMCH) and carbachol revealed that ACh inhibits the majority of acoustically responsive PnC neurons. Neurotoxic lesions of the cholinergic neurons of the PPTg significantly reduced PPI without affecting the ASR amplitude in the absence of prepulses. No effect on long-term habituation of the ASR was observed. The present data indicate that the pathway mediating PPI impinges upon the primary acoustic startle circuit through an inhibitory cholinergic projection from the PPTg to the PnC.

Distribution and origin of noradrenergic and serotonergic fibers in the cochlear nucleus and inferior colliculus of the rat

We examined the monoaminergic innervation of the rat cochlear nucleus (CN) and the inferior colliculus (IC) by using retrograde transport of the fluorescent dye Fluoro-Gold combined with immunohistochemistry. We used antisera against the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), and one against the transmitter serotonin (5-HT). Each substance revealed a distinct pattern of immunoreactive staining in the CN and the IC. In the CN, DBH-immunoreactive (-ir) fibers were present in all subnuclei. The molecular layer of the dorsal CN and the granular layer of the ventral CN, however, were largely devoid of DBH-ir fibers. In contrast, 5-HT-ir fibers were abundant in the molecular layer and the granular cell layer of the CN. In the dorsal CN and the postero- and anteroventral CN, however, this innervation was less dense and evenly distributed across subnuclei. In the IC, the DBH-ir fibers were slightly more numerous in layer 2 of the dorsal cortex than in other subnuclei, while the layer 1 of both the dorsal and the external cortex contained only a few fibers. In contrast, the 5-HT-ir fibers formed a dense network in both the dorsal and external cortices of the IC, while they were less abundant in the remaining subnuclei. PNMT-ir fibers were not found in any of the auditory brainstem nuclei. Following Fluoro-Gold injections into the CN or IC, retrogradely labeled DBH-ir neurons were found in the A6 noradrenergic cell group (locus coeruleus). The CN received additional projections from the A5 noradrenergic cell group, as well as sparse projections from the A4 and A7 cell groups. The serotonergic innervation of the CN and IC originated largely in the B7 serotonergic cell group (dorsal raphe nucleus). Serotonergic neurons in other groups of the raphe nuclei were only occasionally labeled. Our data indicate that both noradrenaline and serotonin may play a role in central auditory processing. Their differential distribution in the IC and CN subnuclei suggests that these transmitter systems might influence different functional circuits.

The Kölliker‐Fuse nucleus gates the postinspiratory phase of the respiratory cycle to control inspiratory off‐switch and upper airway resistance in rat

Lesion or pharmacological manipulation of the dorsolateral pons can transform the breathing pattern to apneusis (pathological prolonged inspiration). Apneusis reflects a disturbed inspiratory off‐switch mechanism (IOS) leading to a delayed phase transition from inspiration to expiration. Under intact conditions the IOS is irreversibly mediated via activation of postinspiratory (PI) neurons within the respiratory network. In parallel, populations of laryngeal premotoneurons manifest the IOS by a brief glottal constriction during the PI phase. We investigated effects of pontine excitation (glutamate injection) or temporary lesion after injection of a GABA‐receptor agonist (isoguvacine) on the strength of PI‐pool activity determined from respiratory motor outputs or kinesiological measurements of laryngeal resistance in a perfused brainstem preparation. Glutamate microinjections into distinct parts of the pontine Kölliker‐Fuse nucleus (KF) evoked a tonic excitation of PI‐motor activity or sustained laryngeal constriction accompanied by prolongation of the expiratory phase. Subsequent isoguvacine microinjections at the same loci abolished PI‐motor or laryngeal constrictor activity, triggered apneusis and established a variable and decreased breathing frequency. In summary, we revealed that excitation or inhibition of defined areas within the KF activated and blocked PI activity and, consequently, IOS. Therefore, we conclude, first, that descending KF inputs are essential to gate PI activity required for a proper pattern formation and phase control within the respiratory network, at least during absence of pulmonary stretch receptor activity and, secondly, that the KF contains large numbers of laryngeal PI premotor neurons that might have a key role in the regulation of upper airway resistance during reflex control and vocalization.

Spinal afferents to functionally distinct periaqueductal gray columns in the rat: an anterograde and retrograde tracing study.

The segmental and laminar organization of spinal projections to the functionally distinct ventrolateral (vlPAG) and lateral periaqueductal gray (lPAG) columns was examined by using retrograde and anterograde tracing techniques. It was found 1) that spinal input to both vlPAG and lPAG columns arose predominantly from neurons in the upper cervical (C1–4) and sacral spinal cord; 2) that there was a topographical separation of vl‐PAG projecting and lPAG‐projecting neurons within the upper cervical spinal cord; but 3) that below spinal segment C4, vlPAG‐projecting and lPAG‐projecting spinal neurons were similarly distributed, predominantly within contralateral lamina I, the nucleus of the dorsolateral fasciculus (the lateral spinal nucleus) and the lateral (reticular) part of lamina V. Consistent with the retrograde results, the greatest density of anterograde label, within both the vlPAG and lPAG, was found after tracer injections made either in the superficial or deep dorsal horn of the upper cervical spinal cord. Tracer injections made within the thoraco‐lumbar spinal cord revealed that the vlPAG column received a convergent input from both the superficial and deep dorsal horn. However, thoraco‐lumbar input to the lPAG was found to arise uniquely from the superficial dorsal horn; whereas the deep dorsal horn was found to innervate the “juxta‐aqueductal” PAG region rather than projecting to the IPAG.

Auditory projections from the cochlear nucleus to pontine and mesencephalic reticular nuclei in the rat

We investigated projections from the cochlear nucleus in the rat using the anterograde tracer Phaseolus vulgaris-leucoagglutinin. We focused on nuclei in the brainstem which are not considered to be part of the classical auditory pathway. In addition to labeling in auditory nuclei, we found presumed terminal fibers in 4 pontine and mesencephalic areas: (1) the pontine nucleus (PN), which receives bilateral projections from the antero- and posteroventral cochlear nuclei; (2) the ventrolateral tegmental nucleus (VLTg), which receives a contralateral projection from the rostral portion of the anteroventral cochlear nucleus; (3) the caudal pontine reticular nucleus (PnC), which receives bilateral input originating predominantly in the dorsal cochlear nucleus; and (4) the lateral paragigantocellular nucleus (LPGi), which receives projections from all subdivisions of the cochlear nuclei. In the VLTg and PnC, anterogradely labeled varicose axons were often found in close apposition to the primary dendrites and somata of large reticular neurons. Injections of the retrograde fluorescent tracer Fluoro-Gold into the VLTg demonstrated that the neurons of origin are mainly located contralaterally in the rostral anteroventral cochlear nucleus and in the cochlear root nucleus. The relevance of these auditory projections for short-latency audio-motor behaviors and acoustically elicited autonomic responses is discussed.

The Kolliker-Fuse nucleus mediates the trigeminally induced apnoea in the rat

The present study examined whether the Kölliker-Fuse nucleus (KF) plays a role in mediating the trigeminally induced apnoea which occurs after noxious perturbation of the nasal mucosa. We stimulated the ethmoidal nerve (EN5) electrically and recorded respiratory responses before and after injections of the calcium channel blocker CoCl2 into the KF. Unilateral EN5 stimulations resulted in an apnoea or in a reduction of respiratory frequency and tidal volume. EN5 stimulations immediately after ipsilateral CoCl2 injections into the caudal KF caused only minor respiratory suppression, indicating a blockade of synaptic transmission. Recovery of the respiratory responses was observed 15-120 min after the CoCl2 injection. Our data strongly suggest that the caudal KF is an obligatory relay site for trigeminally induced apnoea.

NMDA and GABAA receptors in the rat Kölliker-Fuse area control cardiorespiratory responses evoked by trigeminal ethmoidal nerve stimulation

1 Electrical stimulation (10 s) of the ethmoidal nerve (EN5) evokes the nasotrigeminal reflex responses, including apnoea, bradycardia and rise in arterial blood pressure. In the present study, we examined the involvement of N‐methyl‐D‐aspartate (NMDA), AMPA/kainate, (γ‐aminobutyric acidA (GABAA) and glycine receptors in the Kölliker‐Fuse (KF) nucleus in the mediation of the nasotrigeminal reflex responses. 2 Unilateral injections (n= 6) of 50‐100 nl of the NMDA receptor antagonist AP5 into the KF area led to a significant blockade of the EN5‐evoked respiratory depression and bradycardia. Injections placed into the midlevel of the KF area were most effective (80‐90 % blockade). The rise in arterial blood pressure remained unaffected. 3 Unilateral injections (n= 6) of the AMPA/kainate receptor antagonist CNQX into the KF area failed to block EN5‐evoked autonomic responses significantly. 4 Unilateral injections (n= 5) of the GABAA receptor antagonist bicuculline enhanced the EN5‐evoked respiratory depression and bradycardia. The effect persisted for up to 30 s after stimulation. Bicuculline injections into the midlevel of the KF area were most effective. The increase in arterial blood pressure remained unaffected. 5 Unilateral injections (n= 5) of the glycine receptor antagonist strychnine into the KF area did not produce any significant effects on EN5‐evoked autonomic responses. 6 Our results suggest that the KF area represents a mandatory relay for the nasotrigeminally induced apnoea and bradycardia which are predominantly mediated by NMDA receptors in the KF. Furthermore, it appears that KF neurons are under a potent GABAergic inhibitory control. The EN5‐evoked rise in arterial blood pressure was not altered by any of the drugs and, therefore, appears not to be mediated via the KF.

Fos expression in the rat parabrachial and Kölliker-Fuse nuclei after electrical stimulation of the trigeminal ethmoidal nerve and water stimulation of the nasal mucosa

Abstract The present study examined the location of neurons in the lateral parabrachial nucleus (PBL), Kölliker-Fuse nucleus (KF), nucleus of the solitary tract (NTS), spinal trigeminal nucleus (Sp5C) and upper cervical cord possibly involved in the transmission of autonomic responses (apnea, bradycardia and rise in arterial blood pressure) elicited by nasotrigeminal stimulation in the rat. To identify these neurons we employed immunocytochemical detection of the transcription factor Fos. To induce the expression of Fos protein, two kinds of stimuli and experimental controls were performed in chloralose/urethane-anesthetized animals: (i) electrical stimulation of the trigeminal ethmoidal nerve (EN5) and, as sham controls, dissection of the EN5 without electrical stimulation, (ii) stimulation of the nasal mucosa with water and, as control experiments, no stimulation. Both forms of stimulation lead to a consistent pattern of Fos-positive neurons in the PBL and KF. Differences could be observed rostrally in the PBL and KF, where significantly higher numbers of Fos-positive neurons were present after EN5 versus water stimulation. The EN5-stimulated group had a significantly higher number of Fos-immunoreactive neurons in the KF than the sham controls, especially in the midlevel region. In the PBL significant differences in the numbers of activated cells could be observed between EN5-stimulated versus sham controls. In the water-stimulated rats compared with the anesthesia controls, a significantly higher number of Fos-immunoreactive neurons was always observed at all rostrocaudal levels in the KF and in the midlevel PBL. Electrical EN5 stimulation induced Fos expression in the Sp5C at the level of the area postrema and caudally in the upper cervical cord. In contrast, after water stimulation Fos-positive neurons were exclusively found in the Sp5C. In addition, all forms of stimuli and controls induced strong expression of Fos in the medial and commissural NTS. Linear correlations were found between the numbers of Fos-immunoreactive neurons in the Sp5C versus the KF and the NTS versus the PBL. The activated neurons may belong to two functionally discrete pathways: the nasotrigeminal reflex circuit, which is activated by nasal sensory afferents running through the EN5 via the Sp5C to the KF, and a pathway activated most likely by baro- and chemoreceptor afferents running through the NTS to the PBL. Our results indicate that the PB/KF plays a pivotal role in the mediation and maintenance of the autonomic responses induced by the nasotrigeminal reflex.

Development of adaptive behaviour of the respiratory network: implications for the pontine Kolliker-Fuse nucleus.

Breathing is constantly modulated by afferent sensory inputs in order to adapt to changes in behaviour and environment. The pontine respiratory group, in particular the Kolliker-Fuse nucleus, might be a key structure for adaptive behaviours of the respiratory network. Here, we review the anatomical connectivity of the Kolliker-Fuse nucleus with primary sensory structures and with the medullary respiratory centres and focus on the importance of pontine and medullary postinspiratory neurones in the mediation of respiratory reflexes. Furthermore, we will summarise recent findings from our group regarding ontogenetic changes of respiratory reflexes (e.g., the diving response) and provide evidence that immaturity of the Kolliker-Fuse nucleus might account in neonates for a lack of plasticity in sensory evoked modulations of respiratory activity. We propose that a subpopulation of neurones within the Kolliker-Fuse nucleus represent command neurones for sensory processing which are capable of initiating adaptive behaviour in the respiratory network. Recent data from our laboratory suggest that these command neurones undergo substantial postnatal maturation.

GABA, GABA transporters, GABAAreceptor subunits, and GAD mRNAs in the rat parabrachial and Kölliker-Fuse nuclei

In the present study, we investigated the key molecules that determine γ‐aminobutyric acid (GABA)ergic signal transduction in the parabrachial/Kölliker‐Fuse complex (PB/KF) by means of immunocytochemistry and in situ hybridization. Our data demonstrate a dense plexus of GABA‐immunoreactive (‐ir) varicosities throughout the nuclei of the PB and the KF. The number of neurons expressing GAD65 or GAD67 mRNA was fairly low in the PB, whereas caudally in the KF an accumulation of GAD‐expressing neurons was observed. The GABA transporter‐3 (GAT‐3) was detected in all parts of the PB/KF, whereas immunolabeling for GAT1 was not observed. All nuclei of the PB and the KF exhibited immunoreactivity for the γ2‐, α2‐, and α3‐subunits of the GABAA receptor. γ2‐ir was strong and similar in all PB/KF nuclei. In contrast, α2‐labeling was particularly intense in the superior lateral PB, and α3‐labeling was most prominent in the external lateral and external medial PB, compared with the remaining nuclei. With respect to the subcellular localization, we found γ2‐ir in cell bodies and higher order dendrites, whereas α2‐ and α3‐ir was predominantly found in cell bodies. Immunolabeling for the β2/3‐ and the α1‐subunit was seen in cell bodies and presumed dendritic profiles. The staining intensity was strongest in the dorsal lateral PB. Most importantly, the external lateral PB and the waist area were totally devoid of β2/3‐ and α1‐ir. Our data suggest that neural processing in the PB/KF is under a strong GABAergic inhibition that is apparently mediated by different types of GABAA receptors in functionally different pathways through the PB/KF. J. Comp. Neurol. 400:229–243, 1998.