Dolores E. López

A Primary Acoustic Startle Pathway: Obligatory Role of Cochlear Root Neurons and the Nucleus Reticularis Pontis Caudalis

Davis et al. (1982) proposed a primary acoustic startle circuit in rats consisting of the auditory nerve, posteroventral cochlear nucleus, an area near the ventrolateral lemniscus (VLL), nucleus reticularis pontis caudalis (PnC), and spinal motoneurons. Using fiber-sparing lesions, the present study reevaluated these and other structures together with the role of neurons embedded in the auditory nerve [cochlear root neurons (CRNs)], recently hypothesized to be involved in acoustic startle. Small electrolytic lesions of the VLL or ventrolateral tegmental nucleus (VLTg) failed to eliminate startle. Large electrolytic lesions including the rostral ventral nucleus of the trapezoid body (rVNTB) and ventrolateral parts of PnC or lesions of the entire PnC blocked startle. However, small NMDA-induced lesions of the rVNTB failed to block startle, making it unlikely that the rVNTB itself is part of the startle pathway. In contrast, NMDA lesions of the full extension of the ventrolateral part of the PnC blocked startle completely, suggesting that the ventrolateral part of the PnC is critically involved. Bilateral kainic acid lesions of CRNs also blocked the startle reflex completely, providing the first direct evidence for an involvement of CRNs in startle. This blockade probably was not caused by damage to the auditory nerve, because the lesioned animals showed intact compound action potentials recorded from the ventral cochlear nucleus. Hence, a primary acoustic startle pathway may involve three synapses onto (1) CRNs, (2) neurons in PnC, and (3) spinal motoneurons.

Morphology of cochlear root neurons in the rat

SummaryThe morphology of large neurons in the cochlear nerve root of albino rat has been studied with a variety of techniques including Nissl and cell-myelin staining, Golgi impregnation, horseradish peroxidase back-filling of severed axons, transmission electron microscopy, and morphometry. The cells, called root neurons, resemble the globular cells of the ventral cochlear nucleus in having an oval cell body, an eccentric nucleus, an axon that projects centrally via the trapezoid body, and in receiving many primary-like axosomatic boutons. The root neurons, however, are larger than globular cells, and they have at least two types of dendrites oriented, respectively, parallel and across the cochlear nerve fibres. The soma, moreover, has less finely dispersed Nissl material, is less completely covered with terminals, and receives a smaller proportion of presumably inhibitory synapses. So far, this particular type of neuron has been observed only in rat and mouse.

Projections of Cochlear Root Neurons, Sentinels of the Rat Auditory Pathway

In certain rodents, the root of the cochlear nerve contains a population of large neurons, known as cochlear root neurons (CRNs), an essential element of the primary acoustic startle pathway. To characterize the projections of the CRNs, we made stereotaxically guided, iontophoretic injections of biotinylated tracers into the cochlear nerve root of albino rats.

GABA-like and glycine-like immunoreactivities of the cochlear root nucleus in rat

SummaryThe cochlear root nucleus is part of the cochlear nuclear complex in small rodents. Its cells, the large root neurons, have a superficial resemblance to the globular neurons of the ventral cochlear nucleus. It has been a matter of debate, therefore, whether the root neurons and globular neurons represent the same or different types of cell. In the present study the two cell types with adjacent neuropil structures were compared by light microscopic, postembedding immunocytochemistry. Pairs of 0.5 μm sections of resin-embedded, glutaraldehyde-fixed material were treated with purified antisera raised against GABA- and glycine-glutaraldehyde-protein conjugates, respectively. Both types of cell were found to be immunonegative. Striking differences, however, occurred in what was interpreted as afferent nerve terminals. The globular cells appeared to receive numerous afferents with GABA- or glycine-like immunoreactivity on their somata. Immunoreactive terminals on the root neurons, on the contrary, were mostly GABA-positive and located on the dendrites. Although of unknown origin, the immunoreactive afferents were clearly different from the primary fibres as demonstrated both by the immunonegativity of the latter and by the different size and distribution of the terminals labelled anterogradely after horseradish peroxidase injections into the spiral ganglion.

THE COCHLEAR ROOT NEURONS IN THE RAT, MOUSE AND GERBIL

The auditory portion of the eighth cranial nerve (the cochlear nerve) contains a neuronal population which has been well documented only in a few species of small rodents belonging to the Muridae family.1

Cholinergic input from the ventral nucleus of the trapezoid body to cochlear root neurons in rats

Brain stem pathways are essential for the modulation of the acoustic startle reflex by sounds; nevertheless, the neural circuits that convey fast auditory information to the primary acoustic startle circuit are still unclear. In the rat, cochlear root neurons (CRNs) comprise the first component of the primary acoustic startle circuit and are critical in the initiation and full expression of the acoustic startle reflex. To determine whether CRNs receive auditory descending inputs, we developed tract‐tracing studies combined with immunohistochemistry, electron microscopy, morphometry, and confocal microscopy. Either FluoroGold or biotinylated dextran amine (BDA) injections in CRNs showed retrogradely labeled neurons in the ventral nucleus of the trapezoid body (VNTB). We verified the projection to CRNs by injecting BDA into the VNTB. Our results showed that neurons from VNTB project bilaterally and directly to CRNs, giving off numerous endings onto cell bodies and preferentially dendrites of CRNs. Electron microscopy analysis of labeled VNTB terminals demonstrated that they made multiple symmetric synapses and contained small round vesicles. Colocalization of the vesicular acetylcholine transporter and fluorescein dextran after injection in the VNTB indicated that these terminals use acetylcholine as neurotransmitter. We also revealed that the inferior colliculus, an important nucleus mediating the auditory prepulse inhibition, projects to VNTB neurons that innervate CRNs. Our data show a novel and short descending auditory pathway from the VNTB to the first nucleus of the primary acoustic startle circuit that might play an important role in the auditory prepulse inhibition of the startle reflex elicited by sounds. J. Comp. Neurol. 505:452–468, 2008.

Electrophysiological responses of cochlear root neurons.

Cochlear root neurons (CRNs) are second-order neurons interspersed among the fibers of the cochlear nerve in certain rodents. They project, among other nuclei, mainly to the pontine reticular nucleus, and participate in the acoustic startle response (ASR), a short-latency motor reflex initiated by sudden intense sounds. The sound-evoked activity of CRNs has not previously been described. Here we describe extracellular responses of CRNs located in the infranuclear portion of the cochlear nerve root. CRNs exhibited secure responses to tone bursts, with first-spike latencies of approximately 2.2 ms. The characteristic frequencies of the recorded CRNs were about 30 kHz, and the best-characterized CRN had a threshold of 10 dB sound pressure level and sharpness of tuning similar to that of cochlear nerve fibers. The peristimulus time histograms were primary-like with notch. The observed response properties were consistent with the suggestion that CRNs provide the short-latency acoustic input to the reticular formation that leads to an ASR.

Origin and function of short-latency inputs to the neural substrates underlying the acoustic startle reflex

The acoustic startle reflex (ASR) is a survival mechanism of alarm, which rapidly alerts the organism to a sudden loud auditory stimulus. In rats, the primary ASR circuit encompasses three serially connected structures: cochlear root neurons (CRNs), neurons in the caudal pontine reticular nucleus (PnC), and motoneurons in the medulla and spinal cord. It is well-established that both CRNs and PnC neurons receive short-latency auditory inputs to mediate the ASR. Here, we investigated the anatomical origin and functional role of these inputs using a multidisciplinary approach that combines morphological, electrophysiological and behavioral techniques. Anterograde tracer injections into the cochlea suggest that CRNs somata and dendrites receive inputs depending, respectively, on their basal or apical cochlear origin. Confocal colocalization experiments demonstrated that these cochlear inputs are immunopositive for the vesicular glutamate transporter 1 (VGLUT1). Using extracellular recordings in vivo followed by subsequent tracer injections, we investigated the response of PnC neurons after contra-, ipsi-, and bilateral acoustic stimulation and identified the source of their auditory afferents. Our results showed that the binaural firing rate of PnC neurons was higher than the monaural, exhibiting higher spike discharges with contralateral than ipsilateral acoustic stimulations. Our histological analysis confirmed the CRNs as the principal source of short-latency acoustic inputs, and indicated that other areas of the cochlear nucleus complex are not likely to innervate PnC. Behaviorally, we observed a strong reduction of ASR amplitude in monaural earplugged rats that corresponds with the binaural summation process shown in our electrophysiological findings. Our study contributes to understand better the role of neuronal mechanisms in auditory alerting behaviors and provides strong evidence that the CRNs-PnC pathway mediates fast neurotransmission and binaural summation of the ASR.

Anatomy of olivocochlear neurons in the hamster studied with FluoroGold.

The golden hamster (Mesocricetus auratus) is often used in auditory research, but little is known about the anatomical organization of its olivocochlear (OC) neurons, the source of the efferent innervation of the organ of Corti. Accordingly, we labeled the OC neurons projecting to one cochlea by means of retrograde axonal transport of FluoroGold. In four animals, all labeled OC neurons were counted and digital images of the labeling were captured and analyzed morphometrically. In one case, a 3D computer reconstruction of the bilateral distribution of OC neurons was made. The largest group of OC neurons was comprised by small, intrinsic lateral OC neurons within the ipsilateral lateral superior olivary nucleus (LSO), almost all of which (97%) were located ipsilaterally. The second largest group consisted of medial OC neurons in the ventral nucleus of the trapezoid body, 75% of which were located contralaterally. The smallest group consisted of shell neurons surrounding the LSO, 80% of which projected ipsilaterally. These three types of neurons are generally similar in morphology and distribution to those previously described in the rat and the chinchilla. However, there were several unique findings, including the fact that the hamster possesses the smallest total number of OC neurons (mean 341) of any rodent yet studied.

The selective neurotoxin DSP-4 impairs the noradrenergic projections from the locus coeruleus to the inferior colliculus in rats

The inferior colliculus (IC) and the locus coeruleus (LC) are two midbrain nuclei that integrate multimodal information and play a major role in novelty detection to elicit an orienting response. Despite the reciprocal connections between these two structures, the projection pattern and target areas of the LC within the subdivisions of the rat IC are still unknown. Here, we used tract-tracing approaches combined with immunohistochemistry, densitometry, and confocal microscopy (CM) analysis to describe a projection from the LC to the IC. Biotinylated dextran amine (BDA) injections into the LC showed that the LC-IC projection is mainly ipsilateral (90%) and reaches, to a major extent, the dorsal and lateral part of the IC and the intercollicular commissure. Additionally, some LC fibers extend into the central nucleus of the IC. The neurochemical nature of this projection is noradrenergic, given that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) colocalize with the BDA-labeled fibers from the LC. To determine the total field of the LC innervations in the IC, we destroyed the LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then studied the distribution and density of TH- and DBH-immunolabeled axons in the IC. In the DSP-4 treated animals, the number of axonal fibers immunolabeled for TH and DBH were deeply decreased throughout the entire rostrocaudal extent of the IC and its subdivisions compared to controls. Our densitometry results showed that the IC receives up to 97% of its noradrenergic innervations from the LC neurons and only 3% from non-coeruleus neurons. Our results also indicate that TH immunoreactivity in the IC was less impaired than the immunoreactivity for DBH after DSP-4 administration. This is consistent with the existence of an important dopaminergic projection from the substantia nigra to the IC. In conclusion, our study demonstrates and quantifies the noradrenergic projection from the LC to the IC and its subdivisions. The re-examination of the TH and DBH immunoreactivity after DSP-4 treatment provides insights into the source, extent, and topographic distribution of the LC efferent network in the IC, and hence, contributes to our understanding of the role of the noradrenaline (NA) system in auditory processing.