Restricted loss of olivocochlear but not vestibular efferent neurons in the senescent gerbil (Meriones unguiculatus)

Abstract

Degeneration of hearing and vertigo are symptoms of age-related auditory and vestibular disorders reflecting multifactorial changes in the peripheral and central nervous system whose interplay remains largely unknown. Originating bilaterally in the brain stem, vestibular and auditory efferent cholinergic projections exert feedback control on the peripheral sensory organs, and modulate sensory processing. We studied age-related changes in the auditory and vestibular efferent systems by evaluating number of cholinergic efferent neurons in young adult and aged gerbils, and in cholinergic trigeminal neurons serving as a control for efferents not related to the inner ear. We observed a significant loss of olivocochlear (OC) neurons in aged compared to young adult animals, whereas the overall number of lateral superior olive (LSO) cells was not reduced in aging. Although the loss of lateral and medial olivocochlear (MOC) neurons was uniform and equal on both sides of the brain, there were frequency-related differences within the lateral olivocochlear (LOC) neurons, where the decline was larger in the medial limb of the superior olivary nucleus (high frequency representation) than in the lateral limb (middle-to-low frequency representation). In contrast, neither the number of vestibular efferent neurons, nor the population of motor trigeminal neurons were significantly reduced in the aged animals. These observations suggest differential effects of aging on the respective cholinergic efferent brainstem systems.

Keywords: aging; auditory; brainstem; cholinergic efferent systems; olivocochlear neurons; superior olivary complex; trigeminal; vestibular.

Figure 1

Distribution of cholinergic neurons in the superior olivary complex (SOC) of the gerbil (M. unguiculatus). Reference series shows ventral outlines of the brainstem (solid lines) and the subdivisions of the SOC (dotted contours) at 10 rostrocaudal (rc) positions from facial nucleus (Facial Ncl) to the ventral nucleus of the lateral lemniscus (VNLL). Rc-level 1 indicates the most caudal and rc-level 10 the most rostral position. Dots: small cholinergic neurons comprising LOC neurons. Stars: large cholinergic neurons, including MOC neurons. The density of symbols represents roughly the relative frequency of occurrence of the respective cholinergic neurons within each subdivision.

Figure 2

Triple-stained example section of the SOC region of a young adult gerbil. The section corresponds to the reference series at rc-level 6 (E). In (A,B,C) MAP2 stain, ChAT stain and CSPG stain are depicted alone with delineated SOC nuclei. Overlays of ChAT (Alexa Fluor 488, green) and MAP2 (Alexa Fluor 647, red) are shown in (D) and ChAT and CSPG (Cy 3, 570; red) in (F). ChAT positive cells were easily detected in the ChAT stain alone as well as in overlay with both overview stains (MAP and CSPG). Scale bar in (A–F): 500 µm.

Figure 3

Lipofuscin granules in medial superior olive (MSO) neurons of an aged gerbil. MSO neurons are MAP2 immunostained (Alexa Fluor 647, red). Lipofuscin granules have been excited with the DAPI excitation wavelengths and appear blue. Confocal images show a maximum projection of image stacks in (A) and a single optical image of 0.3 µm thickness in the enlargement in (B). Scale bar in (A): 50 µm and 20 µm in (B).

Figure 4

OC neurons in SOC of young adult and aged gerbils. ChAT (Alexa Fluor 488, green) and CSPG (Cy 3, red) immunolabeling is shown. Arrowheads point towards a CSPG-positive perineuronal net in (C,G). Note that OC neurons are not ensheathed in perineuronal nets. Autofluorescent lipofuscin granules appear yellow (arrowhead in D,H). LOC neurons: section at middle of SOC (rc-level 5) in young adult and aged gerbil (A,B) with enlargement of rectangular areas in medial LSO in (C,D). MOC neurons: section at rostral SOC (rc-level 9) in young adult and aged gerbil (E,F). VNTB/LNTB area within rectangle in (E,F) is enlarged in (G,H). Stars in (C,D) mark presumed MOC neurons in DPO/DLPO. The presumed MOC soma in (D) is half-filled with lipofuscin granules (yellow). Scale bar in (A,B,E,F): 200 µm; in (C,D,G,H): 50 µm.

Figure 5

Rostrocaudal distribution of olivocochlear neurons in young adult and aged gerbils. Number of LOC (upper) and MOC (lower) neurons are shown as a function of rostrocaudal location in the SOC. The slice numbers (abscissa) refer to the slices in Figure 1, and the values for young adult and aged animals are represented as stars and triangles, respectively. Values give the mean number of neurons in the respective slice of 40 µm thickness as counted in all five animals of each category.

Figure 6

Loss of olivocochlear (A,B) and LSO neurons in general (C) in aged compared to young adult animals. (A) The mean number of LOC and MOC neurons in one hemisphere is represented for young adult and aged animals. The decrease in OC numbers in aged animals relative to young adult animals is significant in LOC and MOC neurons (LOC: 24%: t = 4.436; df = 18; p < 0.001); MOC: 31%, t = 4.357; df = 18; p < 0.001). (B) LOC neuron numbers in medial and lateral part of LSO in young adult and aged animals. The aged vs. young adult decrease of LOC neurons is −36% in the medial LSO portion (t = 4.888; df = 18; p < 0.001) and −23.4% in the lateral portion (t = 1.96; df = 18; n.s.). The difference of LOC numbers in the medial LSO vs. lateral LSO is not significant neither in young adult (t = 2,608; df = 18; p = 0.0178, n.s.) nor in aged (t = 0.657, df = 18; n.s.) animals. (C) Total number of LSO neurons within an area of interest of the medial and lateral portion of the LSO for young adult and aged animals. The left pair of columns gives the counts in the medial, the right one the counts in the lateral portion of LSO. LSO neurons exhibit only a moderate loss in both subdivisions with 10% in the medial LSO (t = 1.891; df = 6; n.s.) and 13% in the lateral LSO (t = 1.471; df = 6; n.s.). Neuron numbers are given as mean ± SEM. Dark gray columns: young adult animals, light gray columns: aged animals. ***p < 0.001.

Figure 7

Cholinergic neurons of the efferent vestibular system (A–D) and of the motor trigeminal nucleus (E,F) in young adult and aged gerbils. Micrographs were taken from the same series of a pair of young adult and aged gerbils as for the OC examples in Figure 4. ChAT (Alexa Fluor 488, green) and CSPG (Cy 3, red) immunolabeling is shown. The group of vestibular efferents in young adult and aged gerbil is accentuated by rectangles in (A,B). (C,D) show enlargements of the rectangles in (A,B), respectively. The groups of ChAT-positive and CSPG-negative efferent vestibular neurons are encircled. (E,F) give an overview of the motor trigeminal nucleus. ChAT-positive neurons of the young adult gerbil (E) are ensheathed in CSPG-positive perineuronal nets (arrowhead in E) whereas most cholinergic neurons of the aged gerbil (F) show degraded perineuronal nets and a comparably low amount of lipofuscin granules (yellow). N6: abducens nucleus; g7: genu of the facial nerve. Scale bar in (A,B,E,F): 200 µm; in (C,D): 50 µm.

Figure 8

Comparison of age related neuron loss in three cholinergic efferent systems. There is considerable loss in the olivocochlear efferent system (left) of 26% (t = 4,984; df = 18; p < 0.0001), whereas the vestibular efferent system (middle) is not affected by age and the trigeminal motor system (right) shows only a moderate neuronal loss of 12% (t = 2.155; df = 18; n.s.). Neuron numbers are given as mean ± SEM. Dark gray columns: young adult animals, light gray columns: aged animals. **** p < 0.0001.