Effects of brainstem lesions on amino acid levels in the rat cochlear nucleus

Abstract

There is evidence for glutamate, γ-amino butyric acid (GABA), and glycine as neurotransmitters of centrifugal pathways to the cochlear nucleus, but the quantitative extent of their contributions to amino acid neurotransmission in cochlear nucleus regions has not been known. We used microdissection of freeze-dried tissue sections of rat cochlear nucleus, with mapping of sample locations, combined with a high performance liquid chromatography (HPLC) assay, to measure amino acid levels in cochlear nucleus subregions of rats with unilateral lesions of centrifugal pathways to the cochlear nucleus. In rats with lesions transecting all or almost all pathways to the cochlear nucleus from brain stem regions, GABA, aspartate, and glutamate levels were reduced, compared to contralateral values, in almost all ipsilateral cochlear nucleus regions. The largest reductions, in dorsal (DCN), anteroventral (AVCN), and posteroventral (PVCN) cochlear nucleus regions, approached 50 % for GABA, 40 % for aspartate, and 30 % for glutamate. In contrast, glutamine and taurine levels were typically higher in lesioned-side cochlear nucleus regions than contralaterally. Effects on glycine levels were mixed but usually included increased lesioned-side values compared to contralateral, probably reflecting a balance between increases during protein breakdown and decreases of free glycine in transected pathways. More limited lesions transecting just dorsal pathways showed much less effect on amino acid levels. Lesion of the ipsilateral trapezoid body connection plus ipsilateral superior olivary nuclei resulted in decreases of GABA, aspartate, and glutamate levels especially in ventral cochlear nucleus regions. No clear contralateral effects of this lesion could be shown. The results most strongly support centrifugal GABAergic pathways to the cochlear nucleus, providing almost half of GABAergic neurotransmission in most regions. Our results support and extend previously published measurements of lesion effects on GABA uptake and release in cochlear nucleus subdivisions.

1. Introduction

Amino acids include some of the major neurotransmitters of the central nervous system, including the central auditory system. There is evidence for glutamate as neurotransmitter of major excitatory pathways and γ-amino butyric acid (GABA) and glycine as neurotransmitters of major inhibitory pathways. In the first brain center of the auditory system, the cochlear nucleus, there is considerable evidence for glutamate as neurotransmitter of the auditory nerve innervation, carrying information about sounds from the auditory portion of the inner ear, the cochlea (Wenthold, 1978, 1985; Caspary et al., 1985; Martin, 1985; Potashner et al., 1985b; Juiz et al., 1993; Wenthold et al., 1993; Zhou et al., 2007; Godfrey et al, 2014, 2015). In addition, there is evidence for glutamate as neurotransmitter of the most prevalent cochlear nucleus interneurons, the granule cells (Oliver et al., 1983; Potashner et al., 1985b; Juiz et al., 1993; Wenthold et al., 1993; Osen et al., 1995; Waller et al., 1996; Godfrey et al., 1997; Chen et al., 1999), and of many of the neurons with somata in the cochlear nucleus that project to higher auditory centers (Godfrey et al., 1988; Caspary and Faingold, 1989; Suneja et al., 1995). There is evidence for GABA and glycine as neurotransmitters of interneurons within the cochlear nucleus (Mugnaini, 1985; Moore, 1988; Osen et al., 1990; Kolston et al., 1992; Altschuler et al., 1993; Godfrey et al., 1988, 1997; Oertel and Wickesberg, 1993; Saint Marie et al., 1993; Juiz et al., 1996; Gates et al., 1996; Golding and Oertel, 1996; Doucet et al., 1999; Apostolides and Trussell, 2013), and there is evidence for glycine as a neurotransmitter of cochlear nucleus innervation deriving from the contralateral cochlear nucleus (Wenthold, 1987; Benson and Potashner, 1990; Kolston et al., 1992; Potashner et al., 1993; Saint Marie et al., 1993). Besides the contralateral cochlear nucleus (Cant and Gaston, 1982; Shore et al., 1992), the cochlear nucleus also receives many centrifugal pathways deriving from a wide variety of locations including the superior olivary complex (Rasmussen, 1967; Elverland, 1977; Shore et al., 1991; Warr and Beck, 1996; Spangler and Warr, 1991), nuclei of the lateral lemniscus (Kane and Finn, 1977; Kane and Conlee, 1979), inferior colliculus (Rasmussen, 1967; Lorente de Nó, 1981; Shore et al., 1991; Saldaña, 1993; Milinkeviciute et al., 2017), auditory cortex (Weedman and Ryugo, 1996), spinal trigeminal nucleus (Itoh et al., 1987; Young et al., 1997; Haenggeli et al., 2005), cuneate nucleus (Itoh et al., 1987; Weinberg and Rustioni, 1987; Wright and Ryugo, 1996; Young et al., 1997), cerebellum (Rasmussen, 1967; Rossi et al., 1967; Gacek, 1973), vestibular system (Burian and Gstoettner, 1988; Bukowska, 2002), lateral reticular nucleus (Zhan and Ryugo, 2007), pontine nuclei (Ohlrogge et al., 2001), and pedunculopontine and laterodorsal tegmental nuclei (Mellott et al., 2011). Evidence suggests considerable convergence of a variety of projections to cochlear nucleus granular regions (Ryugo et al., 2003). Amino acid neurotransmitters have been implicated in some of these centrifugal pathways to the cochlear nucleus. There is considerable evidence for glycine and GABA as neurotransmitters of pathways from the superior olive to the cochlear nucleus (Potashner et al., 1993; Saint Marie et al., 1993). There is evidence for glutamate as a neurotransmitter of cochlear nucleus innervation from the cuneate nucleus (Wright and Ryugo, 1996; Zeng et al., 2011, 2012) and spinal trigeminal nucleus (Zhou et al., 2007; Zeng et al., 2011, 2012). There is some evidence for glutamate as a neurotransmitter of innervation of the DCN deriving from the inferior colliculus (Milinkeviciute et al., 2017), but there is also evidence against this possibility (Bergman et al., 1989).

Although there are evidences for involvement of amino acids as neurotransmitters in centrifugal pathways to the cochlear nucleus, the relative contributions of these pathways to amino acid neurotransmission in cochlear nucleus subregions have needed further study beyond the available information (Fisher and Davies, 1973; Davies, 1981; Godfrey et al., 1988; Potashner et al., 1985a, 1993). One approach toward providing this information is to measure the levels of the amino acid neurotransmitters in cochlear nucleus subregions after transections of centrifugal pathways for comparison to control values. We previously measured the effects of transections of centrifugal pathways to the rat cochlear nucleus on its cholinergic innervation by a microdissection and quantitative assay approach (Godfrey et al., 1983, 1985, 1987a,b). In the present study, sections from some of the same rats were microdissected for measurement of amino acid levels. Levels in the lesioned-side cochlear nucleus were compared to contralateral values as well as previously reported values for control rats (Godfrey et al., 2000).

The amino acids measured included glutamate, the major excitatory neurotransmitter of the central nervous system; aspartate, closely related metabolically to glutamate and possibly also a neurotransmitter; glutamine, a major precursor for glutamate synthesis in the glutamine cycle (Shank and Aprison, 1981; Dingledine and McBain, 1994); GABA and glycine, major inhibitory neurotransmitters in the central nervous system, which are sometimes co-localized (Osen et al., 1990; Altschuler et al., 1993; Jonas et al., 1998); and taurine, which is sometimes colocalized with GABA (Ottersen et al., 1988) and can act as an agonist at GABA and glycine receptors (Albrecht and Schousboe, 2005; Song et al., 2012). There is evidence that both taurine and glutamine tend to be associated with glial cells more than neurons (Patel and Hunt, 1985; Ottersen et al., 1992; Hassel et al., 1995). Arginine was also included in our analysis as an amino acid less closely related to neurotransmission, although it can be metabolized toward synthesis of glutamate (Lehninger, 1975) and nitric oxide (He and Wu, 2020). Unlike the other amino acids included in this study, it has as high values in fiber tracts as in neuropil regions on a dry weight basis, higher on a lipid-free dry weight basis (Godfrey et al., 2000).

A preliminary report of some of the results of this study has been presented (Shannon-Hartman et a., 1993).

2. Methods

2.1. Preparation of sections

This study used sections from 8 rats that were prepared for previous studies on the effects of brainstem lesions on the cholinergic system in the rat cochlear nucleus (Godfrey et al., 1983, 1985, 1987a, b). The freeze-dried sections were stored under vacuum at −20 °C in vacuum tubes, under which conditions amino acid levels are stable for many years (Godfrey et al., 2000).

In order to obtain the sections in the original experiments, the rats were decapitated and the brain portions containing the cochlear nuclei isolated and frozen within 30 minutes or less in Freon (‘Freeze-it’) cooled to its freezing point (−130 °C) with liquid nitrogen. Frozen transverse sections through the rostral-caudal extent of the cochlear nuclei were cut at 20 μm thickness in a cryostat maintained at −20 °C. Every third or fourth of these sections were freeze-dried (Lowry and Passonneau, 1972), whereas alternate ones were melted onto glass slides for staining of Nissl substance with thionin or for acetylcholinesterase (AChE) activity (Karnovsky and Roots, 1964; El-Badawi and Schenk, 1967).

2.2. Microdissection of samples for assay

In a room with temperature maintained close to 20 °C and relative humidity below 50 %, freeze-dried sections chosen at intervals through the major cochlear nucleus regions were microdissected, using hand-made razor-blade knives and hairpoints (Lowry and Passonneau, 1972), into samples for assay on the plexiglas stage (precleaned with absolute alcohol) of a Wild dissecting microscope, with a drawing tube attachment, at 25 times magnification. The drawing tube was utilized to compare freeze-dried sections with adjacent or nearby Nissl- or AChE-stained sections to confirm regional boundaries and to record the locations of all cuts made. Once the cochlear nucleus portion of a section was completely dissected into samples, the recordings of all the cuts resulted in a map showing the location of each sample and its cut borders (Godfrey and Matschinsky, 1976). In order to minimize any possibility that differences between lesioned-side samples and contralateral samples resulted from differences across assays, samples from comparable locations of both lesioned-side and contralateral cochlear nucleus were always included in the same dissections and assays.

Dry weights of the cut samples were measured on quartz-fiber microbalances (Lowry and Passonneau, 1972). They ranged from 0.06 to 2 μg, averaging about 0.4 μg. The samples were loaded into 300 μL-capacity glass tubes for assay.

2.3. Measurement of tissue density

Tissue density was measured as dry weight per volume by adding the dry weights of all samples in a particular region and dividing by the volume of that region contained in the dissected section. Volumes were measured by scanning dissection maps into a Neurolucida program and tracing around all the samples contained in each region so that the program could calculate the area, then multiplying by 20 μm, the nominal section thickness. Errors in volume measurements could result especially from deviations of section thickness from the microtome setting.

2.4. Measurement of amino acid levels

Levels of free amino acids in samples were measured by high performance liquid chromatography (HPLC), using an assay based on a previous publication (Hill et al., 1979), which we have used in many studies (Ross et al., 1995; Godfrey et al., 1988, 2000, 2012, 2014, 2015). Fifteen μL of 50 % (vol/vol) methanol were added to each sample tube to extract the free amino acids from the tissue, and to some empty tubes to serve as blanks. The methanol contained an internal standard amino acid not present in the tissue (β-[2-thienyl]-DL-serine) to correct for any variations in injection volume. A WISP autosampler (Waters Corporation, Milford, MA, USA) withdrew 8 μL of the methanol containing the extracted amino acids and mixed them with 8 μL of ortho-phthaldialdehyde solution to derivatize the amino acids so that they could be measured by fluorescence. Amino acids were separated by reversed-phase chromatography and gradient elution using a C8 column, and the eluate was passed through a fluorescence detector. Chromatograms were analyzed by MAXIMA software.

Some data were lost because of technical problems with assays, and data for some of the amino acids for a few samples were discarded because of occasional contamination, probably of tubes (Godfrey et al., 2012). Such contamination was recognized by abnormally high serine levels. Glycine, aspartate, and arginine were sometimes affected by contamination, glutamate and glutamine less often, and GABA and taurine almost never. Based on some preliminary experiments, we developed the impression that the contamination profile approximately matched the prevalence of the various amino acids on human skin.

2.5. Data analysis

Amino acid levels are expressed as millimoles per kg dry weight of tissue (mmol/kg dry wt). Averaged data are expressed as mean ± standard error of the mean (SEM). Differences between lesioned-side and contralateral data were evaluated for statistical significance, when there were at least 3 samples in each set, by two-sided t-tests. Differences were considered as possibly significant only if P < 0.05. Because of the large number of comparisons, it is likely that some differences with P < 0.05 are false positives. Values of P < 0.01 or 0.005 or 0.001 are also specified where they occurred and are more likely to be truly significant. Consistency of findings was also taken into account in deciding whether there was a significant lesion effect, especially since the fewer samples for some smaller regions of the cochlear nucleus could easily result in false negatives as well as false positives.

2.6. Materials

Vacuum tubes were obtained from Ace Glass Inc., Vineland, NJ, USA. Microdissection tools and aluminum racks for storing freeze-dried tissue sections were constructed in the lab as needed (Lowry and Passonneau, 1972). Neurolucida software was obtained from MBF Bioscience, Williston, VT, USA, and MAXIMA software from Waters, Milford, MA, USA. Ortho-phthaldialdehyde solution was obtained from Sigma, St. Louis, MO, USA, and amino acid standard solution from Pierce, Rockford, IL, USA. Columns for HPLC were obtained from Dupont or Mac-Mod Analytical, Chadds Ford, PA, USA. Other chemicals were from Sigma or Fisher.

3. Results

3.1. Lesion locations

The locations of the lesions in the 8 rats have been shown in detail in previous publications (Godfrey et al., 1983, 1985, 1987a, b), except for that in rat 020180B, which is shown in Fig. 1. The lesion locations for all 8 rats are summarized in Fig. 2 and Table 1. The lesions in all the rats were unilateral, on the right sides of all except rat 110382D, in which the lesion was placed on the left side. The approach was from the dorsal brain surface in all rats except rat 110382D, for which the approach was from the ventral surface. In rats 013180A, B, and C, the lesion separated the cochlear nucleus almost completely from its brainstem connections and also damaged the medial part of the DCN, except that the caudalmost trapezoid body connection was spared in rat 013180A and the rostralmost trapezoid body connection in rat 013180C. The lesion in rat 020180B passed through the VCN, producing major damage and separating it from DCN connections. The DCN remained connected dorsally to the brain stem but was separated from any connections via the trapezoid body. In rats 070181H and 070982A and D, the lesion cut dorsally located connections of the cochlear nucleus but spared the trapezoid body connection. In rat 110382D, the lesion cut the entire trapezoid body and also destroyed the LSO on the same side and the VNTB as far as could be assessed from the stained sections, which did not include its rostralmost extent.

Figure 1.

Tracings of transverse sections of rat 020180B to show the location of the lesion in relation to the right cochlear nucleus and lateral brainstem structures. Lesioned tissue is shaded in yellow. Section numbers are shown next to each section (s followed by number), with lower numbers more caudal. Scale bar also indicates lateral (L) and medial (M) directions. Dorsal is up. Regional abbreviations are: A, anteroventral cochlear nucleus; Cb, cerebellum; D, dorsal cochlear nucleus; G, granular region; LV, lateral vestibular nucleus; P, posteroventral cochlear nucleus; R, restiform body; S, spinal trigeminal tract; V, vestibular ganglion; x, choroid plexus;?, unidentified tissue.

Figure 2.

Sketches of lesion locations relative to the cochlear nucleus in the 8 rats. Dorsal is up and lateral to the left in each sketch. The cochlear nucleus (CN) is shaded in blue, and the lesion damage (L) in yellow. Other abbreviations are Cb, cerebellum, and T, trapezoid body (ventral acoustic stria). The name of each rat is to the lower left of each sketch, and a 2 mm scale bar is included.

Table 1.

Lesion characteristics

Rat	Survival	Damage
013180A	7 days	Complete separation of the right cochlear nucleus from brainstem connections except for the caudalmost 0.2 mm of the trapezoid body, some damage to the medial part of the DCN
013180B	7 days	Complete separation of the right cochlear nucleus from brainstem connections, significant damage to the medial part of the DCN
013180C	7 days	Complete separation of the right cochlear nucleus from brainstem connections except for the rostralmost 0.5 mm of the trapezoid body, minor damage to the medial part of the DCN
020180B	7 days	More lateral location completely separating VCN from brainstem connections and also greatly damaging VCN, leaving DCN mostly intact medial to lesion and separated from VCN except for some caudodorsal PVCN, trapezoid body connection to cochlear nucleus completely removed but dorsal connections of DCN and some caudodorsal PVCN intact
070181H	2 days	Complete transection of the right centrifugal labyrinthine bundle and dorsal and intermediate acoustic striae, sparing the trapezoid body
070982A	7 days	Complete transection of the right centrifugal labyrinthine bundle and dorsal acoustic stria, sparing the trapezoid body and intermediate acoustic stria
070982D	7 days	Complete transection of the right centrifugal labyrinthine bundle and dorsal and intermediate acoustic striae, sparing the trapezoid body
110382D	7 days	Complete transection of the left trapezoid body, complete destruction of the left LSO and nearby periolivary regions, partial damage of the left MNTB, complete destruction of left VNTB in saved sections (sections rostral to rostralmost AVCN, which would include the rostralmost VNTB, were not saved)

3.2. Effects of the lesions on tissue density

Measurements of dry weight per volume, presumably because of variations in section thickness, gave rather inconsistent values for individual regions in individual cochlear nuclei. However, when data were averaged and ratios of lesioned side to contralateral were calculated, there was no clear effect of the lesions on the tissue density in the rats with the largest lesions (013180A, B, and C). The ratios of lesioned side to contralateral were, as mean ± SEM (number of regions), 0.98 ± 0.14 (3) for granular regions, 0.96 ± 0.05 (3) for DCN layers, and 0.96 ± 0.06 (5) for VCN regions.

3,3. Effects of the lesions on amino acid levels

The distribution of GABA levels in maps of the lesioned-side and contralateral cochlear nucleus of rat 013180A (Fig. 3) shows decreases throughout the lesioned side compared to contralateral levels, with the exception of the interstitial nucleus (IN), which had very low GABA levels on either side. The data from maps such as these are summarized for the 8 rats in bar graphs for 7 amino acids (Figs. 4–10 and Table 2).

Figure 3.

Maps of the distributions of GABA levels in microdissected lesioned-side and contralateral cochlear nucleus transverse sections of rat 013180A. Thin lines are cut edges of samples. Thick lines are regional boundaries, solid for outside boundary, dashed for inside boundary seen in the freeze-dried section itself, and dotted for inside boundary traced from an adjacent stained section. Section numbers are shown next to each section (s followed by number), with lower numbers more caudal. GABA levels are represented by colors as shown in the code at the left. The millimeter scale bar also shows dorsal (D), ventral (V), medial (M), and lateral (L) directions. Regional abbreviations are: A, anteroventral cochlear nucleus; G, granular region; I, interstitial nucleus (auditory nerve root); P, posteroventral cochlear nucleus; S, acoustic striae (dorsal and intermediate); m, f, and d, molecular, fusiform soma, and deep layers of dorsal cochlear nucleus. Data were not obtained for the two samples marked with x, one in section 79 and one in section 31; they were colored to blend with adjacent samples having data.

Figure 4.

Vertical bar graphs of GABA levels in lesion-side (gray bars) and contralateral (blue bars) cochlear nucleus regions of the 8 rats in this study. Standard error (SEM) bars are included for regions with more than 2 samples. Number of samples for each bar is listed in Table 2. Probabilities of statistical significance, based on 2-sided t-tests, for comparisons between sides are indicated by asterisks: * for p < 0.05, ** for p < 0.01, *** for p < 0.005, and **** for p < 0.001.

Figure 10.

Vertical bar graphs of arginine levels in lesion-side (gray bars) and contralateral (brown bars) cochlear nucleus regions of the 8 rats. Details as in Figure 4.

Table 2.

Numbers of samples providing data for each region of each rat^a

Region	013180A		013180B		013180C		020180B		070181H		070982A		070982D		110382D
	Con	Les	Con	Les	Con	Les	Con	Les	Con	Les	Con	Les	Con	Les	Con	Les
Grd	4	0	8	4	4	2–3	0	0	0–4	0–2	2	3	1	1	3	3
Grl	6	6	7	4	3	3	1–2	1	1–3	2–7	8	8–9	2	1	5	6
Gcl	0	1	3	4	3	3	0–1	1	2	1	2	2	2	3	1	2
Dm	3	2	5	3–4	5	3	5	1–3	4	3–4	3	5	6	4	6	5
Df	3–4	6	5	4	6	4–5	6	3	4	4	4	3	7	8	6–7	5–6
Dd	6	3	1	2	2	3–4	3	2	3	3	1	3	3	7	5	7
P	7	7	4	9	12	8–9	6	1	8	7	10	9	7	6	9	8
I	1	5–6	8	5	5	2	1	0	3	7	9	9	0	0	4	6
Acv	3	4	2	0	6	3–4	0	0	0–5	0–4	6	6	0	0	3	3
Acd	7	8	4	0	6	7	0	0	0–5	0–6	6	6	0	0	6	5
Ar	9	9	8	17	4	4	9	6	8	6	20–21	21–23	10	11	8–9	9
AS	4	1	3	0	5	0	0	2	3	3	0	0	3	4	5	7
TB	0	0	0	0	1	2	0	0	0	0	0	0	0	0	7	6

^aNumbers are for contralateral (Con) and lesioned (Les) sides of each rat, not including PVCN samples of 020180B within lesioned area. The regional abbreviations are the same as for Figures 4–10: Grd, granular region dorsal to anteroventral cochlear nucleus (AVCN); Grl, granular region lateral to AVCN; Gcl, granular region lateral to posteroventral cochlear nucleus (PVCN); Dm, dorsal cochlear nucleus (DCN) molecular layer; Df, DCN fusiform soma layer; Dd, DCN deep layer; P, PVCN; I, interstitial nucleus (auditory nerve root); Acv, caudoventral part of AVCN; Acd, caudodorsal part of AVCN; Ar, rostral part of AVCN; AS, acoustic striae; TB, trapezoid body (ventral acoustic stria). Where there is a range of numbers, some data were lost because of technical problems with the assay. In by far the most cases, these resulted from contamination, which especially affect glycine and sometimes also aspartate and arginine. In one experiment for rat 070181H, the glycine values were discarded, resulting in ranges that included zero for Grd, Acv, and Acd, and 1 and 2 for Grl.

GABA levels were consistently reduced in regions of the lesioned-side cochlear nucleus, as compared to contralateral, in the 4 rats with large lateral lesions – 013180A-C and 020180B, except for some granular and caudoventral AVCN regions of rat 013180C (Fig. 4). Some differences did not reach statistical significance, usually because of insufficient numbers of samples. Contrastingly, there were few differences between the lesioned-side cochlear nucleus regions and contralateral in the rats with dorsal lesions, transecting the centrifugal labyrinthine bundle but not the trapezoid body – 070181H and 070982A and D. In rat 110382D, in which the trapezoid body was cut and the LSO and VNTB severely damaged on the lesioned side, GABA levels were consistently reduced in lesioned-side cochlear nucleus regions compared to contralateral, but not as greatly as in the rats with the large lateral lesions. The largest differences, on a percentage basis, were in the trapezoid body, lateral to the lesion, and rostral and caudodorsal AVCN.

Unlike GABA, glycine levels in the cochlear nucleus of the 4 rats with large lateral lesions were usually higher on the lesioned side than contralaterally, although rarely significantly (Fig. 5). Lesioned-side increases were prominent in the DCN, PVCN, and acoustic striae of rat 070181H, with only 2 days survival after transection of dorsal pathways including the centrifugal labyrinthine bundle, but not in rats 070982A and D, with 7 days survival after a similar lesion. There was a small but highly statistically significant decrease of glycine level in the trapezoid body lateral to its transection in rat 110382D.

Figure 5.

Vertical bar graphs of glycine levels in lesion-side (gray bars) and contralateral (green bars) cochlear nucleus regions of the 8 rats. Details as in Figure 4.

Taurine levels were consistently increased in lesioned-side cochlear nucleus regions of the rats with large lateral lesions (Fig. 6). They were also increased on the lesioned side in caudal parts of the cochlear nucleus – DCN layers, acoustic striae, and PVCN of rat 070181H, with the 2-days survival after transection of dorsal pathways. Differences between the cochlear nuclei on the two sides were much less in the two rats with 7-days survival after transection of dorsal pathways (070982A and D) or trapezoid body (110382D). In rat 020180B, samples obtained from within the lesioned portion of the VCN, where there was dense gliosis, had especially high taurine levels, whereas there were decreased levels of most other amino acids (Table 3).

Figure 6.

Vertical bar graphs of taurine levels in lesion-side (gray bars) and contralateral (turquoise bars) cochlear nucleus regions of the 8 rats. Details as in Figure 4.

Table 3.

Amino acid levels in lesioned-side and contralateral PVCN of rat 020180B

Amino acid	Contralateral (6)	Not within lesion (1)	Within lesion (13)
Aspartate	8.9 ± 0.6	3.2	3.2 ± 0.3****
Glutamate	23.3 ± 1.3	12.6	10.2 ± 0.7****
Glutamine	14.9 ± 1.4	22.6	14.7 ± 1.3
Glycine	11.1 ± 0.5	15.5	8.4 ± 0.7**
GABA	2.9 ± 0.3	1.6	1.5 ± 0.3**
Taurine	4.5 ± 0.4	13.8	28.4 ± 2.4****
Serine	3.7 ± 0.3	7.4	5.0 ± 0.3**
Arginine	1.8 ± 0.2	2.7	1.9 ± 0.2

^**P < 0.01 vs. contralateral

^****P < 0.0001 vs. contralateral

Aspartate levels were consistently lower in lesioned-side cochlear nucleus regions as compared to contralateral values in the 4 rats with large lateral lesions, except for some granular regions of rat 013180C (Fig. 7). Effects of the dorsal lesions in rats 070181H and 070982A and D were inconsistent. As for the rats with large lateral lesions, aspartate levels were consistently reduced in lesioned-side cochlear nucleus regions of rat 110382D, with trapezoid body transection and superior olivary damage.

Figure 7.

Vertical bar graphs of aspartate levels in lesion-side (gray bars) and contralateral (orange bars) cochlear nucleus regions of the 8 rats. Details as in Figure 4.

Glutamate levels were usually lower in lesioned-side cochlear nucleus regions than contralaterally in the 4 rats with large lateral lesions, but differences were not as large or consistent as for aspartate (Fig. 8). As for aspartate, lesioned-side glutamate levels were consistently reduced after the ventral lesion of the trapezoid body and superior olive, whereas effects of the dorsal lesions in rats 070181H and 070982A and D were inconsistent, including some lesioned side increases in the VCN of rat 070982A.

Figure 8.

Vertical bar graphs of glutamate levels in lesion-side (gray bars) and contralateral (red bars) cochlear nucleus regions of the 8 rats. Details as in Figure 4.

Glutamine levels were almost always higher on the lesioned side than contralaterally in cochlear nucleus regions of the rats with large lateral lesions, in rat 070181H, with 2-days survival after a dorsal lesion, and in rat 110382, with the ventral lesion of the trapezoid body and superior olive (Fig. 9). Results for rats 070982A and D, with dorsal lesions and 7 days survival, were inconsistent and differed from those of the other rats, in that some regions had lower levels on the lesioned side than contralaterally.

Figure 9.

Vertical bar graphs of glutamine levels in lesion-side (gray bars) and contralateral (purple bars) cochlear nucleus regions of the 8 rats. Details as in Figure 4.

Arginine levels were usually higher on the lesioned side than contralaterally in cochlear nucleus regions of the 4 rats with large lateral lesions but not in the other 4 rats (Fig. 10). There were few statistically significant differences between lesioned-side and contralateral levels in any region, the most significant being a somewhat higher level on the lesioned side in the trapezoid body lateral to its cut in rat 110382D.

Since the lesion in rat 110382D damaged the superior olivary complex as well as cutting the trapezoid body on the left side, we checked for effects on amino acid levels in the contralateral (right) cochlear nucleus, which receives some crossed innervation from the superior olivary complex (Potashner et al., 1993; Saint Marie et al., 1993), by comparing regional averages of amino acid levels to the contralateral averages for the other 7 rats, which did not have damage of the superior olivary complex, and those of control rats from our previously published study (Godfrey et al., 2000). There was no clear evidence of an effect on amino acid levels in the contralateral cochlear nucleus of rat 110382D (Table 4), although the comparison is limited by the variations among individual animals. Also, since there is evidence for some connections between the cochlear nuclei on either side (Cant and Gaston, 1982; Shore et al., 1992), which may be mostly or entirely glycinergic (Wenthold, 1987; Benson and Potashner, 1990; Potashner et al., 1993; Saint Marie et al., 1993), the averages for the contralateral cochlear nucleus regions can be compared to those for the control rats from the previous study. Not too surprisingly, since those connections are not numerous, we did not measure any clear differences in transmitter-related amino acid levels between the two groups.

Table 4.

Comparison of contralateral average amino acid levels (mmol/kg dry wt) of rat 110382D with the averages for the other 7 rats and with control rat levels^a

	Gran rd	Gran rl	Gran cl	DCNm	DCNf	DCNd	PVCN	IN	AVCNcv	AVCNcd	AVCNr	AS	TB
Aspartate
110382D	5.3±0.7	9.6±1.3	9.0	13.0±0.5	11.7±0.8	10.3±1.2	11.1±1.7	5.0±0.6	9.8±1.0	9.1±0.5	12.9±0.5	7.2±0.7	5.3±0.6
Other rats	8.3±1.1	10.4±1.0	7.0±0.7	10.0±0.8	10.2±0.5	8.5±0.6	8.7±0.5	4.7±0.4	9.4±0.9	9.8±0.7	11.5±0.6	6.3±0.5	5.3
Control rats		11.4±0.4	9.8±0.4	10.7±0.3	13.7±0.8	11.5±0.6	11.2±0.8	5.0±0.5			13.5±0.5	7.8±0.5	5.5±0.3
Glutamate
110382D	23.1±1.9	33.1±2.7	39.3	47.0±3.2	35.4±2.7	28.8±2.8	29.6±4.3	16.8±1.6	25.8±2.6	26.0±0.6	29.3±0.9	22.1±0.8	14.8±1.4
Other rats	23.6±1.6	30.3±1.5	28.2±1.3	39.1±1.0	31.2±1.4	24.9±1.2	22.6±1.1	13.3±1.7	22.6±2.0	22.5±2.0	24.5±1.5	21.4±1.4	12.1
Control rats		39.3±1.5	36.1±2.5	49.2±2.6	39.5±1.7	29.9±1.7	26.6±1.2	15.4±1.9			29.4±1.5	22.2±1.4	13.8±0.9
Glutamine
110382D	9.7±1.3	13.9±1.1	14.7	17.0±1.3	12.2±1.1	9.4±0.9	11.1±1.5	6.6±0.2	9.5±1.3	10.3±0.4	10.4±0.4	7.2±0.2	6.8±0.5
Other rats	13.7±1.3	18.7±1.1	19.2±1.1	23.4±1.1	17.9±1.1	14.1±0.6	13.6±0.6	9.6±1.0	13.4±1.4	11.7±1.0	13.6±0.6	10.8±1.1	7.4
Control rats		19.7±1.4	17.5±1.6	21.5±1.5	17.9±0.9	14.0±0.8	13.0±0.7	7.9±0.4			13.4±0.6	10.0±0.7	7.5±0.3
Glycine
110382D	10.0±1.5	17.1±1.3	18.4	23.6±0.7	20.2±1.1	17.4±2.1	13.6±1.9	4.7±0.6	13.4±0.5	15.0±0.8	16.3±0.6	11.4±1.3	6.5±0.2
Other rats	17.0±2.4	19.5±1.0	20.2±1.9	24.1±0.9	22.3±0.8	18.3±1.0	13.4±1.0	7.2±1.0	15.5±1.5	15.5±1.5	15.7±0.8	14.1±1.8	8.6
Control rats		20.0±0.6	18.2±0.8	23.9±1.5	25.0±0.6	21.3±0.9	13.7±0.6	5.8±0.6			18.0±0.6	15.6±1.6	6.7±0.2
GABA
110382D	3.6±0.9	6.3±0.7	9.6	10.6±0.8	7.5±0.7	6.1±1.1	4.5±0.7	0.9±0.2	4.6±0.8	4.2±0.4	5.1±0.4	4.5±1.1	1.2±0.3
Other rats	5.0±0.4	7.5±0.7	6.1±0.3	11.2±0.5	8.9±0.6	5.9±0.3	3.3±0.2	1.1±0.2	4.2±0.9	4.5±0.5	4.9±0.2	4.4±0.6	0.8
Control rats		7.3±0.7	6.7±0.5	11.1±0.8	9.0±0.7	5.7±0.6	4.1±0.3	1.0±0.06			5.5±0.4	4.7±0.5	1.1±0.06
Taurine
110382D	6.5±1.2	9.1±1.3	11.3	13.2±1.5	7.4±0.7	5.8±0.5	7.8±1.0	4.6±0.3	5.8±0.9	5.0±0.4	6.6±0.5	4.5±0.3	4.8±0.3
Other rats	6.5±0.5	10.3±1.0	10.2±1.2	12.7±0.7	8.3±0.7	5.8±0.4	6.2±0.7	4.8±0.3	5.9±0.8	5.0±0.4	5.9±0.4	4.6±0.4	3.6
Control rats		14.7±1.3	13.0±1.5	17.0±1.6	10.6±0.9	7.6±0.6	8.6±0.7	5.9±0.7			7.9±0.7	7.1±0.6	6.1±0.5
Arginine
110382D	2.5±0.2	3.5±0.3	2.7	3.4±0.4	3.3±0.9	2.4±0.2	2.8±0.4	2.1±0.05	2.7±0.1	2.6±0.1	2.7±0.2	2.5±0.3	2.3±0.1
Other rats	2.0±0.3	2.6±0.4	1.9±0.3	1.8±0.3	1.9±0.3	1.7±0.3	1.9±0.4	2.3±0.5	1.9±0.4	2.2±0.4	1.8±0.1	2.3±0.4	2.7
Control rats		2.7±0.3	2.9±0.5	2.7±0.6	2.4±0.4	2.6±0.4	2.4±0.3	2.8±0.2			2.1±0.1	2.9±0.3	2.3

^aAmino acid levels for rat 110382D are averages based on numbers of samples, listed in Table 2. Amino acid levels for other rats are averages across the rats having data for the regions, as shown in Figures 4–10, ranging from 5 to 7, except just one for TB. Amino acid levels for 5–6 control rats, except 2–4 for arginine, are from Godfrey et al. (2000), converted to a dry weight basis. Abbreviations are: Gran rd, rl, and cl, granular regions dorsal to AVCN, lateral to AVCN, and lateral to PVCN; DCNm,f, and d, dorsal cochlear nucleus molecular, fusiform soma, and deep layers; PVCN, posteroventral cochlear nucleus; IN, interstitial nucleus (auditory nerve root); AVCNcv, cd, and r, caudoventral, caudodorsal, and rostral portions of anteroventral cochlear nucleus; AS, acoustic striae (dorsal and intermediate combined); TB, trapezoid body. For control rats, Gran rd and rl data were combined, but most were for rl, and data for AVCN were most representative of rostral AVCN.

4. Discussion

4.1. Considerations in interpreting the results

Changes of amino acid levels after the lesions could result from several effects of the lesions. These include the loss of free amino acids during degeneration of the distal portions of cut nerve fibers separated from their somata of origin, release of amino acids during breakdown of proteins, transneuronal effects on cochlear nucleus neurons receiving centrifugal innervation, retrograde effects on cochlear nucleus neurons whose axons are cut by the lesions, migration of glial cells to regions of degeneration and their hypertrophy – gliosis, or nonspecific effects of tissue damage (Godfrey et al., 1983).

Since GABA and taurine are not incorporated into protein structures (Lehninger, 1975), they should not be affected by protein breakdown during degeneration of transected centrifugal projections, which could begin within a few days (Cohen et al., 1972; Kane, 1974; Morest et al., 1997), but measured levels of the other amino acids would likely increase.

Major transneuronal effects may not be generally expected at survival times up to one week (Cowan, 1970), as in our study, but they have been reported for rodent olfactory system and central auditory system at times shorter than one week (Heimer and Kalil, 1978; Nadi et al., 1981; Morest et al., 1997).

Retrograde effects should be confined to regions containing neurons whose axons would be cut by the lesions and free amino acids that those neurons contain. The granular regions and DCN molecular layer, since they contain few if any projection neurons (Osen, 1988; Helfert et al., 1991), should not show such effects to much extent, with their chemistry being affected only to the extent that neurons projecting out of the cochlear nucleus send collaterals to those regions or have dendrites extending into them, as with the superficial dendrites of DCN fusiform cells extending into the molecular layer (Osen, 1969; Brawer et al., 1974; Lorente de Nó, 1981) or multipolar cells of the VCN that project to the DCN as well as the inferior colliculus (Adams, 1983).

Gliosis can occur quickly after transection of fiber pathways (De Waele et al., 1996; Li et al., 1999) and could especially lead to increased levels of taurine and also glutamine, since relatively high concentrations of these amino acids have been measured in glia (Patel and Hunt, 1985; Hassel et al., 1995).

Possible nonspecific effects of the lesions have been discussed previously relative to the large lateral lesions (Godfrey et al., 1983). Their possible contribution to our measurements has been assessed in some of the rats by measuring possible effects on cochlear nucleus volume, tissue density (as dry weight per volume), activity of enzymes not closely related to neurotransmission (Godfrey et al., 1983, 1987b), or levels of amino acids not closely related to neurotransmission. Our previous measurements on cochlear nucleus subregion volumes in the rats with the large lateral lesions, which should have the greatest potential for nonspecific effects, found no evidence for effects of the lesions on VCN volume, and some loss of DCN volume was attributed direct lesion damage (Godfrey et al., 1983). Our measurements as part of this present study showed no lesion effects on cochlear nucleus tissue density in these same rats. Measured activities of malate dehydrogenase, a major enzyme of oxidative energy metabolism (Lehninger, 1975), in the rats with large lateral lesions and in rat 110382D, with transection of the trapezoid body and lesion damage of the superior olivary complex on one side, indicated 15–20% decreases in most lesioned-side regions, less in granular region (Godfrey et al., 1983, 1987b), which suggest an effect of the lesions on energy metabolism in the cochlear nucleus. This is also supported by a measured decrease of the activity of lactate dehydrogenase, a major enzyme of glycolytic energy metabolism (Lehninger, 1975), in the lesioned-side AVCN of rat 110382D (Godfrey et al., 1987b). Decreased activity of aspartate aminotransferase, a major enzyme of glutamate and aspartate metabolism (Lehninger, 1975), in the lesioned-side AVCN of rat 110382D (Godfrey et al., 1987b) is consistent with the decreases of aspartate and glutamate levels measured in its lesioned-side AVCN (Figs. 7 and 8). Arginine was included in our study as an amino acid less closely related to neurotransmission in the brain, which also, rather uniquely, has a more uniform distribution among central auditory neuropil (gray matter) and fiber tract (white matter) regions on a dry weight basis (Table 4), higher in fiber tracts on a lipid-free dry weight basis (Godfrey et al., 2000). Effects of the lesions on arginine levels in lesioned-side cochlear nucleus regions were usually not large, and almost all statistically significant differences between sides were increases rather than decreases, which would be consistent with release from proteins during transected fiber degeneration. This suggests that nonspecific effects on amino acid levels may be relatively minor.

4.2. GABA

Our most definitive results are for GABA. The lesion effects on GABA levels in the cochlear nucleus should not involve protein breakdown since GABA is not incorporated into proteins (Lehninger, 1975). They should also not involve retrograde effects since there is no evidence for GABA-containing neurons projecting out of the cochlear nucleus. Also, very little if any GABA has been measured in astrocytes (Patel and Hunt, 1985; Hassel et al., 1995), so that gliosis should not be a major factor affecting GABA levels. Major gliosis, as in the PVCN of rat 020180B, could lead to decreased GABA levels, but there was no difference between GABA levels within the densely gliotic lesioned part of PVCN and the part outside of it in that rat (Table 3), nor was the effect on lesioned-side PVCN GABA level different in rat 020180B than in the other rats (013180A-C) with large lateral lesions (Fig. 4). Effects of the lesions on GABA levels should then represent loss from GABA-containing fibers and terminals cut by the lesion plus possible transneuronal effects on GABA-containing neurons within the cochlear nucleus. Transneuronal effects could result from lost centrifugal fiber input or from loss of collateral fiber input from cochlear nucleus projection neurons whose axons have been cut by a lesion.

Our results for effects of the large lateral lesions on GABA levels in rat cochlear nucleus regions (averages for rats 013180A-C) are comparable to those which we previously published for cat cochlear nucleus with similar lesions (Godfrey et al., 1988). Lesioned-side GABA levels as percentages of contralateral values for comparable regions are: about 55 % in cat and 53 % in rat for rostral AVCN, about 55 % in cat and 58 % in rat for caudal AVCN, about 60 % in cat and 68 % in rat for rostral granular region, about 65 % in cat and 58 % in rat for PVCN, about 55 % in cat and 58 % in rat for DCN molecular layer, about 65 % in cat and 53 % in rat for DCN fusiform soma layer, and about 65 % in cat and 51 % in rat for DCN deep layer.

The results of Potashner et al. (1985a, 1993) for GABA uptake and release after large lateral lesions in guinea pig cochlear nucleus can be compared with our measures of GABA levels in PVCN and DCN, but not in AVCN because their lesion did not extend far enough rostrally to sever the entire trapezoid body. For lesioned side compared to control animal, they measured 70 % and 77 %, respectively, for uptake and release in PVCN and 60 % and 57 % in DCN. These values for GABA uptake and release are not very different from our values for GABA levels in DCN layers but somewhat higher than our values for GABA levels in PVCN.

The dorsally placed lesions angling from dorsomedial to ventrolateral, aimed to cut the olivocochlear bundle but not the trapezoid body, in rats 070181H and 070982A and D, had only minor and rarely statistically significant effects on GABA levels in cochlear nucleus regions, with largest and most consistent effects in rat 070982A, wherein the lesion extended closest to the ventral edge of the brain stem and may have slightly affected the trapezoid body (Fig. 2, and Fig. 1 of Godfrey et al., 1987a). The effects in these 3 rats contrasted with the larger effects in the rats with the large lateral lesions (013180A-C and 020180B) and rat 110382D with the lateral trapezoid body cut. The decreases of GABA levels in the AVCN magnocellular regions (AVCN rostral, caudodorsal and caudoventral) were similar in rat 110382D to those in the rats with the large lateral lesions, suggesting that all effects on GABA levels in these regions result from trapezoid body transection. The lesion effects on GABA levels in DCN layers in rat 020180B, wherein the DCN was not separated from dorsal brainstem connections, but from trapezoid body connections, would support all its GABA level decreases also being associated with transected trapezoid body connections. However, the smaller effects in DCN, especially the fusiform soma and deep layers, and PVCN in rat 110382D than in the rats with large lateral lesions, complicate this explanation. The possibility that the smaller lesion-side vs. contralateral GABA level ratios in the DCN layers and PVCN result from a contralateral effect of the superior olivary damage in this rat is not supported by the similar contralateral GABA levels in these regions of rat 110382D compared to the average for the other rats in this study or with control rat values (Table 4). Evidence indicates that contralateral projections of GABAergic pathways from the superior olivary complex are much fewer than ipsilateral projections (Potashner et al., 1993; Saint Marie et al., 1993). The discrepancy between the results for the rats with large lateral lesions, including rat 020180B, and those for rat 110382D suggest that there may be another fiber pathway, not cut by the lateral trapezoid body lesion but cut by the large lateral lesions, contributing to GABA levels in DCN and PVCN regions. Davies (1981) reported approximately 30 % decrease of the activity of glutamic acid decarboxylase, the enzyme that catalyzes synthesis of GABA, in the guinea pig cochlear nucleus after transection of the dorsal and intermediate acoustic striae, with a 2-mm-deep cut placed just caudolateral to the inferior cerebellar peduncle, a location similar to but not as deep as those of our large lateral cuts in rats 013180A-C. This result, combined with the differences in effects of our large lateral and ventral trapezoid body lesions, suggests that there are GABA-containing fibers entering the caudal part of the cochlear nucleus via the acoustic striae route that were not cut by our more medially placed dorsal lesions. Such fibers would somehow need to follow a route that would not be affected by our more medially placed dorsal cuts or the slightly more medial trapezoid body transection in rat 110382D. Their existence would be consistent with the much higher GABA levels in the acoustic striae than in the trapezoid body (Fig. 4 and Godfrey et al., 2000) and presence of GABA-immunoreactive fibers within the acoustic striae as well as the trapezoid body (Kolston et al., 1992). If they approach from a more rostral direction and follow a caudodorsal route to reach the acoustic striae caudodorsal to the inferior cerebellar peduncle, they could be missed by our more medially placed dorsal cuts and the slightly more medial trapezoid body cut in rat 10382D. They presumably would not be fibers approaching from the cerebellum (Rossi et al., 1967; Gacek, 1973) because the cerebellum was removed before making the cut in the experiments of Davies (1981), and the comparisons were made between the lesioned-side and contralateral cochlear nucleus.

The effects of the various lesions on GABA levels in granular regions were somewhat variable. The significant decreases found in rats 013180A and B but not C may have resulted from the lesion in rat 013180C not cutting the most rostral part of the trapezoid body, which would likely contain most of the GABA-containing fibers innervating rostral cochlear nucleus regions. That the GABA levels in lesioned-side rostral granular regions of rat 110382D were not significantly lower than contralateral values might possibly result from a bilateral effect of the lesion, which damaged the superior olivary complex in this rat. The contralateral rostral granular region GABA levels in rat 110382D were somewhat lower than the average for the other rats and for control rats (Table 4). Because of across-animal variations, however, this can only be tentatively suggested.

We cannot rule out a contribution of transneuronal effects to the decreases of GABA levels in the cochlear nucleus regions after transection of its centrifugal innervation. Such effects on cochlear nucleus neurons might be somewhat proportional to the amount of lost innervation and so could be larger in the rats with large lateral lesions than in the other rats. Such transneuronal effects may have been involved in the small decreases of GABA levels measured in the cochlear nucleus after transection of the auditory nerve by cochlear ablation (Godfrey et al., 2014, 2015). As in those cochlear ablation studies, however, the contribution should be relatively small compared to the loss from transected fibers.

4.3. Glycine

Our results for effects of the lesions on glycine levels are extremely difficult to interpret because, besides loss of free glycine from degenerating glycinergic fibers and terminals, the transection of centrifugal fibers could result in increases of glycine levels because of release from protein breakdown in degenerating fibers and terminals, possible decreases because of retrograde effects on glycinergic neurons that project out of the cochlear nucleus (Wenthold, 1987; Potashner et al., 1993; Saint Marie et al., 1993), changes because of transneuronal effects on glycinergic interneurons receiving innervation from centrifugal pathways, or increases because of gliosis since glycine levels may be similar in neurons and astrocytes (Patel and Hunt, 1985). Some of the most consistent increases of glycine levels among caudal cochlear nucleus (DCN and PVCN) regions, as well as the dorsal and intermediate acoustic striae, were measured in rat 070181H, with only 2 days survival after transection of dorsal pathways, whereas rat 070982A, with a similar lesion but 7 days survival, had small decreases in almost all cochlear nucleus regions. There was a slight trend for increased glycine levels in lesioned-side cochlear nucleus regions after cochlear ablation in rats (Godfrey et al., 2014) but not in chinchillas (Godfrey et al., 2015). The trend for increased glycine levels in cochlear nucleus regions on the lesioned side suggests that, in rats, release of free glycine from protein breakdown may be a major result of the centrifugal fiber transections, obscuring decreases because of loss from degenerating glycinergic centrifugal fibers and terminals. Measurements of glycine uptake and release in cochlear nucleus subdivisions have shown significant decreases following transection of centrifugal pathways (Staatz-Benson and Potashner, 1988; Potashner et al., 1993), which probably derive especially from the superior olivary complex (Potashner et al., 1993; Saint Marie et al., 1993). The significantly decreased glycine level in the trapezoid body lateral to its transection in rat 110382D is consistent with this derivation.

4.4. Taurine

Like GABA, taurine is not incorporated into proteins, so the consistent increases in virtually all cochlear nucleus regions in the rats with large lateral lesions (013180A-C and 020180B) are probably related mostly to gliosis, since glia contain high taurine concentrations (Patel and Hunt, 1985; Hassel et al., 1995). This conclusion is supported by the extremely high taurine levels within the densely gliotic portion of the lesioned-side PVCN of rat 020180B (Table 3). Lesioned side changes of taurine levels were much less in the rats with the dorsal or more specific trapezoid body lesions, wherein the lesions were not as close to the cochlear nucleus. Effects were larger than those after cochlear ablation in rats and chinchillas (Godfrey et al., 2014, 2015).

4.5. Aspartate and glutamate

Besides the role of glutamate in neurotransmission and the close metabolic relationship of aspartate with glutamate, whether or not it functions as a neurotransmitter itself, they are associated with various aspects of cell metabolism. This includes energy metabolism, through linking of their transaminase (aspartate aminotransferase) reaction to the malate-aspartate shuttle (Lehninger, 1975; Dingledine and McBain, 1994). Therefore, some of the decreases of aspartate and glutamate levels in the rats with large lateral lesions (013180A-C and 020180B) may be associated with decreased energy metabolism in the lesioned-side cochlear nucleus of these rats (Godfrey et al., 1983). When the decreases of aspartate and glutamate levels in lesioned-side cochlear nucleus regions (granular region lateral to AVCN, rostral AVCN, DCN molecular and fusiform soma layers, and PVCN, except for PVCN in rat 020180B because of the extensive direct lesion damage to it) relative to contralateral in the rats with large lateral lesions are compared to the previously reported (Godfrey et al., 1983) decreases of malate dehydrogenase activity, as a measure of oxidative energy metabolism (Lehninger, 1975), relatively high correlations are found: 0.83 for aspartate and 0.81 for glutamate vs. malate dehydrogenase. This comparison suggests that much of the decrease of aspartate and glutamate levels after transection of centrifugal pathways to the cochlear nucleus may be associated with decreased oxidative energy metabolism after the lesions. This decreased energy metabolism might represent a nonspecific effect of the lesion, such as by an effect on blood supply, or it could result from the loss of neural activity following the lesion. Also, decreased availability of aspartate and glutamate might lead to decreased energy metabolism. Decreases of aspartate and/or glutamate levels that exceed those of malate dehydrogenase activity might be especially indicative of loss from degenerating centrifugal fibers. Although the decreases of glutamate levels were, overall, similar in magnitude to those of malate dehydrogenase activities, the decreases of aspartate levels exceeded the decreases of malate dehydrogenase activities by about 13 %. In line with evidence that some centrifugal pathways to cochlear nucleus granular regions may use glutamate as neurotransmitter (Wright and Ryugo, 1996; Milinkeviciute et al., 2017), the decreases of glutamate level in the rostrolateral granular region of rats 013180A and 020180B exceed those of malate dehydrogenase activity by 19 % and 39 %, respectively. In line with cochlear nucleus granule cells using glutamate as neurotransmitter (Oliver et al., 1983; Potashner et al., 1985b; Juiz et al., 1993; Wenthold et al., 1993; Osen et al., 1995; Waller et al., 1996; Godfrey et al., 1997; Chen et al., 1999) and projecting to the DCN molecular layer (Mugnaini et al., 1980; Adams, 1983; Osen, 1988), the decrease in lesioned-side DCN molecular layer glutamate level in rat 020180B, wherein the lesion transected granule cell axons projecting from rostral cochlear nucleus to DCN molecular layer, exceeded the decrease in malate dehydrogenase activity by 18 %. In rat 110382D, with transection of the trapezoid body but not dorsal centrifugal pathways, lesioned-side glutamate and aspartate levels in rostral AVCN decreased to about half, whereas malate and lactate dehydrogenase activities, for oxidative and glycolytic energy metabolism, respectively, decreased by about a third relative to contralateral (Godfrey et al., 1987b), and the significant decreases in the trapezoid body lateral to its transection were not accompanied by any change in the enzyme activities (Godfrey et al., 1987b). Retrograde effects on glutamate and aspartate levels could occur since they are associated with neurons whose axons project out of the cochlear nucleus to higher auditory centers (Godfrey et al., 1988; Caspary and Faingold, 1989; Suneja et al., 1995). Lesioned side decreases of glutamate levels in VCN regions, which contain neurons involved in these projections (Warr, 1966, 1969; Helfert et al., 1991), were not large in the rats with large lateral lesions (013180A-C) transecting the cochlear nucleus ascending projections, but they were larger and statistically significant in rat 110382D, with the more specific trapezoid body transection. Effects on lesioned-side aspartate levels were generally larger than those on glutamate levels. Greatly decreased lesioned-side glutamate and aspartate levels in the rostrolateral granular region of rat 020180B might include a retrograde effect on granule cells following transection of their axons by the lesion in that rat.

4.6. Glutamine

Effects of the lesions on glutamine levels could include a mix of decreases because of its close metabolic association with glutamate, especially in the glutamine cycle for replenishment of transmitter glutamate (Shank and Aprison, 1981; Dingledine and McBain, 1994), and increases because of lesioned side gliosis, because, like taurine, it has relatively high concentrations in glia (Patel and Hunt, 1985; Hassel et al., 1995). The results suggest that the effects of gliosis outweigh those resulting from the close relationship to glutamate metabolism since the lesioned-side cochlear nucleus regions routinely had increased glutamine levels relative to contralateral, with relative increases resembling in many regions those of lesioned-side taurine levels. As for taurine, effects were larger than those after cochlear ablation in rats and chinchillas (Godfrey et al., 2014, 2015). The statistically significant lesioned-side increases of taurine and glutamine, as well as glycine, in caudal cochlear nucleus regions at 2 days (rat 070181H) after the dorsal lesion but not at 7 days (rats 070982A and D) are interesting but not easily explainable. They should not be related to a transient gliosis since prominent gliosis was not observed in these regions of the Nissl-stained sections of rat 070181H.

4.7. Comparisons to effects of cochlear ablation

Some comparisons can be made between the effects of transection of centrifugal, descending, pathways to the rat cochlear nucleus and the effects of transection of the ascending auditory nerve input by cochlear ablation (Godfrey et al., 2014). Both types of lesions did not affect cochlear nucleus tissue density (dry weight per volume) at 7 days after the lesion. Since glutamate is well established as a neurotransmitter of auditory nerve fibers (Wenthold, 1978, 1985; Caspary et al., 1985; Martin, 1985; Potashner et al., 1985b; Juiz et al., 1993; Wenthold et al., 1993; Zhou et al., 2007; Godfrey et al, 2014, 2015), whereas GABA shows a strong association with centrifugal fibers, it is of interest to compare results for these two amino acids, as well as for aspartate, which is closely related metabolically to glutamate and shows some preferential association with the auditory nerve (Wenthold, 1985; Godfrey et al., 1988). Reductions of GABA levels, expressed as the ratio of lesioned-side level to contralateral level, were consistently much larger after transection of centrifugal pathways than after transection of the auditory nerve (Table 5). Reductions of glutamate levels in VCN regions were larger after auditory nerve transection, whereas those in DCN layers were larger after centrifugal pathway transection. Effects of auditory nerve transection on aspartate levels were not very different from effects on glutamate levels, whereas effects of centrifugal pathway transection tended to fall between those on GABA and glutamate.

Table 5.

Average lesioned-side/contralateral ratios of amino acid levels in cochlear nucleus regions of rats with brainstem lesions and cochlear ablations^a

Region	GABA		Aspartate		Glutamate
	Brainstem	Cochlea	Brainstem	Cochlea	Brainstem	Cochlea
Granular rostrolateral	0.71	0.97	0.85	0.91	0.87	0.88
DCN molecular	0.58	0.83	0.72	0.91	0.81	0.97
DCN fusiform soma	0.51	0.91	0.68	0.86	0.81	0.91
DCN deep	0.50	0.97	0.53	0.78	0.71	0.80
PVCN	0.58	0.94	0.69	0.63	0.87	0.64
IN (ANR)	0.46	0.92	0.62	0.45	0.87	0.48
AVCN rostral	0.53	1.03	0.66	0.82	0.84	0.74

^aBrainstem values are averages for rats 013180A-C; Cochlea values are calculated from Godfrey et al. (2014) for the rats with 7 days survival after cochlear ablation. Abbreviations are AVCN, anteroventral cochlear nucleus; DCN, dorsal cochlear nucleus; IN (ANR), interstitial nucleus (auditory nerve root); PVCN, posteroventral cochlear nucleus. The rostrolateral granular region of the present study is comparable to the AVCN granular region and rostral AVCN to the combined AVCN main and subgranular regions of Godfrey et al. (2014).

4.8. Conclusions

The effects of the lesions on amino acid levels were smaller than the very large effects on choline acetyltransferase activity in cochlear nucleus regions (Godfrey et al., 1983, 1987a,b). This is consistent with other evidence that the amino acids have more association than acetylcholine with the auditory nerve input from the cochlea and with cochlear nucleus interneurons.

Our results complement previous measurements of uptake and release of GABA after lesions of centrifugal pathways to the cochlear nucleus (Potashner et al., 1985a, 1993). They support a sizeable centrifugal GABAergic projection to the cochlear nucleus, contributing almost half the GABA in some of the DCN layers and rostral AVCN and about a third in granular regions. The DCN results are quite similar to what has been reported based on GABA uptake and release measurements (Potashner et al., 1985a, 1993). Probably the only other factor that might contribute to the reductions of GABA levels after the transection of the centrifugal pathways would be transneuronal effects on GABAergic interneurons within the cochlear nucleus.

Unfortunately, the complicating factors of release during protein breakdown, transneuronal and retrograde effects, gliosis, and nonspecific metabolic effects complicate the interpretation of the lesion effects on levels of other neurotransmitter-related amino acids in cochlear nucleus regions. There is considerable evidence from other studies for involvement of glycine in centrifugal pathways from the superior olivary complex and the contralateral cochlear nucleus (Wenthold, 1987; Staatz-Benson and Potashner, 1988; Potashner et al., 1993; Saint Marie et al., 1993) as well as cochlear nucleus interneurons (Osen et al., 1990; Altschuler et al., 1993; Godfrey et al., 1988, 1997; Oertel and Wickesberg, 1993; Juiz et al., 1996; Gates et al., 1996; Doucet et al., 1999; Apostolides and Trussell, 2013)). Our study provides suggestive evidence for association of aspartate and glutamate with some centrifugal pathways to the cochlear nucleus, but their involvement in energy metabolism, which is affected by the lesions, and possible retrograde effects prevent our findings from being definitive.

Abbreviations in text:

AVCN

anteroventral cochlear nucleus

DCN

dorsal cochlear nucleus

GABA

γ-amino butyric acid

HPLC

high performance liquid chromatography

IN

interstitial nucleus (auditory nerve root)

PVCN

posteroventral cochlear nucleus

SEM

standard error of the mean

VCN

ventral cochlear nucleus