S.J. Potashner

BACLOFEN: EFFECTS ON AMINO ACID RELEASE AND METABOLISM IN SLICES OF GUINEA PIG CEREBRAL CORTEX

Slices of guinea‐pig cerebral cortex were used to investigate the effects of the antispastic drug β‐(p‐chlorophenyl)‐γ‐aminobutyrate (Baclofen, Lioresal) on the release and metabolism of several amino acids. Electrical stimulation of slices evoked (1) a relatively large release, probably from nerve terminals, of 14C‐labelled tissue glumate, aspartate and γ‐aminobutyrate (GABA) synthesized via metabolism of D‐[U‐14C]glucose and (2) a relatively small release, probably not from nerve terminals, of 14C‐labelled tissue alanine and threonine‐serine‐glutamine and of exogenous radiolabeled glutamate, aspartate, GABA and α‐aminoisobutyrate that had been taken up from the medium. Baclofen (4μM) preferentially inhibited the release of 14C‐labelled tissue glutamate and aspartate. It had no effect on the concentrations and specific radio‐activities of most of the labelled tissue amino acids in the slices. However, it increased the turnover of 14C‐labelled tissue glycine approx 4‐fold and elevated the specific radio activity of tissue alanine by 40%. It was concluded that Baclofen affects transmission not by modulating the release of the inhibitory amino acid GABA, but by selectively suppressing the release of the excitatory amino acids glutamate and aspartate from nerve terminals. Provided that this action obtains in the spinal cord, it may at least partly underlie the antispastic action of Baclofen as glutamate and aspartate are presumed to be the transmitters released from terminals of non‐nociceptive primary afferent fibers and excitatory interneurons, respectively. The Baclofen‐induced increase in glycine turnover suggests an additional effect on inhibitory glycinergic interneurons in the spinal cord.

Plastic changes in glycine and GABA release and uptake in adult brain stem auditory nuclei after unilateral middle ear ossicle removal and cochlear ablation

[i] In young adult guinea pigs, the effects of unilateral ossicle removal and unilateral cochlear ablation were determined on [14C]glycine or [14C]GABA release and uptake measured in subdivisions of the cochlear nucleus (CN), the superior olivary complex, and the auditory midbrain, after 2 or 5, 59, and 145 postlesion days. Activities were compared to those of age-matched, unlesioned controls. [ii] [14C]Glycine release declined bilaterally in the anteroventral and dorsal CN after ossicle removal and in the dorsal CN after cochlear ablation. [iii] Transient elevations of release occurred at 59 days in the ipsilateral posteroventral CN ([14C]glycine) and bilaterally in the ventral nucleus of the lateral lemniscus ([14C]GABA) after ossicle removal, and bilaterally in the medial superior olive ([14C]glycine) after cochlear ablation. [iv] In the medial nucleus of the trapezoid body, [14C]GABA release was depressed bilaterally 5 days after ossicle removal, but was elevated at 145 days contralaterally after ossicle removal and ipsilaterally after cochlear ablation. [v] In the contralateral central nucleus of the inferior colliculus, [14C]GABA release was elevated persistently after ossicle removal. After cochlear ablation, release was elevated at 5 days, near the control at 59 days, and elevated again at 145 days. [vi] After both lesions, [14C]glycine uptake was elevated bilaterally in the CN and medial superior olive. [14C]GABA uptake became depressed by 59 or 145 days bilaterally in the auditory midbrain. [vii] These changes may stem from regulation and may contribute to mechanisms that generate symptoms such as loudness recruitment and tinnitus, which often accompany hearing loss.

Evidence for a Glutamatergic Pathway from the Guinea Pig Auditory Cortex to the Inferior Colliculus

Abstract: We attempt to provide evidence that the projection from the guinea pig auditory cortex (AC) to the inferior colliculus (IC) may contain glutamatergic or GABAergic fibers. Seven days after unilateral AC aspiration, histological studies indicated almost complete AC destruction and preterminal degeneration of fibers and terminal fields in the dorsal cortex (DCIC), external cortex (ECIC), and central nucleus (CNIC) of the IC ipsilateral to the ablated AC. Contralaterally, degeneration appeared in the DCIC. AC ablation depressed the electrically evoked Ca2+‐dependent release of d‐[3H]aspartate (d‐[3H]Asp) in the ipsilateral DCIC, ECIC, and CNIC, and d‐[3H]Asp uptake in the CNIC. Together with other evidence that the corticocollicular pathway is excitatory, these findings suggest that this projection may contain glufamatergic and/or aspartatergic (Glu/Asp‐ergic) fibers. Glutamic acid decarboxylase immunoreactivity was not apparent in presumed pyramidal cells of layer V of the AC retrogradely labeled with biotinylated dextran injected into the ipsilateral IC. Thus, corticocollicular neurons probably do not synthesize GABA and may not be GABAergic. However, AC ablation depressed [14C]GABA release from the ipsilateral DCIC and ECIC, and [14C]GABA uptake in the DCIC. These findings are consistent with the atrophy or down‐regulation of some subcortical neurons that mediate GABAergic transmission in the IC.

Glycine Receptors in Adult Guinea Pig Brain Stem Auditory Nuclei: Regulation after Unilateral Cochlear Ablation

In young adult guinea pigs, the effects of unilateral cochlear ablation were determined on the specific binding of [3H]strychnine measured in subdivisions of the cochlear nucleus (CN), the superior olivary complex, and the auditory midbrain, after 2, 7, 31, 60, and 147 postlesion days. Changes in binding relative to that in age-matched controls were interpreted as altered activity and/or expression of synaptic glycine receptors. Postlesion binding declined ipsilaterally in most of the ventral CN and in the lateral superior olive (LSO). Binding was modestly deficient in the ipsilateral dorsal CN and in the anterior part of the contralateral anteroventral CN. Binding was elevated in the contralateral LSO. Transient changes also occurred. Binding was elevated transiently, between 2 and 31 days, contralaterally in parts of the anteroventral CN, bilaterally in the medial superior olive (MSO), and bilaterally in most of the midbrain nuclei. Binding was deficient transiently, at 60 days, in most of the contralateral CN and bilaterally in the midbrain nuclei. The present findings, together with previously reported postlesion changes in glycine release, were consistent with persistently weakened glycinergic inhibitory transmission ipsilaterally in the ventral CN and the LSO and bilaterally in the dorsal CN. Glycinergic inhibitory transmission was strengthened in the contralateral LSO and transiently strengthened in the MSO bilaterally. A hypothetical model of the findings suggested that glycine receptor regulation may depend on excitatory and glycinergic input to auditory neurons. The present changes in glycine receptor activity may contribute to altered auditory functions, which often accompany hearing loss.

Regulation ofd-Aspartate Release and Uptake in Adult Brain Stem Auditory Nuclei after Unilateral Middle Ear Ossicle Removal and Cochlear Ablation☆

In young adult guinea pigs, the effects of unilateral ossicle removal and cochlear ablation were determined on transmitter release from glutamatergic presynaptic endings and glutamate inactivation via uptake. (i) D-[3H]Aspartate release and uptake were measured in subdivisions of the cochlear nucleus (CN) and in nuclei of the superior olive (SOC) and auditory midbrain (MB) up to 145 days after placing the lesions. Activities were compared to those from age-matched unlesioned controls. Fiber degeneration was visualized histologically. (ii) In the ipsilateral CN, changes in release and uptake were governed by the type of lesion. Ossicle removal produced sparse pruning of fibers only after 112 days and decreased release and uptake at 145 days, consistent with regulatory weakening of excitatory glutamatergic transmission. Cochlear ablation deafferented the CN, producing deficient release and uptake at 2 days and abundant fiber degeneration at 7 days. Subsequently, the residual release and uptake increased in magnitude, consistent with strengthening of excitatory glutamatergic transmission. (iii) In the contralateral CN, after either lesion, changes in release and uptake usually matched those in the ipsilateral CN. Thus, the auditory pathway associated with the lesioned ear probably provided cues for the regulation of synaptic strength in the contralateral CN. (iv) Both lesions increased release in the SOC and MB, and uptake in the SOC, consistent with strengthening of excitatory glutamatergic transmission. Sparse fiber degeneration, suggesting axonal pruning, appeared in the SOC and MB after cochlear ablation. (v) The strengthening of excitatory glutamatergic transmission may facilitate and maintain symptoms such as loudness recruitment and tinnitus which often accompany hearing loss.

SYNAPTOPHYSIN IMMUNOREACTIVITY IN THE COCHLEAR NUCLEUS AFTER UNILATERAL COCHLEAR OR OSSICULAR REMOVAL

This study determined if unilateral cochlear removal in adult guinea pigs led to synaptic loss followed by synaptogenesis in the cochlear nucleus (CN) and if unilateral middle ear ossicle removal led to synaptic loss in the CN. Synaptic endings were identified immunohistochemically, using a monoclonal antibody to synaptophysin. Immunolabeling was quantified densitometrically in the CN 4–161 days after cochlear removal and 161 days after ossicle removal. Fiber degeneration was visualized with the Nauta‐Rasmussen silver method. Tissue shrinkage was measured from drawings of CN sections.

New Growth of Axons in the Cochlear Nucleus of Adult Chinchillas after Acoustic Trauma

This study determined the effect of acoustic overstimulation of the adult cochlea on axons in the cochlear nucleus. Chinchillas were exposed to an octave-band noise centered at 4 kHz at 108 dB sound pressure level for 1.75 h. One chinchilla was never exposed to the noise, and several others had one ear protected by an ear plug or prior removal of the malleus and incus. Exposure of unprotected ears caused loss of inner and outer hair cells and myelinated nerve fibers, mostly in the basal half of the cochlea. Cochlear nerve fiber degeneration, ipsilateral to the exposed ears, was traced to regions of the cochlear nucleus representing the damaged parts of the cochlea. In silver impregnations of a deafferented zone in the posteroventral cochlear nucleus, the concentration of axons decreased by 43% after 1 month and by 54% after 2 months. However, by 8 months, the concentration of thinner axons, with diameters of less than 0.46 microm, increased by 46-90% over that at 2 months. The concentration of axons with larger diameters did not change. Between 2 and 8 months small axonal endings appeared next to neuronal cell bodies. This later increase of thinner axons and endings is consistent with a reactive growth of new axons of relatively small diameter. The emergence of small perisomatic boutons suggests that the new axons formed synaptic endings, which might contribute to an abnormal reorganization of the central auditory system and to the pathological changes that accompany acoustic overstimulation.

Altered glycinergic synaptic activities in guinea pig brain stem auditory nuclei after unilateral cochlear ablation.

This paper reviews efforts to determine if a unilateral hearing loss altered inhibitory glycinergic synapses in the cochlear nucleus (CN) and the superior olive. In young adult guinea pigs, 2-147 days after unilateral cochlear ablation, we quantified the electrically evoked release and the high-affinity uptake of [(14)C]glycine as measures of transmitter release from glycinergic presynaptic endings and glycine removal from extracellular spaces. The specific binding of [(3)H]strychnine was quantified to measure synaptic glycine receptor activity and/or expression. Three types of post-lesion change were observed. First, several tissues exhibited changes consistent with a persistent deficiency in glycinergic inhibitory transmission. Deficient binding prevailed on the ablated side in the anterior and caudal anteroventral CN, the posteroventral CN and the lateral superior olive (LSO), while glycine release was near normal and uptake was elevated (except in the LSO). However, deficient release prevailed in the dorsal CN, bilaterally, and was accompanied by elevated uptake. Second, the LSO on the intact side exhibited changes consistent with strengthened glycinergic inhibition, as binding was elevated while release and uptake were near normal. Third, several tissues exhibited various transient changes in activity. These types of post-lesion change might contribute to altered auditory functions, which often accompany hearing loss.

Evidence for a GABAergic Projection from the Dorsal Nucleus of the Lateral Lemniscus to the Inferior Colliculus

Abstract: This study attempts to determine whether the pathways from the guinea pig dorsal nucleus of the lateral lemniscus (DNLL) to the inferior colliculus (IC) use γ‐aminobutyric acid (GABA) as a transmitter. Injections of kainic acid (KA) were used to destroy neurons in the left DNLL. Two to 4 days after the injection, Nissl‐stained sections through the lesion site showed destruction of the DNLL neurons. The lesions varied in size; 12–100% of the DNLL neurons were destroyed on the injected side without damage to the ipsilateral IC. Two to 4 days after the injection, the electrically evoked, Ca2+‐dependent release and high‐affinity uptake of [3H]GABA were measured in dissected pieces of the left and right IC. These activities were compared with those in the IC taken from unlesioned controls and from sham controls, which received injections of saline instead of KA. Each IC was divided into a dorsal piece, which contained the dorsal cortex and dorsomedial nucleus, and a ventral piece, which contained the central and lateral nuclei. Lesions of the left DNLL depressed the release and uptake of [3H]GABA in the ventral pieces of the IC, but there was a greater depression in the ventral IC contralateral to the lesioned DNLL. There were good correlations between the percentage of neuronal loss in the left DNLL and deficits in [3H]GABA release and uptake activities in the ipsi‐ and contralateral ventral IC. By contrast, there was no depression of [3H]GABA release and uptake in the dorsal pieces of the IC. The localization of the deficits in release and uptake appears to match the distribution of the synaptic endings of the DNLL pathways in the IC. This correspondence associates GABA release and uptake activities with the DNLL projections to the IC and, therefore, suggests that GABA may be a transmitter of these pathways. The release and uptake of [14C]glycine was also measured to determine whether glycine might be a transmitter of the DNLL pathways to the IC. Lesions of the left DNLL failed to alter the Ca2+‐dependent release or the uptake of [14C]glycine, suggesting that DNLL neurons are unlikely to use this compound as a transmitter.

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA‐PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca2+‐deprivation and high‐Mg2+ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [14C]‐glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose‐free solutions and stimulated electrically.