Sanoj K. Suneja

The fatty acid chain elongation system of mammalian endoplasmic reticulum.

Much has been learned about FACES of the endoplasmic reticulum since its discovery in the early 1960s. FACES consists of four component reactions, requires the fatty acid to be activated in the form of a CoA derivative, utilizes reducing equivalents in the form of NADH or NADPH, is induced by a fat-free diet, resides on the cytoplasmic surface of the endoplasmic reticulum, appears to function in concert with the desaturase system and appears to exist in multiple forms (either multiple condensing enzymes connected to a single pathway or multiple pathways). FACES has been found in all tissues investigated, namely, liver, brain, kidney, lung, adrenals, retina, testis, small intestine, blood cells (lymphocytes and neutrophils) and fibroblasts, with one exception--the heart has no measurable activity. Yet, much more needs to be learned. The critical, inducible and rate-limiting condensing enzyme has resisted solubilization and purification; the purification of the other components has met with limited success. We know nothing about the site of synthesis of each component of FACES. How is each component enzyme integrated into the endoplasmic reticulum membrane? Is there a single mRNA directing synthesis of all four components or are there four separate mRNAs? How are elongation and desaturation coordinated? What is (are) the physiological regulator(s) of FACES--ADP, AMP, IP3, G-proteins, phosphorylation, CoA, Ca2+, cAMP, none of these? The molecular biology of FACES is only in the fetal stage of development. We are only scratching the surface--it is an undiscovered country.

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.

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.

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 Glutamatergic Projections from the Cochlear Nucleus to the Superior Olive and the Ventral Nucleus of the Lateral Lemniscus

Abstract: This study attempts to determine if projections ascending from the guinea pig cochlear nucleus (CN) could be glutamatergic and/or aspartatergic. Multiple radio frequency lesions were made to ablate the right CN. The ablation was verified histologically. To identify the principal targets of CN efferents, silver impregnation methods were used to localize the preterminal degeneration of fibers in transverse sections of the brainstem 5 and 7 days after CN ablation. CN efferents projected heavily to the lateral superior olive (LSO) ipsilaterally, the medial superior olive (MSO) bilaterally, and contralaterally to the medial (MNTB) and ventral (VNTB) nuclei of the trapezoid body, the ventral (VNLL) and intermediate nuclei of the lateral lemniscus and the central nucleus of the inferior colliculus (ICc). There were smaller projections to the lateral nucleus of the trapezoid body ipsilaterally, the dorsal and dorsomedial periolivary nuclei bilaterally, and the dorsal nucleus of the lateral lemniscus contralaterally. There were sparse projections to the VNLL and ICc ipsilaterally and the CN contralaterally, and a very sparse projection to the contralateral LSO. To determine if CN efferents were glutamatergic and/or aspartatergic, the fresh brainstem was sectioned transversely and samples of the LSO, MSO, MNTB, VNLL, and ICc were taken to measure the electrically evoked release and the uptake of d‐[3H]Asp and [14C]Gly or [14C]GABA 3–5 days after the CN ablation. The release studies suggest that only certain of the histologically identified projections ascending from the CN may be glutamatergic and/or aspartatergic. CN ablation depressed d‐[3H]Asp release in the MSO bilaterally and in the contralateral MNTB and VNLL, suggesting that the CN efferents to these nuclei may use glutamate or aspartate as a transmitter. It was unclear whether a marginal depression of d‐[3H]Asp release in the ipsilateral LSO reflected the presence of glutamatergic CN projections to this nucleus. d‐[3H]Asp release in the ICc was unaffected, suggesting that CN efferents to this nucleus may not be glutamatergic. There were no deficits in d‐[3H]Asp uptake. [14C]Gly release from the LSO and MSO was unchanged. [14C]Gly uptake was unchanged in the MSO and depressed only in the contralateral LSO, possibly reflecting subnormal uptake activity in endings contributed by contralateral MNTB cells that had lost their CN efferents. [14C]GABA uptake in the MNTB, VNLL, and ICc was unchanged. [14C]GABA release was unchanged in the VNLL and ICc. [14C]GABA release was depressed only in the contralateral MNTB, possibly reflecting the loss of a small complement of GABAergic CN efferents and the reaction of GABAergic projections from the contralateral VNTB to their loss of CN efferents.

AMPA Receptor Binding in Adult Guinea Pig Brain Stem Auditory Nuclei after Unilateral Cochlear Ablation

This study determined if an asymmetric hearing loss, due to unilateral cochlear ablation, could induce the regulation of intracellular AMPA receptors in brain stem auditory nuclei. In young adult guinea pigs, the high-affinity specific binding of [(3)H]AMPA was measured in the cochlear nucleus (CN), the superior olivary complex (SOC), and the auditory midbrain at 2-147 postlesion days. After correction for tissue shrinkage, changes in specific binding relative to that in age-matched unlesioned controls were interpreted as altered numbers and/or activity of intracellular AMPA receptors. In the CN, transient elevations and/or deficits in binding were evident in most regions, which usually recovered by 147 days. However, persistently deficient binding was evident ipsilaterally in the anterior part of the anteroventral CN (AVCNa). In the SOC, transient elevations in binding were evident at 2 days in the medial limb of the lateral superior olive (LSOmed) and the medial superior olive. Between 7 and 147 days, most SOC nuclei exhibited transient, temporally synchronized postlesion deficits in binding. However, late in the survival period, deficits persisted ipsilaterally in the LSOmed and the lateral (LSOlat) limb of the lateral superior olive. In the midbrain, transient elevations and/or deficits in binding were evident in the dorsal nucleus of the lateral lemniscus as well as in the central and dorsal nucleus of the inferior colliculus. A persistent deficit was evident in the intermediate nucleus of the lateral lemniscus. The findings implied that auditory neurons contain regulatory mechanisms that control the numbers and/or activity of intracellular AMPA receptors. Regulation was induced by cochlear nerve destruction and probably by changes in the excitation of glutamatergic neurons. Many of the regulatory changes were transient, except in the ipsilateral AVCNa and LSO, where postlesion downregulations were persistent. The downregulation in the ipsilateral AVCNa was probably induced directly by the loss of cochlear nerve endings. However, other regulatory changes may have been induced by signals carried on pathways emerging from the ipsilateral CN and on centrifugal auditory pathways.

Uptake and Release of d‐Aspartate, GABA, and Glycine in Guinea Pig Brainstem Auditory Nuclei

Abstract: This study attempts to determine if the medial (MSO) and lateral superior olive (LSO), medial nucleus of the trapezoid body (MNTB), ventral nucleus of the lateral lemniscus (VNLL), and central nucleus of the inferior colliculus (ICc) contain glutamatergic synaptic endings. Micropunch and microdissection procedures provided fresh samples of these auditory nuclei for the measurement of the high‐affinity uptake and electrically evoked release of exogenous d‐[3H]ASP. The study also determined if the LSO and MSO contain glycinergic synaptic endings by measuring uptake and release of [14C]‐Gly in these nuclei, and whether the MNTB, VNLL, and ICc contain GABAergic endings by assessing the uptake and release of [14C]GABA in these structures. Several strategies optimized the evoked Ca2+‐dependent release of the labeled amino acids. These included the enhancement of high‐affinity uptake during loading of the markers into the tissues, inhibition of uptake during the subsequent measurement of release, and use of an electrical stimulus current that evoked maximal Ca2+‐dependent release. Each of these nuclei manifested the high‐affinity uptake and the evoked Ca2+‐dependent release of d‐[3H]Asp, suggesting the presence of synaptic endings that may use Glu or Asp as a transmitter. Similar findings suggest the presence of glycinergic synaptic endings in the LSO and MSO, and of GABAergic synaptic endings in the MNTB, VNLL, and ICc.

Regulation of NT-3 and BDNF levels in guinea pig auditory brain stem nuclei after unilateral cochlear ablation

Injury to areas of the central nervous system can alter neurotrophin levels, which may influence postlesion neuronal survival and plasticity. To determine if sensorineural hearing loss induces such changes, we used an enzyme‐linked immunosorbent assay (ELISA) to measure neurotrophin‐3 (NT‐3) and brain‐derived neurotrophic factor (BDNF) levels in adult guinea pig brain stem auditory nuclei 3–60 days after a unilateral cochlear ablation (UCA). After UCA, which destroyed the cochlea and cochlear nerve on one side, NT‐3 levels were usually depressed at 3 days by 22–44% but became elevated transiently at 7 days by 28–124%. BDNF levels were elevated transiently by 50% on the ablated side in the anteroventral (AVCN) and posteroventral (PVCN) cochlear nucleus at 3 days and may have signaled support for the survival of deafferented neurons. Coincident elevation at 3 and 7 days of BDNF or NT‐3 and phosphorylated extracellular signal‐regulated protein kinase 2 (ERK2‐P) suggested a relationship to stimulated signal transduction activity. Elevated neurotrophin levels may have contributed to synaptogenesis in the AVCN and the superior olive and to changes in the synaptic biochemistry in the auditory nuclei after UCA. In contrast, deficiencies or failure to elevate neurotrophin levels within several days of the UCA correlated with upregulation of phosphorylated stress‐activated protein kinase (SAPK‐P), suggesting a relationship with stress‐activated signal transduction and with the sparse degeneration of fibers observed in some of the auditory nuclei after UCA.