Enzymes of acetylcholine metabolism in the rat inferior colliculus.

Although there is strong evidence for cholinergic projections to the rat inferior colliculus, especially from the pedunculopontine tegmental nucleus (Noftz et al., 2020), there is a lack of information about the quantitative prevalence of the enzymes of acetylcholine metabolism in its various portions. We have used microdissection of freeze-dried sections combined with radiometric assays to map the distributions in the rat inferior colliculus of the activities of choline acetyltransferase (ChAT), which catalyzes synthesis of acetylcholine, and acetylcholinesterase (AChE), which catalyzes its breakdown by hydrolysis. Both enzyme activities were present throughout the inferior colliculus. Average ChAT activity was consistently somewhat higher in the external cortex, excluding its most superficial layer, than in the dorsal cortex or central nucleus. Within the external cortex, ChAT activity was about half as high laterally in its most superficial layer as elsewhere. The distribution of AChE activity was more uniform than that of ChAT. Overall, ChAT activity in the rat inferior colliculus was relatively low, about a fifth of that in whole brain of rat and lower than in other central auditory regions, whereas AChE activity was about two-thirds that of rat whole brain and about average for central auditory regions. The results are compared to previous measurements for cat and hamster inferior colliculus. They are consistent with a modest role for cholinergic neurotransmission in the inferior colliculus, to modulate the activity of its major neuronal types.

Analysis of ERK1/2 kinases in the inferior colliculus of rats genetically prone to audiogenic seizures during postnatal development.

The activity of ERK1/2 kinases in the quadrigemina inferior colliculus of Krushinsky-Molodkina rats of different age, which are characterized by an increased seizure readiness compared to Wistar rats, was analyzed. An increased (probably genetically determined) activity of these enzymes during the development of epileptiform activity in ontogeny was found, which may be the cause of abnormalities in the neurotransmitter system functioning.

[Effect of Electroacupuncture on Expression of Catechol-O-methyltransferase in the Inferior Colliculus and Auditory Cortex in Age-related Hearing Loss Guinea Pigs].

OBJECTIVE: To observe the expression of catechol-O-methyltransferase (COMT) in inferior colliculus and auditory cortex of guinea pigs with age-related hearing loss(AHL) induced by D-galactose, so as to explore the possible mechanism of electroacupuncture(EA) underlying preventing AHL. METHODS: Thirty 3-month-old guinea pigs were randomly divided into control group, model group and EA group(n=10 in each group), and ten 18-month-old guinea pigs were allocated as elderly group. The AHL model was established by subcutaneous injection of D-galactose. EA was applied to bilateral "Yifeng"(SJ 17) and "Tinggong"(SI 19) for 15 min in the EA group while modeling, once daily for 6 weeks. After treatment, the latency of auditory brainstem response(ABR) Ⅲ wave was measured by a brain-stem evoked potentiometer. The expressions of COMT in the inferior colliculus and auditory cortex were detected by Western blot. RESULTS: Compared with the control group, the latencies of ABR Ⅲ wave were significantly prolonged and the expressions of COMT in the inferior colliculus and auditory cortex were significantly decreased in the model group and the elderly group(P<0.05). After the treatment, the latency of ABR Ⅲ wave was significantly shortened and the expressions of COMT in the inferior colliculus and auditory cortex were significantly increased in the EA group in comparison with the model group (P<0.05). CONCLUSIONS: EA at "Yifeng" (SJ 17) and "Tinggong" (SI 19) can improve the hearing of age-related deafness in guinea pigs, which may contribute to its effect in up-regulating the expression of COMT in the inferior colliculus and auditory cortex.

NADPH-diaphorase-positive neurons in the human inferior colliculus: morphology, distribution and clinical implications.

Using the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction with nitroblue tetrazolium, we provided a detailed investigation of the distribution, dimensional characteristics and morphology of NADPH-d-positive neurons in the three main subdivisions of the human inferior colliculus (IC): central nucleus, pericentral nucleus, and external nucleus. In accordance with their perikaryal diameter, dendritic and axonal morphology, these neurons were categorized as large (averaging up to 45 µm in diameter), medium (20-30 µm), small (13-16 µm) and very small (7-10 µm). Their morphological differences could contribute to varying functionality and processing capacity. Our results support the hypothesis that large and medium NADPH-d-positive cells represent projection neurons, while the small cells correspond to interneurons. Heretofore, the very small NADPH-d-positive neurons have not been described in any species. Their functions-and if they are, indeed, the smallest neurons in the IC of humans-remain to be clarified. Owing to their location, we posit that they are interneurons that connect the large NADPH-d-positive neurons and thereby serve as an anatomical substrate for information exchange and processing before feeding forward to higher brain centers. Our results also suggest that the broad distribution of nitric oxide (NO) synthesis in the human IC is closely tied to the neuromodulatory action of NO on collicular neurotransmitters such as GABA and glutamate, and to calcium-binding proteins such as parvalbumin. A deeper understanding of the relationship between NADPH-d-positive fibers in all IC connections and their co-localization with other neurotransmitters and calcium-binding proteins will assist in better defining the function of NO in the context of its interplay with the cerebral cortex, the sequelae of the aging process and neurodegenerative disorders.

Alcohol Withdrawal Increases Protein Kinase A Activity in the Rat Inferior Colliculus.

BACKGROUND: Cyclic AMP-dependent protein kinase A (PKA) signaling is a key target for the action of alcohol and may therefore play a role in the pathophysiology of alcohol withdrawal seizures (AWSs). Here, we investigated the role of PKA activity with respect to increased seizure susceptibility in rats that were subjected to alcohol withdrawal. METHODS: Adult male Sprague Dawley rats received 3 daily doses of ethanol (EtOH) (or vehicle) for 4 consecutive days. Rats were then tested for susceptibility to acoustically evoked AWSs 3, 24, and 48 hours after the last alcohol dose. In separate experiments, the inferior colliculus (IC) was collected at these same time points from rats subjected to alcohol withdrawal and control rats following alcohol withdrawal. PKA activity, catalytic Cα (PKACα ) protein, regulatory RIIα (PKARIIα ) protein, and RIIß (PKARIIß ) protein were measured in the IC. Lastly, in situ pharmacological studies were performed to evaluate whether inhibiting PKA activity in the IC suppressed AWSs. RESULTS: In the EtOH-treated group, AWSs were observed at the 24-hour time point, but not at the 3-hour or 48-hour time points. In the IC, PKA activity was significantly higher both 3 hours (i.e., before AWS susceptibility) and 24 hours after the last alcohol dose (when AWS susceptibility peaked) than in control rats. Consistent with these findings, protein levels of the PKACα subunit were significantly increased in the IC both 3 and 24 hours after the last alcohol dose. Lastly, in situ inhibition of PKA activity within the IC suppressed AWSs. CONCLUSIONS: The increase in PKA activity and PKACα protein expression in the IC preceded the occurrence of AWSs, and inhibiting PKA activity within the IC suppressed acoustically evoked AWSs. Together, these findings suggest that altered PKA activity plays a key role in the pathogenesis of AWSs.

Developmental PCB Exposure Increases Audiogenic Seizures and Decreases Glutamic Acid Decarboxylase in the Inferior Colliculus.

Previously, we observed that developmental polychlorinated biphenyl (PCB) exposure resulted in an increase in audiogenic seizures (AGSs) in rats. However, the rats were exposed to loud noise in adulthood, and were not tested for AGS until after 1 year of age, either of which could have interacted with early PCB exposure to increase AGS susceptibility. This study assessed susceptibility to AGS in young adult rats following developmental PCB exposure alone (without loud noise exposure) and investigated whether there was a decrease in GABA inhibitory neurotransmission in the inferior colliculus (IC) that could potentially explain this effect. Female Long-Evans rats were dosed orally with 0 or 6 mg/kg/day of an environmentally relevant PCB mixture from 28 days prior to breeding until the pups were weaned at postnatal day 21. One male-female pair from each litter was retained for the AGS study whilst another was retained for Western blot analysis of glutamic acid decarboxylase (GAD) and GABAAα1 receptor in the IC, the site in the auditory midbrain where AGS are initiated. There was a significant increase in the number and severity of AGSs in the PCB groups, with females somewhat more affected than males. GAD65 was decreased but there was no change in GAD67 or GABAAα1 in the IC indicating decreased inhibitory regulation in the PCB group. These results confirm that developmental PCB exposure alone is sufficient to increase susceptibility to AGS, and provide the first evidence for a possible mechanism of action at the level of the IC.

The expression of mitogen-activated protein kinases and brain-derived neurotrophic factor in inferior colliculi after acoustic trauma.

Acoustic trauma is well known to cause peripheral damage with subsequent effects in the central auditory system. The inferior colliculus (IC) is a major auditory center for the integration of ascending and descending information and is involved in noise-induced tinnitus and central hyperactivity. Here we show that the early effects of acoustic trauma, that eventually result in permanent damage to auditory system, lead to a transient activation of BDNF and mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in the IC. In contrast, the early effects of acoustic trauma that result in a temporary damage produced a reversible activation only of p38. The transient activation of MAPK and BDNF in the IC after permanent acoustic trauma is attributed to the plastic changes triggered by a decreased signal input from the damaged periphery. The pattern of MAPK and BDNF activation in the IC is different from that previously described for the cochlea from this laboratory. The differences in the pattern of MAPK and BDNF expression in the IC highlight unique molecular mechanisms underlying temporary and permanent acoustic damage to the central auditory system.

Evidence for increased NADPH-diaphorase-positive neurons in the central auditory system of the aged rat.

CONCLUSIONS: The age-related increase in the production of nitric oxide (NO) suggests that this increase was related to neuron aging. Additional studies may provide information regarding aging-related changes in the central auditory system. OBJECTIVES: Although NO has been associated with aging, it is unclear whether specific areas of the central auditory system are involved. We therefore assayed aging-related changes in NADPH-diaphorase (NADPH-d), a selective histochemical marker for NO, in the neurons of the central auditory system and other brain regions. MATERIALS AND METHODS: The numbers of NADPH-d-stained neurons and the area and staining density of cell bodies were examined in aged (24 months old) and younger (4 months old) Wistar rats. RESULTS: The number of NADPH-d-positive neurons in the inferior colliculus was significantly increased in aged rats (p<0.05), whereas the area of NADPH-d-positive neurons in all areas did not differ significantly between aged and younger rats (p>0.05). The staining densities of NADPH-d-positive neurons in the inferior colliculus, the auditory cortex, and the visual cortex were significantly greater in aged compared with younger rats (p<0.05).

The distribution of nitric oxide synthase in the inferior colliculus of guinea pig.

The modulation of neuronal activity by the gas nitric oxide is one of the most novel discoveries in neuroscience. In the auditory pathway, the highest expression of nitric oxide synthase is found in the inferior colliculus (IC), an important center for the convergence of parallel ascending pathways traveling in the brainstem, and descending projections from the auditory cortex. Here we use immunocytochemistry with an antibody for neuronal nitric oxide synthase (nNOS), or NOS Type 1, to map the distribution of nNOS expression in the IC of the guinea pig. The results show that nNOS is differentially expressed by both cell bodies and neuropil across its different subdivisions. The highest levels of neuronal staining are seen in the dorsal and lateral cortices, and the commissural nucleus, making them readily distinguishable from the ventro-lateral part of the central nucleus where nNOS expression in neuropil and somata is minimal. Dorso-medially, and caudally, however, the region of nNOS expression extends from the dorsal cortex into the area normally designated as the central nucleus, and nNOS is expressed by neurons characteristic of this subdivision. Our findings support the idea of a gradual transition in cell properties rather than a distinct boundary between the central nucleus and the dorsal cortex. This transition zone may provide a cytoarchitectonic substrate for functional interaction between these two subdivisions.

The selective neurotoxicity produced by 3-chloropropanediol in the rat is not a result of energy deprivation.

The biochemical mechanism of toxicity of the experimental astrocyte neurotoxicant and food contaminant S-3-chloro-1,2-propanediol (3-CPD) has been proposed to be via inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have confirmed this action in liver, which shows inhibition to 6.0+/-0.7% control at the neuropathic dose of 140 mg/kg. However, GAPDH activity in brain only fell to a minimum of 54+/-24% control, and the concentrations of lactate and pyruvate (the downstream products of GAPDH), showed no pre-neuropathic decreases in 3-CPD susceptible brain tissue. There was no inhibition of GAPDH activity in primary astrocyte cultures at sub-cytotoxic exposures. We therefore sought alternative mechanisms to explain its toxicity to astrocytes. We were able to show that 3-CPD is a substrate for glutathione-S-transferase and also that, after bioactivation by alcohol dehydrogenase, it generates an irreversible inhibitor of glutathione reductase. In addition, incubation of brain slices from the 3-CPD-vulnerable inferior colliculus produces a depletion of glutathione and an inhibition of glutathione-S-transferase that is not seen in equivalent slices taken from the 3-CPD-resistant occipital neocortex. A smaller but significant and similarly regionally selective decrease in glutathione content is also seen in vivo. We conclude that 3-CPD does not produce its astrocytic toxicity via energy deprivation, and suggest that selective bioactivation and consequent disruption of redox state is a more likely mechanism.

Tyrosine hydroxylase in rat auditory midbrain: distribution and changes following deafness.

Tyrosine hydroxylase (TH), a key enzyme in the catecholaminergic pathway, allows for the differentiation of dopaminergic neurons. We previously showed decreases in TH gene expression in the rat inferior colliculus (IC) 3 and 21 days following deafness. In the present study, we characterized the normal distribution of TH as well as changes following deafness (bilateral cochlear ablation) in the IC and nuclei of the lateral lemniscus. Immunostaining was compared in three groups of rats: normal hearing (n=8), 21 day deaf (n=5) and 90 days following deafening (n=5). Many TH immunoreactive fibers and puncta were identified in the IC and nuclei of the lateral lemniscus of normal hearing animals and labeling was most dense in the external cortex of the IC. We also identified immunolabeling for fibers and puncta for another catecholaminergic enzyme, dopamine beta hydroxylase (DBH), but not phenylethanolamine-N-methyltranferase (PNMT). Neurons immunopositive for TH but not DBH or PNMT were observed in the dorsal cortex and dorsal horn of the central nucleus of the IC and ventral and intermediate lemniscus. In the central nucleus of the IC and dorsal lateral lemniscus many lightly labeled TH neurons were also DBH positive. Although the number of immunopositive cells in the IC and lemniscus declined 3 weeks and 3 months after deafening, the decline was not significant at three weeks in the VNLL nor after three months in the dorsal cortex. Immunolabeling for TH decreased significantly in IC and lemniscus 3 weeks and 3 months following deafening. These results suggest a role for dopaminergic neurons and fibers in deafness-related plasticity.

Consequences of noise- or styrene-induced cochlear damages on glutamate decarboxylase levels in the rat inferior colliculus.

Both noise and styrene can injure the cochlea, resulting in a reduction of incoming inputs from the cochlea to the central nervous system. In addition, styrene is known to have neurotoxic properties at high doses. The loss of inputs caused by noise has been shown to be compensated by a new equilibrium between excitatory and inhibitory influences within the inferior colliculus (IC). The main goal of this study was to determine whether styrene-induced hearing loss could also be counterbalanced by a GABAergic adjustment in the IC. For this purpose, rats were exposed to noise (97 dB SPL octave band noise centered at 8 kHz), or to a non-neurotoxic dose of styrene for 4 weeks (700 ppm, 6 h/day, 5 days/week). Auditory sensitivity was tested by evoked potentials, and cochlear damage was assessed by hair cell counts. Glutamate decarboxylase (GAD) was dosed in the IC by indirect competitive enzyme-linked immunosorbent assay. Both noise and styrene caused PTSs that reached 27.0 and 14.6 dB respectively. Outer hair cell (OHC) loss caused by noise did not exceed 9% in the first row, on the other hand OHC loss induced by styrene reached 63% in the third row. Only the noise caused a decrease of GAD of 37% compared to that measured in the controls. No significant modification of GAD concentration has been shown after styrene exposure. Thus, central compensation for cochlear damage may depend on the nature of the ototoxic agent. Unless styrene directly affects IC function, it is reasonable to assume that noise causes a modification of inhibitory neurotransmission within the structure because of impairment of afferent supply to the auditory brainstem. The present findings suggest that central compensation for cochlear damage can preferably occur when afferent fibers are altered.

Carboplatin-induced oxidative injury in rat inferior colliculus.

Carboplatin is currently being used as an anticancer drug against human cancers. However, high dose of carboplatin chemotherapy resulted in ototoxicity in cancer patients. Carboplatin-induced ototoxicity was related to oxidative stress to the cochlea and inner hair cell loss in animals. It is likely that initial oxidative injury spreads throughout the neuroaxis of the auditory system later. The study aim was to evaluate carboplatin-induced hearing loss and oxidative injury to the central auditory system (inferior colliculus) of the rat. Male Wistar rats were divided into two groups of seven animals each and treated as follows: (1) control (normal saline, intraperitoneal [i.p.]) and (2) carboplatin (256 mg/kg, i.p.). Auditory brain-evoked responses (ABRs) were recorded before and 4 days after treatments. The animals were sacrificed on the 4th day and inferior colliculus from brain stem and cerebellum were isolated and analyzed. Carboplatin significantly elevated the hearing threshold shifts at clicks, 2-, 4-, 8-, 16-, and 32-kHz tone burst stimuli. Carboplatin significantly increased nitric oxide and lipid peroxidation, xanthine oxidase, and manganese superoxide dismutase activities in the inferior colliculus, but not in the cerebellum, indicating an enhanced flux of free radicals in the central auditory system. Carboplatin significantly depressed the reduced to oxidized glutathione ratio, antioxidant enzyme activities, such as copper-zinc superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, and enzyme protein expressions in the inferior colliculus, but not in the cerebellum, 4 days after treatment. The data suggest that carboplatin induced oxidative injury specifically in the inferior colliculus of the rat leading to hearing loss.

Transplantation of M213-2O cells with enhanced GAD67 expression into the inferior colliculus alters audiogenic seizures.

The purpose of the present study was to examine the effects of GABA-producing cell transplants on audiogenic seizures (AGS). The M213-2O cell line was derived from fetal rat striatum and has GABAergic properties. This cell line was further modified to express human GAD(67) and produce elevated levels of GABA. The present study compares the effects of parent M213-2O cell transplants with those of GAD(67)-modified M213-2O cells in AGS-prone Long-Evans rats. Two weeks following implantation of engineered cells, latency to AGS-typical wild running was increased compared to nonimplanted subjects. Survival of the transplanted cells was confirmed by immunochemical labeling of GAD(67) and Epstein-Barr virus nuclear antigen. These findings support the use of GABA-producing cell lines to modify seizure activity.

Conductive hearing loss results in changes in cytochrome oxidase activity in gerbil central auditory system.

Conductive hearing loss (CHL) restricts auditory input to an intact peripheral auditory system. Effects of deprivation on the central auditory system (CAS) have been debated, although a number of studies support the hypothesis that CHL can cause modification of CAS structure and function. The present study was designed to test the hypothesis that unilateral CHL results in a decrease in cytochrome oxidase (CO) activity in CAS nuclei that receive major afferent input from the affected ear. Gerbils at postnatal day 12 (P21) or 6-8 weeks underwent left unilateral CHL (malleus removal), cochlear ablation, or a sham surgical procedure. After a survival time of 48 hours or 3 weeks, animals were sacrificed and tissue was processed for cytochrome oxidase histochemistry. Optical density (OD) measurements were made from individual neurons in the anteroventral cochlear nucleus (AVCN) and from medial and lateral dendritic fields in the medial superior olivary nucleus (MSO), the lateral superior olivary nucleus, and the inferior colliculus. The width of the CO-stained neuropil in MSO was also measured as an estimate of dendritic length. OD measures were corrected to neutral areas of the brain. Cochlear ablation caused significant decreases in CO activity in left lower brainstem nuclei, particularly in adult animals. Following CHL, a significant decrease in CO activity was observed in the ipsilateral AVCN and a significant increase was observed in the contralateral AVCN. Cochlear ablation resulted in decreased width of MSO neuropil containing dendrites that receive primary input from the ablated ear. CHL resulted in a significant increase in the width of MSO neuropil on both sides of the brain in the P21 animals that survived 3 weeks but not in P21 animals that survived only 48 hours or in the adult animals. Unilateral CHL is associated with changes in CO activity in the AVCN and may affect MSO dendritic length in younger animals.

Tyrosine hydroxylase-like immunoreactive synaptic boutons in the inferior colliculus of the cat.

The data on the distribution of catecholaminergic cells and fibers in such a significant subcortical relay auditory center as the inferior colliculus (IC) are both few and controversial, and ultrastructural data are lacking. Young adult mongrel cats of both sexes were used. Following routine preparation procedures, the ultrathin sections were prepared for the ultrastructural examination of tyrosine hydroxylase (TH)-like immunoreactivity. TH-positive neuronal perikarya were not detected in the IC. On the other hand, an appreciable number of TH-immunoreactive unmyelinated axons and synaptic boutons were found in all subdivisions of the IC, most often in the nucleus externus, followed by the nucleus pericentralis, and a few were seen in the dorsomedial part of the central nucleus. The boutons measured 0.5-1.8 microns, contained pleomorphic synaptic vesicles, and established symmetrical synaptic contacts almost exclusively with dendrites of small caliber.

Effect of repeated seizure experiences on tyrosine hydroxylase immunoreactivities in the brain of genetically epilepsy-prone rats.

The genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy, and has functional noradrenergic deficiencies that act as partial determinants for the seizure predisposition and expression. The present study investigated the effect of repeated seizure experiences by acoustic stimulation (110 dB, 10 times) on the immunoreactivities of tyrosine hydroxylase (TH), a rate-determining enzyme in the synthesis of norepinephrine, in brain regions of GEPRs. TH immunoreactivity in locus coeruleus, the major noradrenergic nucleus in brain, was lower in GEPRs than control Sprague-Dawley rats. It was also decreased in several regions including inferior colliculus of GEPRs. Repeated experiences of audiogenic seizures further decreased TH immunoreactivities in locus coeruleus and inferior colliculus of GEPRs. The results from the present study suggest that the lower immunoreactivities of TH in locus coeruleus and inferior colliculus contribute, at least in part, to the noradrenergic deficits in GEPRs, and repeated seizure experiences further intensified these noradrenergic deficits, which may be related to the altered seizure expression by repetitive audiogenic seizure in GEPRs.

Detection of glutamate decarboxylase isoforms in rat inferior colliculus following acoustic exposure.

The inferior colliculus is a central auditory structure which serves as a site for the integration of ascending and descending auditory information. Changes in central auditory structures may occur with acoustic exposure, which cannot be explained by alterations in cochlear function alone. Rats were exposed to a 10-kHz tone at 100 dB SPL for 9 h. Auditory brainstem response measures showed an initial 25-30-dB threshold shift across all tested frequencies. By 30 days post-exposure, thresholds for clicks and most frequencies returned to near control levels; however, thresholds remained elevated at 10 and 20 kHz. Inner hair cell loss was confined to apical and basal ends of the cochlea, and did not exceed 20%. Inferior colliculus levels of the two isoforms of the GABA synthetic enzyme glutamate decarboxylase (65,000 and 67,000 mol. wt forms) were measured immediately post-exposure (0 h) and at two and 30 days post-exposure using quantitative immunocytochemical and western blotting techniques. Zero-hour measures revealed a significant increase in the level of glutamate decarboxylase (mol. wt 67,000) protein (118%), as well as in the optical density (35%) of immunolabeled cells. By 30 days post-exposure, inferior colliculus protein levels of both glutamate decarboxylase isoforms were significantly below unexposed controls (39% and 21% for the 65,000 and 67,000 mol. wt forms, respectively). These studies describe increased markers for GABA immediately following acoustic exposure, followed by a decline to below control levels from two to 30 days post-exposure. It remains to be determined whether noise trauma-induced changes in glutamate decarboxylase levels in the inferior colliculus reflect protective up-regulation in response to intense stimulation, followed by the establishment of new neurotransmitter equilibrium levels.