Kamakshi V. Gopal

Neurotoxic effects of mercury on auditory cortex networks growing on microelectrode arrays: a preliminary analysis.

Mercury is known to cause sensorineural hearing loss and impaired speech perception. However, there is still a lack of a quantitative description of mercury toxicity on central auditory structures. This is a preliminary study using the novel technique of microelectrode array (MEA) recordings to evaluate acute and chronic neurotoxic effects of mercury on auditory cortex networks (ACNs) in vitro. Morphological and electrophysiological effects of mercuric chloride (HgCl(2)) were studied. Neurons dissociated from auditory cortices of 14-day-old mouse embryos were grown on photoetched MEAs containing 64 transparent indium-tin oxide (ITO) electrodes. For acute electrophysiological experiments, the spontaneous spiking and bursting activity from ACNs were compared before and after application of HgCl(2). For chronic electrophysiological experiments, auditory cortex cultures were treated with various concentrations of HgCl(2) from the day of seeding, and were tested 4 weeks later for the presence of spontaneous activity. Morphological analysis was conducted on 8-day-old ACNs treated with HgCl(2) for 3 days. Results of acute experiments indicated that <75 mM of HgCl(2) had an excitatory effect of variable magnitude on the spontaneous activity of ACNs; however, concentrations above 100 microM completely and irreversibly inhibited spike and burst activity. Chronic exposure of ACNs to 10 microM HgCl(2) completely blocked the spontaneous activity. Morphological analysis indicated that 10 microM HgCl(2) caused neuronal cell death in 3 days. It is concluded that HgCl(2) has a more toxic effect on auditory networks when exposed chronically, and the levels of mercury showing toxic effects on ACNs are within the dose range shown to cause neurologic symptoms in humans.

BINAURAL INTERACTION COMPONENT IN CHILDREN AT RISK FOR CENTRAL AUDITORY PROCESSING DISORDERS

The binaural interaction component (BIC) occurring in the latency range of peak V of the auditory brainstem responses (ABR) was investigated in nine normal children, comprising the control group, and nine children at risk for central auditory processing disorders (CAPD), comprising the CAPD group. All children tested had normal hearing, normal intelligence and normal ABR thresholds. Averaged BIC obtained from the difference between the summed and binaural ABR waveforms was compared between the two groups for amplitude and latency measures. Results indicated a significant reduction in the amplitude of the BIC occurring in the latency domain of ABR peak V, in the CAPD group.

Differential acute effects of fluoxetine on frontal and auditory cortex networks in vitro.

Primary cultures of neuronal networks grown on microelectrode arrays were used to quantify acute effects of fluoxetine (Prozac) on spontaneous spike and burst activity. For frontal cortex cultures, fluoxetine showed consistent inhibitory effects and terminated activity at 10-16 microM. IC(50) mean+/-S.E. for spike rates was 5.4+/-0.7 microM (n=15). For auditory cortex cultures, fluoxetine caused excitation at 1-10 microM, initial inhibition at 15 microM, and activity cessation at 20-25 microM. The spike rate IC(50) was 15.9+/-1.0 microM (n=11). Fluoxetine did not change the action potential waveform shape. However, at high concentrations, it caused total cessation of spike activity on all channels. The inhibition caused by fluoxetine was reversible for both tissues. Based on the results, we conclude that cultures showed repeatable, concentration-dependent sensitivities to fluoxetine but demonstrated tissue-specific responses for frontal and auditory cortex networks. These responses may not be due to the interference with serotonin reuptake, but may be due to a secondary effect on ionic channels.

Auditory cortical neurons in vitro: cell culture and multichannel extracellular recording.

Self organization, pattern generation, and pattern processing in local cortical circuits are difficult to study in vivo. The complexities of cortical circuits require simplified systems for study. We have developed a simplified model of auditory cortical neurons growing as monolayer networks in culture. Neurons dissociated from auditory cortex of 14-day mouse embryos were grown on photoetched microelectrode array containing 64 transparent indium-tin oxide electrodes. Cultures were maintained in incubators for up to 113 days. Neurons developed processes and made synaptic connections. All cultures were spontaneously active and exhibited complex temporal burst patterns. In a data set of 12 cultures, the number of active channels varied from culture to culture and ranged from 6-17. Signal/noise ratios ranged from 3:1 to a maximum of 16:1. No significant correlations were found between age of the culture and number of active channels, or signal/noise ratios. Spontaneous firing patterns recorded from various channels showed complex bursting patterns in all cultures. Within a culture, coordinated synchronous bursting were found among some channels, and independent bursting on others. Preliminary histological analysis of cultures using the Loots-modified Bodian stain showed neurons with axonal and dendritic profiles growing extensively on top of the glial carpet. Neuronal processes crossing the electrodes singly or in small groups were also observed. Pyramidal and non-pyramidal cells could be identified. In a pool of 2,093 neurons in a 49-day-old culture, the average size of the somata was found to be 16 microns, with a mode of 12 microns.

Slope analysis of Auditory Brainstem Responses in children at risk of central auditory processing disorders.

The method of slope vectors was used to quantify Auditory Brainstem Responses (ABR) obtained from nine normal children and nine children at risk for central auditory processing disorders (CAPD) with language impairment, for monaural and binaural stimulation conditions. Slopes thus obtained were subjected to K-Means Cluster Analysis. Distinction between the two groups was obtained only for binaural stimulation conditions, wherein all normal children were grouped under cluster 1 with higher slope values and 6 out of 9 CAPD children were grouped under cluster 2 with lower slopes. The results suggest that there may be several subcategories among children who are found to be at risk for CAPD. One of the subcategories may comprise children who exhibit poor ABR morphology, especially during binaural stimulation conditions, which could be due to binaural interference.

Audiological findings in individuals exposed to organic solvents: case studies.

Millions of people around the world are exposed to industrial organic solvents such as toluene and xylene in the manufacturing sectors. Solvents are neurotoxic substances that are detrimental to the functioning of the nervous system, including the central auditory nervous system (CANS). This study investigated hearing and auditory processing in seven individuals with a history of exposure to industrial solvents. A battery of audiological tests was administered to all subjects: pure tone, speech, and impedance audiometry, otoacoustic emissions tests, auditory brainstem responses, middle latency responses, as well as the SCAN-A and R-SPIN tests with low predictability sentence lists. All individuals in this study exhibited findings consistent with retrocochlear and/or central abnormality. Two of the seven subjects in this study had normal pure tone thresholds at all frequencies bilaterally, yet showed abnormal retrocochlear/central results on one or more tests. The auditory test battery approach used in this study appears to be valuable in evaluating the pathological conditions of the CANS in solvent-exposed individuals.

Acute and sub-chronic functional neurotoxicity of methylphenidate on neural networks in vitro

SummaryMethylphenidate (MPH) is the drug of choice in the treatment of attention deficit and hyperactivity disorders. Although a popular drug, concentration-dependent electrophysiological alteration or impairment (functional toxicity) and reversibility, have not been quantified. This study used spontaneously active neuronal networks growing on microelectrode arrays (MEA) to investigate functional neurotoxicity of MPH by assessing its acute and sub-chronic electrophysiologic effects on auditory cortex networks (ACN) and frontal cortex networks (FCN) at concentrations that reflect clinical doses and overdoses. Acute exposure to 1–300 µM MPH induced concentration-dependent inhibition of spontaneous activity with spike rate IC50 values (concentration inducing 50% inhibition) of 112.9 ± 18.6 and 108.0 ± 18.9 µM for ACNs and FCNs respectively. Sub-chronic exposure to 1 mM MPH for 24 h blocked all activity followed by partial spontaneous recovery after 15 h. Tyrosine hydroxylase immunocytochemistry analysis indicated positive staining of neurons, confirming the presence of catecholaminergic neurons in cultured cortex networks.

Unique responses of auditory cortex networks in vitro to low concentrations of quinine

The anti-malarial drug quinine has several side effects including tinnitus. The aim of the study was to determine if cultured auditory networks growing on microelectrode arrays exhibited unique dynamic states when exposed to quinine. Eight auditory cortex networks (ACN), eight frontal cortex networks (FCN), and five inferior colliculus networks (ICN) were used in this study. Response of ACNs to quinine was biphasic, with an excitatory phase followed by inhibition. FCNs and ICNs revealed only inhibitory responses. The concentrations at which the spike rate was inhibited by 50% (IC50 mean +/- SE) were 42.5 +/- 3.9, 28.7 +/- 4.8 and 23.9 +/- 2.1 microM for ACNs, FCNs, and ICNs, respectively. Quinine increased the regularity and coordination of bursting in all three tissues. The increased burst pattern regularity of ICNs coupled with the excitatory responses seen only in ACNs between 1 and 10 microM show a unique susceptibility of auditory tissues to quinine that may be related to the underlying mechanism that triggers tinnitus-like activity.

Auditory cortical neurons in vitro : Initial pharmacological studies

Dissociated embryonic tissue from murine auditory cortex formed spontaneously active monolayer networks in culture that were maintained for up to 113 days in vitro (div). As a first step in determining whether neurons retain histiotypic properties, we subjected a set of 10 cultures to a sequence of 4 synaptically active substances. The test sequence consisted of 50 microM bicuculline, 10 microM strychnine, 5 microM NMDA, and 20 microM GABA. Recordings were made for 5-30 min under each condition followed by complete medium changes. Six to 14 channels with the best signal-to-noise ratios were selected for analysis that consisted of continual chart recordings of integrated burst data and further analysis of short data segments after digitizing and processing. All networks showed spontaneous activity, but had greatly varying native activity ranging from organized, quasi-periodic bursting on all channels to more complex spatio-temporal patterns with less coordination among channels. Bicuculline triggered oscillatory activity, simplified bursting, increased burst amplitude, and enhanced burst regularity among electrodes. Strychnine also changed the burst activity to a simpler pattern and enhanced the burst amplitude, indicating presence of glycine receptors in cortical tissue. Application of NMDA increased burst frequencies, but reduced burst regularity and coordination among channels. 20 microM of GABA inhibited all bursting activity in the networks. These results suggest that monolayer networks cultured on multi-electrode arrays retain some basic histiotypic pharmacological responses and may provide useful platforms for the study of network dynamics in the auditory cortex.

Emerging Histiotypic Properties of Cultured Neuronal Networks

In the past decade, substantial progress has been made with substrate-integrated micro-electrode arrays and the growth of viable networks on such arrays. However, such progress has not been accompanied by the acceptance of dissociated (primary) neuronal cultures as reliable systems for pharmacological and toxicological studies. Given that such quasi-monolayer cultures provide strong long-term adhesion stability, optical access to major components of the network circuitry, and high signal-to-noise ratios for multi-site recording of spatiotemporal action potential (spike) patterns, it is essential that such systems be validated as representative of the parent tissue. In addition, action potential waveshapes can also be monitored quantitatively over long periods of time for assessment of channel pharmacology and statistical surveys of discharges from different neuronal compartments. This chapter summarizes characteristic tissue specificities of native activity and histiotypic pharmacological and toxicological responses in order to demonstrate that appropriate dissociation and culture maintenance techniques can generate spontaneously active networks with remarkable similarity to parent tissue responses in vivo. Work with neuronal tissue in culture can be classified as part of two major mechanistic domains: (1) receptor-dependent studies and (2) circuit-dependent studies. The second domain has received limited attention, however, the first domain is less complex, has been explored by several laboratories, and is supported by a rapidly growing database attesting to the histiotypic nature of network responses in culture. In this chapter, we show that different tissues from the murine CNS have different native activity states and may also differ quantitatively in their pharmacological responses. Nevertheless, the overall pharmacological responses agree well with in vivo data. These results imply that, compared to the parent tissue in situ, primary cultures retain the same general ratio of cells (neurons and glia), receptor properties, synaptic mechanisms, and inherent cellular spike generation characteristics. The lower synaptic density and shallow three-dimensional tissue layer do not seem to impair or alter the type and character of pharmacological responses.