Neural Plasticity in Tinnitus Mechanisms.

Basic work into neuroplasticity mechanisms in both invertebrate and vertebrate brains, followed by the development of the first animal model of tinnitus, and coupled with clinical studies of tinnitus, meant that, by 1990, Jastreboff [...].

The large numbers of minicolumns in the primary visual cortex of humans, chimpanzees and gorillas are related to high visual acuity.

Minicolumns are thought to be a fundamental neural unit in the neocortex and their replication may have formed the basis of the rapid cortical expansion that occurred during primate evolution. We sought evidence of minicolumns in the primary visual cortex (V-1) of three great apes, three rodents and representatives from three other mammalian orders: Eulipotyphla (European hedgehog), Artiodactyla (domestic pig) and Carnivora (ferret). Minicolumns, identified by the presence of a long bundle of radial, myelinated fibers stretching from layer III to the white matter of silver-stained sections, were found in the human, chimpanzee, gorilla and guinea pig V-1. Shorter bundles confined to one or two layers were found in the other species but represent modules rather than minicolumns. The inter-bundle distance, and hence density of minicolumns, varied systematically both within a local area that might represent a hypercolumn but also across the whole visual field. The distance between all bundles had a similar range for human, chimpanzee, gorilla, ferret and guinea pig: most bundles were 20-45 µm apart. By contrast, the space between bundles was greater for the hedgehog and pig (20-140 µm). The mean density of minicolumns was greater in tangential sections of the gorilla and chimpanzee (1,243-1,287 bundles/mm2) than in human (314-422 bundles/mm2) or guinea pig (643 bundles/mm2). The minicolumnar bundles did not form a hexagonal lattice but were arranged in thin curving and branched bands separated by thicker bands of neuropil/somata. Estimates of the total number of modules/minicolumns within V-1 were strongly correlated with visual acuity.

Quantifying spatial non-stationarity in the relationship between landscape structure and the provision of ecosystem services: An example in the New Zealand hill country.

Knowing how landscape structure affects the provision of ecosystem services (ES) is an important first step toward better landscape planning. Because landscape structure is often heterogenous across space, modelling the relationship between landscape structure and the provision of ES must account for spatial non-stationarity. This paper examines the relationship between landscape structure and the provision of ES using a hill country and steep-land case farm in New Zealand. Indicators derived from land cover and topographical data such as Largest Patch Index (LPI), Contrast Class Edge (CCE), Edge Density (ED), and Terrain slope (SLOPE) were used to examine the landscape's structure and pattern. Measures of pasture productivity, soil erosion control, and water supply were derived with InVEST tools and spatial analysis in a GIS. Multiscale Geographically Weighted Regression (MGWR) was used to evaluate the relationship between indicators of landscape structure and the provisioning of ES. Other regression models, including Ordinary Least Square (OLS) and Geographically Weighted Regression (GWR), were carried out to evaluate the performance of MGWR. Results showed that landscape patterns significantly affect the supply of all mapped ES, and this varies across the landscape, dependent on the pattern of topographical features and land cover pattern and structure. MWGR outperformed other OLS and GWR in terms of explanatory power of the ES determinants and had a better ability to deal with the presence of spatial autocorrelation. Spatially and quantitatively detailed variations of the relationship between landscape structure and the provision of ES provide a scientific basis to inform the design of sustainable multifunctional landscapes. Information derived from this analysis can be used for spatial planning of farmed landscapes to promote multiple ES which meet multiple sustainable development objectives.

Juxtacellular Labeling of Stellate, Disk and Basket Neurons in the Central Nucleus of the Guinea Pig Inferior Colliculus.

We reconstructed the intrinsic axons of 32 neurons in the guinea pig inferior colliculus (IC) following juxtacellular labeling. Biocytin was injected into cells in vivo, after first analyzing physiological response properties. Based on axonal morphology there were two classes of neuron: (1) laminar cells (14/32, 44%) with an intrinsic axon and flattened dendrites confined to a single fibrodendritic lamina and (2) translaminar cells (18/32, 56%) with axons that terminated in two or more laminae in the central nucleus (ICc) or the surrounding cortex. There was also one small, low-frequency cell with bushy-like dendrites that was very sensitive to interaural timing differences. The translaminar cells were subdivided into three groups of cells with: (a) stellate dendrites that crossed at least two laminae (8/32, 25%); (b) flattened dendrites confined to one lamina and that had mainly en passant axonal swellings (7/32, 22%) and (c) short, flattened dendrites and axons with distinctive clusters of large terminal boutons in the ICc (3/32, 9%). These terminal clusters were similar to those of cortical basket cells. The 14 laminar cells all had sustained responses apart from one offset response. Almost half the non-basket type translaminar cells (7/15) had onset responses while the others had sustained responses. The basket cells were the only ones to have short-latency (7-9 ms), chopper responses and this distinctive temporal response should allow them to be studied in more detail in future. This is the first description of basket cells in the auditory brainstem, but more work is required to confirm their neurotransmitter and precise post-synaptic targets.

Salicylate decreases the spontaneous firing rate of guinea pig auditory nerve fibres.

Tinnitus has similarities to chronic neuropathic pain where there are changes in the firing rate of different types of afferent neurons. We postulated that one possible cause of tinnitus is a change in the distribution of spontaneous firing rates in at least one type of afferent auditory nerve fibre in anaesthetised guinea pigs. In control animals there was a bimodal distribution of spontaneous rates, but the position of the second mode was different depending upon whether the fibres responded best to high (> 4 kHz) or low (≤4 kHz) frequency tonal stimulation. The simplest and most reliable way of inducing tinnitus in experimental animals is to administer a high dose of sodium salicylate. The distribution of the spontaneous firing rates was different when salicylate (350 mg/kg) was administered, even when the sample was matched for the distribution of characteristic frequencies in the control population. The proportion of medium spontaneous rate fibres (MSR, 1≤ spikes/s ≤20) increased while the proportion of the highest, high spontaneous firing rate fibres (HSR, > 80 spikes/s) decreased following salicylate. The median rate fell from 64.7 spikes/s (control) to 35.4 spikes/s (salicylate); a highly significant change (Kruskal-Wallis test p < 0.001). When the changes were compared with various models of statistical probability, the most accurate model was one where most HSR fibres decreased their firing rate by 32 spikes/s. Thus, we have shown a reduction in the firing rate of HSR fibres that may be related to tinnitus.

Systematic approach to selecting licensed drugs for repurposing in the treatment of progressive multiple sclerosis.

OBJECTIVE: To establish a rigorous, expert-led, evidence-based approach to the evaluation of licensed drugs for repurposing and testing in clinical trials of people with progressive multiple sclerosis (MS). METHODS: We long-listed licensed drugs with evidence of human safety, blood-brain barrier penetrance and demonstrable efficacy in at least one animal model, or mechanistic target, agreed by a panel of experts and people with MS to be relevant to the pathogenesis of progression. We systematically reviewed the preclinical and clinical literature for each compound, condensed this into a database of summary documents and short-listed drugs by scoring each one of them. Drugs were evaluated for immediate use in a clinical trial, and our selection was scrutinised by a final independent expert review. RESULTS: From a short list of 55 treatments, we recommended four treatments for immediate testing in progressive MS: R-α-lipoic acid, metformin, the combination treatment of R-α-lipoic acid and metformin, and niacin. We also prioritised clemastine, lamotrigine, oxcarbazepine, nimodipine and flunarizine. CONCLUSIONS: We report a standardised approach for the identification of candidate drugs for repurposing in the treatment of progressive MS.

Nitric oxide increases gain in the ventral cochlear nucleus of guinea pigs with tinnitus.

Previous work has led to the hypothesis that, during the production of noise-induced tinnitus, higher levels of nitric oxide (NO), in the ventral cochlear nucleus (VCN), increase the gain applied to a reduced input from the cochlea. To test this hypothesis, we noise-exposed 26 guinea pigs, identified evidence of tinnitus in 12 of them and then compared the effects of an iontophoretically applied NO donor or production inhibitor on VCN single unit activity. We confirmed that the mean driven firing rate for the tinnitus and control groups was the same while it had fallen in the non-tinnitus group. By contrast, the mean spontaneous rate had increased for the tinnitus group relative to the control group, while it remained the same for the non-tinnitus group. A greater proportion of units responded to exogenously applied NO in the tinnitus (56%) and non-tinnitus groups (71%) than a control population (24%). In the tinnitus group, endogenous NO facilitated the driven firing rate in 37% (7/19) of neurons and appeared to bring the mean driven rate back up to control levels by a mechanism involving N-methyl-D-aspartic acid (NMDA) receptors. By contrast, in the non-tinnitus group, endogenous NO only facilitated the driven firing rate in 5% (1/22) of neurons and there was no facilitation of driven rate in the control group. The effects of endogenous NO on spontaneous activity were unclear. These results suggest that NO is involved in increasing the gain applied to driven activity, but other factors are also involved in the increase in spontaneous activity.

Nitric oxide regulates the firing rate of neuronal subtypes in the guinea pig ventral cochlear nucleus.

The gaseous free radical, nitric oxide (NO) acts as a ubiquitous neuromodulator, contributing to synaptic plasticity in a complex way that can involve either long term potentiation or depression. It is produced by neuronal nitric oxide synthase (nNOS) which is presynaptically expressed and also located postsynaptically in the membrane and cytoplasm of a subpopulation of each major neuronal type in the ventral cochlear nucleus (VCN). We have used iontophoresis in vivo to study the effect of the NOS inhibitor L-NAME (L-NG-Nitroarginine methyl ester) and the NO donors SIN-1 (3-Morpholinosydnonimine hydrochloride) and SNOG (S-Nitrosoglutathione) on VCN units under urethane anaesthesia. Collectively, both donors produced increases and decreases in driven and spontaneous firing rates of some neurones. Inhibition of endogenous NO production with L-NAME evoked a consistent increase in driven firing rates in 18% of units without much effect on spontaneous rate. This reduction of gain produced by endogenous NO was mirrored when studying the effect of L-NAME on NMDA(N-Methyl-D-aspartic acid)-evoked excitation, with 30% of units showing enhanced NMDA-evoked excitation during L-NAME application (reduced NO levels). Approximately 25% of neurones contain nNOS and the NO produced can modulate the firing rate of the main principal cells: medium stellates (choppers), large stellates (onset responses) and bushy cells (primary-like responses). The main endogenous role of NO seems to be to partly suppress driven firing rates associated with NMDA channel activity but there is scope for it to increase neural gain if there were a pathological increase in its production following hearing loss.

Variation in Saturated Hydraulic Conductivity at the Outcrop Scale, the Whanganui Basin, New Zealand.

Groundwater flow and contaminant transport are strongly influenced by hydrogeological spatial variation. Understanding the textural heterogeneity of aquifer and aquitard units is critical for predicting preferential flow pathways, but is often hindered by sparse hydrogeological data, widely spaced data points, and complex stratigraphy. Here, we demonstrate the application of a relatively new air permeameter technology, providing a cost-effective, rapid alternative for characterizing hydrostratigraphic units in the field. The aim of this research is to (1) characterize the variation of saturated hydraulic conductivity across shallow-marine hydrostratigraphic units of the Whanganui Basin, New Zealand, and (2) assess the variation of saturated hydraulic conductivity within individual hydrostratigraphic units and relate these changes to facies and depositional environments. Results suggest heterogeneity within fine-grained aquitard units is controlled by bioturbation, whereby burrowing, ingestion and defecation results in grain size segregation and differential micrite cementation. Coarse-grained heterolithic aquifer facies display sharp changes in permeability across planar to cross-bedded sets, related to current and wave energy fluctuations within shallow-marine depositional settings. Bedding plane orientation creates high permeability zones that promotes down dip subsurface flow. Down dip gradation of coarse-grained nearshore facies into fine-grained shelf facies along the paleo shoreline-shelf transect is suggested to promote lateral and vertical groundwater flow within the basin fill. Air permeameter techniques have potential for application within groundwater basins around the world, providing datasets that facilitate greater understanding of groundwater systems, informing practices and policies for targeted water quality management.

The role of mouse tumour models in the discovery and development of anticancer drugs.

Our understanding of cancer biology has increased substantially over the past 30 years. Despite this, and an increasing pharmaceutical company expenditure on research and development, the approval of novel oncology drugs during the past decade continues to be modest. In addition, the attrition of agents during clinical development remains high. This attrition can be attributed, at least in part, to the clinical development being underpinned by the demonstration of predictable efficacy in experimental models of human tumours. This review will focus on the range of models available for the discovery and development of anticancer drugs, from traditional subcutaneous injection of tumour cell lines to mice genetically engineered to spontaneously give rise to tumours. It will consider the best time to use the models, along with practical applications and shortcomings. Finally, and most importantly, it will describe how these models reflect the underlying cancer biology and how well they predict efficacy in the clinic. Developing a line of sight to the clinic early in a drug discovery project provides clear benefit, as it helps to guide the selection of appropriate preclinical models and facilitates the investigation of relevant biomarkers.

Gap-induced inhibition of the post-auricular muscle response in humans and guinea pigs.

A common method for measuring changes in temporal processing sensitivity in both humans and animals makes use of GaP-induced Inhibition of the Acoustic Startle (GPIAS). It is also the basis of a common method for detecting tinnitus in rodents. However, the link to tinnitus has not been properly established because GPIAS has not yet been used to objectively demonstrate tinnitus in humans. In guinea pigs, the Preyer (ear flick) myogenic reflex is an established method for measuring the acoustic startle for the GPIAS test, while in humans, it is the eye-blink reflex. Yet, humans have a vestigial remnant of the Preyer reflex, which can be detected by measuring skin surface potentials associated with the Post-Auricular Muscle Response (PAMR). A similar electrical potential can be measured in guinea pigs and we aimed to show that the PAMR could be used to demonstrate GPIAS in both species. In guinea pigs, we compare the GPIAS measured using the pinna movement of the Preyer reflex and the electrical potential of the PAMR to demonstrate that the two are at least equivalent. In humans, we establish for the first time that the PAMR provides a reliable way of measuring GPIAS that is a pure acoustic alternative to the multimodal eye-blink reflex. Further exploratory tests showed that while eye gaze position influenced the size of the PAMR response, it did not change the degree of GPIAS. Our findings confirm that the PAMR is a sensitive method for measuring GPIAS and suggest that it may allow direct comparison of temporal processing between humans and animals and may provide a basis for an objective test of tinnitus.

Weka Trainable Segmentation Plugin in ImageJ: A Semi-Automatic Tool Applied to Crystal Size Distributions of Microlites in Volcanic Rocks.

Crystals within volcanic rocks record geochemical and textural signatures during magmatic evolution before eruption. Clues to this magmatic history can be examined using crystal size distribution (CSD) studies. The analysis of CSDs is a standard petrological tool, but laborious due to manual hand-drawing of crystal margins. The trainable Weka segmentation (TWS) plugin in ImageJ is a promising alternative. It uses machine learning and image segmentation to classify an image. We recorded back-scattered electron (BSE) images of three volcanic samples with different crystallinity (35, 50 and ≥85 vol. %), using scanning electron microscopes (SEM) of variable image resolutions, which we then tested using TWS. Crystal measurements obtained from the automatically segmented images are compared with those of the manual segmentation. Samples up to 50 vol. % crystallinity are successfully segmented using TWS. Segmentation at significantly higher crystallinities fails, as crystal boundaries cannot be distinguished. Accuracy performance tests for the TWS classifiers yield high F-scores (>0.930), hence, TWS is a successful and fast computing tool for outlining crystals from BSE images of glassy rocks. Finally, reliable CSD's can be derived using a low-cost desktop SEM, paving the way for a wide range of research to take advantage of this new petrological method.

Mammalian behavior and physiology converge to confirm sharper cochlear tuning in humans.

Frequency analysis of sound by the cochlea is the most fundamental property of the auditory system. Despite its importance, the resolution of this frequency analysis in humans remains controversial. The controversy persists because the methods used to estimate tuning in humans are indirect and have not all been independently validated in other species. Some data suggest that human cochlear tuning is considerably sharper than that of laboratory animals, while others suggest little or no difference between species. We show here in a single species (ferret) that behavioral estimates of tuning bandwidths obtained using perceptual masking methods, and objective estimates obtained using otoacoustic emissions, both also employed in humans, agree closely with direct physiological measurements from single auditory-nerve fibers. Combined with human behavioral data, this outcome indicates that the frequency analysis performed by the human cochlea is of significantly higher resolution than found in common laboratory animals. This finding raises important questions about the evolutionary origins of human cochlear tuning, its role in the emergence of speech communication, and the mechanisms underlying our ability to separate and process natural sounds in complex acoustic environments.

Communication calls produced by electrical stimulation of four structures in the guinea pig brain.

One of the main central processes affecting the cortical representation of conspecific vocalizations is the collateral output from the extended motor system for call generation. Before starting to study this interaction we sought to compare the characteristics of calls produced by stimulating four different parts of the brain in guinea pigs (Cavia porcellus). By using anaesthetised animals we were able to reposition electrodes without distressing the animals. Trains of 100 electrical pulses were used to stimulate the midbrain periaqueductal grey (PAG), hypothalamus, amygdala, and anterior cingulate cortex (ACC). Each structure produced a similar range of calls, but in significantly different proportions. Two of the spontaneous calls (chirrup and purr) were never produced by electrical stimulation and although we identified versions of chutter, durr and tooth chatter, they differed significantly from our natural call templates. However, we were routinely able to elicit seven other identifiable calls. All seven calls were produced both during the 1.6 s period of stimulation and subsequently in a period which could last for more than a minute. A single stimulation site could produce four or five different calls, but the amygdala was much less likely to produce a scream, whistle or rising whistle than any of the other structures. These three high-frequency calls were more likely to be produced by females than males. There were also differences in the timing of the call production with the amygdala primarily producing calls during the electrical stimulation and the hypothalamus mainly producing calls after the electrical stimulation. For all four structures a significantly higher stimulation current was required in males than females. We conclude that all four structures can be stimulated to produce fictive vocalizations that should be useful in studying the relationship between the vocal motor system and cortical sensory representation.

Gap-induced reductions of evoked potentials in the auditory cortex: A possible objective marker for the presence of tinnitus in animals.

Animal models of tinnitus are essential for determining the underlying mechanisms and testing pharmacotherapies. However, there is doubt over the validity of current behavioural methods for detecting tinnitus. Here, we applied a stimulus paradigm widely used in a behavioural test (gap-induced inhibition of the acoustic startle reflex GPIAS) whilst recording from the auditory cortex, and showed neural response changes that mirror those found in the behavioural tests. We implanted guinea pigs (GPs) with electrocorticographic (ECoG) arrays and recorded baseline auditory cortical responses to a startling stimulus. When a gap was inserted in otherwise continuous background noise prior to the startling stimulus, there was a clear reduction in the subsequent evoked response (termed gap-induced reductions in evoked potentials; GIREP), suggestive of a neural analogue of the GPIAS test. We then unilaterally exposed guinea pigs to narrowband noise (left ear; 8-10 kHz; 1 h) at one of two different sound levels - either 105 dB SPL or 120 dB SPL - and recorded the same responses seven-to-ten weeks following the noise exposure. Significant deficits in GIREP were observed for all areas of the auditory cortex (AC) in the 120 dB-exposed GPs, but not in the 105 dB-exposed GPs. These deficits could not simply be accounted for by changes in response amplitudes. Furthermore, in the contralateral (right) caudal AC we observed a significant increase in evoked potential amplitudes across narrowband background frequencies in both 105 dB and 120 dB-exposed GPs. Taken in the context of the large body of literature that has used the behavioural test as a demonstration of the presence of tinnitus, these results are suggestive of objective neural correlates of the presence of noise-induced tinnitus and hyperacusis.

Effects of the cannabinoid CB1 agonist ACEA on salicylate ototoxicity, hyperacusis and tinnitus in guinea pigs.

Cannabinoids have been suggested as a therapeutic target for a variety of brain disorders. Despite the presence of their receptors throughout the auditory system, little is known about how cannabinoids affect auditory function. We sought to determine whether administration of arachidonyl-2'-chloroethylamide (ACEA), a highly-selective CB1 agonist, could attenuate a variety of auditory effects caused by prior administration of salicylate, and potentially treat tinnitus. We recorded cortical resting-state activity, auditory-evoked cortical activity and auditory brainstem responses (ABRs), from chronically-implanted awake guinea pigs, before and after salicylate + ACEA. Salicylate-induced reductions in click-evoked ABR amplitudes were smaller in the presence of ACEA, suggesting that the ototoxic effects of salicylate were less severe. ACEA also abolished salicylate-induced changes in cortical alpha band (6-10 Hz) oscillatory activity. However, salicylate-induced increases in cortical evoked activity (suggestive of the presence of hyperacusis) were still present with salicylate + ACEA. ACEA administered alone did not induce significant changes in either ABR amplitudes or oscillatory activity, but did increase cortical evoked potentials. Furthermore, in two separate groups of non-implanted animals, we found no evidence that ACEA could reverse behavioural identification of salicylate- or noise-induced tinnitus. Together, these data suggest that while ACEA may be potentially otoprotective, selective CB1 agonists are not effective in diminishing the presence of tinnitus or hyperacusis.

Reductions in cortical alpha activity, enhancements in neural responses and impaired gap detection caused by sodium salicylate in awake guinea pigs.

Tinnitus chronically affects between 10-15% of the population but, despite its prevalence, the underlying mechanisms are still not properly understood. One experimental model involves administration of high doses of sodium salicylate, as this is known to reliably induce tinnitus in both humans and animals. Guinea pigs were implanted with chronic electrocorticography (ECoG) electrode arrays, with silver-ball electrodes placed on the dura over left and right auditory cortex. Two more electrodes were positioned over the cerebellum to monitor auditory brainstem responses (ABRs). We recorded resting-state and auditory evoked neural activity from awake animals before and 2 h following salicylate administration (350 mg/kg; i.p.). Large increases in click-evoked responses (> 100%) were evident across the whole auditory cortex, despite significant reductions in wave I ABR amplitudes (in response to 20 kHz tones), which are indicative of auditory nerve activity. In the same animals, significant decreases in 6-10 Hz spontaneous oscillations (alpha waves) were evident over dorsocaudal auditory cortex. We were also able to demonstrate for the first time that cortical evoked potentials can be inhibited by a preceding gap in background noise [gap-induced pre-pulse inhibition (PPI)], in a similar fashion to the gap-induced inhibition of the acoustic startle reflex that is used as a behavioural test for tinnitus. Furthermore, 2 h following salicylate administration, we observed significant deficits in PPI of cortical responses that were closely aligned with significant deficits in behavioural responses to the same stimuli. Together, these data are suggestive of neural correlates of tinnitus and oversensitivity to sound (hyperacusis).

Neuroanatomical Alterations in Tinnitus Assessed with Magnetic Resonance Imaging.

Previous studies of anatomical changes associated with tinnitus have provided inconsistent results, with some showing significant cortical and subcortical changes, while others have found effects due to hearing loss, but not tinnitus. In this study, we examined changes in brain anatomy associated with tinnitus using anatomical scans from 128 participants with tinnitus and hearing loss, tinnitus with clinically normal hearing, and non-tinnitus controls with clinically normal hearing. The groups were matched for hearing loss, age and gender. We employed voxel- and surface-based morphometry (SBM) to investigate gray and white matter volume and thickness within regions-of-interest (ROI) that were based on the results of previous studies. The largest overall effects were found for age, gender, and hearing loss. With regard to tinnitus, analysis of ROI revealed numerous small increases and decreases in gray matter and thickness between tinnitus and non-tinnitus controls, in both cortical and subcortical structures. For whole brain analysis, the main tinnitus-related significant clusters were found outside sensory auditory structures. These include a decrease in cortical thickness for the tinnitus group compared to controls in the left superior frontal gyrus (SFG), and a decrease in cortical volume with hearing loss in left Heschl's gyrus (HG). For masked analysis, we found a decrease in gray matter volume in the right Heschle's gyrus for the tinnitus group compared to the controls. We found no changes in the subcallosal region as reported in some previous studies. Overall, while some of the morphological differences observed in this study are similar to previously published findings, others are entirely different or even contradict previous results. We highlight other discrepancies among previous results and the increasing need for a more precise subtyping of the condition.

Histological Basis of Laminar MRI Patterns in High Resolution Images of Fixed Human Auditory Cortex.

Functional magnetic resonance imaging (fMRI) studies of the auditory region of the temporal lobe would benefit from the availability of image contrast that allowed direct identification of the primary auditory cortex, as this region cannot be accurately located using gyral landmarks alone. Previous work has suggested that the primary area can be identified in magnetic resonance (MR) images because of its relatively high myelin content. However, MR images are also affected by the iron content of the tissue and in this study we sought to confirm that different MR image contrasts did correlate with the myelin content in the gray matter and were not primarily affected by iron content as is the case in the primary visual and somatosensory areas. By imaging blocks of fixed post-mortem cortex in a 7 T scanner and then sectioning them for histological staining we sought to assess the relative contribution of myelin and iron to the gray matter contrast in the auditory region. Evaluating the image contrast in [Formula: see text]-weighted images and quantitative [Formula: see text] maps showed a reasonably high correlation between the myelin density of the gray matter and the intensity of the MR images. The correlation with T1-weighted phase sensitive inversion recovery (PSIR) images was better than with the previous two image types, and there were clearly differentiated borders between adjacent cortical areas in these images. A significant amount of iron was present in the auditory region, but did not seem to contribute to the laminar pattern of the cortical gray matter in MR images. Similar levels of iron were present in the gray and white matter and although iron was present in fibers within the gray matter, these fibers were fairly uniformly distributed across the cortex. Thus, we conclude that T1- and [Formula: see text]-weighted imaging sequences do demonstrate the relatively high myelin levels that are characteristic of the deep layers in primary auditory cortex and allow it and some of the surrounding areas to be reliably distinguished.

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing. The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).