The presence of fixative-induced and cellular-derived artifactual autofluorescences (AAFs) presents a challenge in histological analysis involving immunofluorescence. We have established a simple and highly effective method for the reduction of AAFs that are ubiquitous in fixed mammalian brain and other tissues. A compact AAF-quenching photo-irradiation device was constructed using a commercially available light emitting diode (LED) array, cooling unit, and supporting components. The LED panel is comprised of an array of multispectral high intensity LEDs which serve as the illumination source for the photo-irradiation process. Rabbit and cat brain specimens of 5 μm- and 40 μm-thicknesses, respectively, were photo-irradiated for various durations. The AAFs were reduced to near tissue background levels after 24 h of treatment for both deparaffinized and paraffinized rabbit brain specimens, and for the free-floating cat brain specimens. Subsequent immunofluorescence staining using primary antibodies against GFAP, NeuN, Iba-1, and MAP-2, and the corresponding Qdot® and Alexafluor® fluoroconjugates confirmed that the LED photo-irradiation treatment did not compromise the efficiency of the immunofluorescence labeling. The use of the device is not labor intensive, and only requires minimal tissue processing and experimental set-up time, with very low maintenance and operating costs. Finally, multiple specimens, in both slide and well-plate format, can be simultaneously photo-irradiated, thus, allowing for scalable batch processing.
We developed and validated silicon-based neural probes for neural stimulating and recording in long-term implantation in the brain. The probes combine the deep reactive ion etching process and mechanical shaping of their tip region, yielding a mechanically sturdy shank with a sharpened tip to reduce insertion force into the brain and spinal cord, particularly, with multiple shanks in the same array. The arrays’ insertion forces have been quantified in vitro. Five consecutive chronically-implanted devices were fully functional from 3 to 18 months. The microelectrode sites were electroplated with iridium oxide, and the charge injection capacity measurements were performed both in vitro and after implantation in the adult feline brain. The functionality of the chronic array was validated by stimulating in the cochlear nucleus and recording the evoked neuronal activity in the central nucleus of the inferior colliculus. The arrays’ recording quality has also been quantified in vivo with neuronal spike activity recorded up to 566 days after implantation. Histopathology evaluation of neurons and astrocytes using immunohistochemical stains indicated minimal alterations of tissue architecture after chronic implantation.
Persons lacking functional auditory nerves cannot benefit from cochlear implants, but an auditory brainstem implant (ABI) utilizing stimulating electrodes adjacent to or on their cochlear nucleus (CN) can restore some hearing. We are investigating the feasibility of supplementing these surface electrodes with penetrating microstimulating electrodes within the ventral CN (VCN), and how the two types of electrodes can be used synergistically. Multiunit neuronal responses evoked by VCN electrical stimulation with surface electrodes and microelectrodes were recorded in the inferior colliculus (ICC) of five cats. The findings are consistent with those from patients with type II neurofibromatosis who received ABIs with both surface and microelectrodes. The patients described percepts from their microelectrodes as more similar to pure tones than those from their surface electrodes, consistent with the greater tonotopic selectivity of microelectrodes in the cats' VCN. Also, the patients describe percepts from their surface electrodes as louder than those from the microelectrodes, while in the cat, the neuronal activity evoked in the ICC by the surface electrodes tended to be greater. This concordance helps to validate our cat model as a means of investigating the synergistic use of surface and penetrating electrodes in a clinical ABI.
We present versatile multifunctional programmable controller with bidirectional data telemetry, implemented using existing commercial microchips and standard Bluetooth protocol, which adds convenience, reliability, and ease-of-use to neuroprosthetic devices. Controller, weighing 190 g, is placed on animal's back and provides bidirectional sustained telemetry rate of 500 kb/s, allowing real-time control of stimulation parameters and viewing of acquired data. In continuously-active state, controller consumes ∼420 mW and operates without recharge for 8 h. It features independent 16-channel current-controlled stimulation, allowing current steering; customizable stimulus current waveforms; recording of stimulus voltage waveforms and evoked neuronal responses with stimulus artifact blanking circuitry. Flexibility, scalability, cost-efficiency, and a user-friendly computer interface of this device allow use in animal testing for variety of neuroprosthetic applications. Initial testing of the controller has been done in a feline model of brainstem auditory prosthesis. In this model, the electrical stimulation is applied to the array of microelectrodes implanted in the ventral cochlear nucleus, while the evoked neuronal activity was recorded with the electrode implanted in the contralateral inferior colliculus. Stimulus voltage waveforms to monitor the access impedance of the electrodes were acquired at the rate of 312 kilosamples/s. Evoked neuronal activity in the inferior colliculus was recorded after the blanking (transient silencing) of the recording amplifier during the stimulus pulse, allowing the detection of neuronal responses within 100 μs after the end of the stimulus pulse applied in the cochlear nucleus.
The findings have implications for improved sound processors for present and future ABIs. The performance of ABIs may benefit from using pulse rates greater than those presently used in most ABIs, and by sound processing strategies that enhance the modulation depth of the electrical stimulus while preserving dynamic range.
Background: Previous studies have shown that neurons of the cerebral cortex can be injured by implantation of, and stimulation with, implanted microelectrodes. Objectives: Objective 1 was to determine parameters of microstimulation delivered through multisite intracortical microelectrode arrays that will activate neurons of the feline cerebral cortex without causing loss of neurons. Objective: 2 was to determine if the stimulus parameters that induced loss of cortical neurons differed for all cortical neurons vs. the subset of inhibitory neurons expressing parvalbumin. Methods: The intracortical microstimulation was applied for 7 h/day for 20 days (140 h). Microelectrode site areas were 2000 and 4000 mm 2 , Q was 2e8 nanocoulombs (nC) at 50 Hz, and QD was 50 e400 mcoulombs/cm 2 .Results: Neuron loss due to stimulation was minimal at Q ¼ 2 Ncp, but at 8 Ncp, 20%e50% of neurons within 250 mm of the stimulated microelectrodes were lost, compared to unstimulated microelectrodes.Loss was greatest in tissue facing electrode sites. Stimulation-induced loss was similar for neurons labeled for NeuN and for inhibitory neurons expressing parvalbumin. Correlation between neuron loss and QD was not significant. Electrodes in the medullary pyramidal tract recorded neuronal activity evoked by stimulation in the cerebral cortex. The pyramidal neurons were activated by intracortical stimulation of 2 nC/phase. 140 h of microstimulation at 2 nC/phase and 50 Hz induced minimal neuron loss.
Auditory brainstem implants (ABIs) can restore useful hearing to persons with deafness who cannot benefit from cochlear implants. However, the quality of hearing restored by ABIs rarely is comparable to that provided by cochlear implants in persons for whom those are appropriate. In an animal model, we evaluated elements of a prototype of an ABI in which the functions of macroelectrodes on the surface of the dorsal cochlear nucleus would be integrated with the function of multiple penetrating microelectrodes implanted into the ventral cochlear nucleus. The surface electrodes would convey most of the range of loudness percepts while the intranuclear microelectrodes would sharpen and focus pitch percepts. In the present study, stimulating electrodes were implanted chronically on the surface of the animal's dorsal cochlear nucleus (DCN) and also within their ventral cochlear nucleus (VCN). Recording microelectrodes were implanted into the central nucleus of the inferior colliculus (ICC). The electrical stimuli were sinusoidally modulated stimulus pulse trains applied on the DCN and within the VCN. Temporal encoding of neuronal responses was quantified as vector strength (VS) and as full-cycle rate of neuronal activity in the ICC. VS and full-cycle AP rate were measured for 4 stimulation modes; continuous and transient amplitude modulation of the stimulus pulse trains, each delivered via the macroelectrode on the surface of the DCN and then by the intranuclear penetrating microelectrodes. In the proposed clinical device the functions of the surface and intranuclear microelectrodes could best be integrated if there is minimal variation in the neuronal responses across the range of modulation depth, modulation frequencies, and across the four stimulation modes. In this study VS did vary as much as 34% across modulation frequency and modulation depth within a stimulation mode, and up to 40% between modulation modes. However, these intra- and inter-mode variances differed for different stimulation rates, and at 500 Hz the inter-mode differences in VS and across the range of modulation frequencies and modulation depths was
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