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Long-term recordings from afferent taste fibers
Physiology & Behavior 80 (2003) 309 – 315
Long-term recordings from afferent taste fibers Yuichi Shimatania, Stefan A. Niklesb, Khalil Najafib, Robert M. Bradleyc,d,* a
b
Department of Physiology, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan Center for Integrated Sensors and Circuits, Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI 48109-2122, USA c Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA d Department of Molecular and Integrative Physiology, Medical School, University of Michigan, Ann Arbor, MI 48109-0622, USA Received 2 May 2003; received in revised form 7 July 2003; accepted 14 August 2003
Abstract The receptor cells of taste buds have a life span of about 10 days but it is not known if response characteristics of these receptors alter during the turnover cycle. To examine taste cell responses over time, a micromachined polyimide sieve electrode array was implanted between the cut ends of the rat chorda tympani nerve, which then regenerated through the electrode array. Long-term stable recordings from regenerated single afferent fibers innervating taste buds were possible using this technique for up to 21 days. Responses to taste stimuli recorded from the same fiber changed with time. The changes occurred in both the magnitude of response and the relative response profiles to four chemical stimuli, NaCl, sucrose, HCl, and quinine HCl. These changes in response characteristics were hypothesized to result from changes in the taste receptor cells as the receptor cells turnover in the taste buds. D 2003 Elsevier Inc. All rights reserved. Keywords: Taste; Chronic recording; Chorda tympani; Electrophysiology; Neural implant; Regeneration
1. Introduction Taste receptor cells are very dynamic structures that constantly turnover with a life span of about 10 days [1]. During turnover, the taste cells migrate from the periphery to the center of the taste buds and then die by apoptosis [17 – 19]. Taste cells are innervated by afferent sensory fibers travelling in the facial (VII), glossopharyngeal (IX), and vagus (X) nerves. Terminal fibers of these nerves branch as they approach the taste buds and synapse with the taste cells [2]. This anatomical arrangement of dynamically changing receptor cells and a relatively stable innervation poses an interesting series of questions [1]. What happens to the primary afferent nerve connections during turnover? Does an afferent fiber connect to a single taste receptor cell for its entire life span or do the cells get passed from fiber to fiber as the receptor cells migrate from the periphery to the center of
the taste bud? Do taste cells have the same response properties throughout their life cycle during the time they are connected to an afferent fiber? Does a single fiber change its response properties with time by disconnecting and reconnecting to cells of different response characteristics, or does it maintain the same properties by making connections repeatedly to cells of the same response properties? To answer these questions, we have taken advantage of modern microfabrication techniques combined with the regenerative properties of the peripheral taste system and implanted a sieve-like array of small holes surrounded by electrode sites between the cut ends of the chorda tympani nerve. The nerve then regenerates through the holes to reinnervate the taste buds [3]. The electrode sites are connected to external recording equipment via a cable and percutaneous headcap connector.
2. Methods * Corresponding author. Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Room 6228, Ann Arbor, MI 48109-1078, USA. Tel.: +1-734-763-1080; fax: +1-734-6472110. E-mail address: [email protected] (R.M. Bradley). 0031-9384/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2003.08.009
2.1. Electrode fabrication Sieve electrodes were microfabricated using a process similar to that described by Heiduschka et al. [4]. Briefly, a
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4-Am layer of polyimide (PI2611D-HD Microsystems) was cured on a silicon carrier wafer and metal layers evaporated onto the polyimide surface patterned to form the recording sites, interconnecting lines, and headcap contact pads. A second layer of polyimide completely insulated the metal layers. Selective etching was used to uncover the recording sites, holes, contact pads, and to define the electrode perimeter. The exposed recording sites and contact pads were then electroplated with 1 Am gold. The electrodes contained nine holes, 10 Am in diameter (Fig. 1A), four of which were electroplated gold electrode sites while the remaining five holes were included to increase the overall ‘‘transparency’’ of the electrode. The recording sites were connected to the contact pads on the headcap end via the metal interconnecting lines. Each electrode was then connected to a percutaneous headcap machined from stainless steel, with a base flange contoured to fit the skull curvature (Fig. 1B) and drilled for screws used to fasten the headcap to the skull. The Teflon connector, with four embedded gold pins was bonded to connector pads that terminate the interconnecting lines.
Small polyimide guide tubes (200 Am inner diameter and 200 Am long) were cemented to opposing faces of the electrode diaphragm. 2.2. Surgery Female Sprague – Dawley rats weighing 240– 260 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and laid on a heating pad to maintain body temperature. The skull was exposed by an incision through the skin and the periosteum carefully removed. Screw holes were made with a dental burr and the headcap fixed to the skull with titanium screws (1 mm diameter, 4 mm long; W. Lorenz, Jacksonville, FL). The chorda tympani nerve was exposed in the neck, carefully separated from surrounding tissue, and then cut. The cut ends of the chorda tympani were then inserted and sutured into short lengths of polyamide tubing concentric in diameter to the guide tubes. Via blunt dissection, a tunnel was made along the skull inside the zygomatic arch towards the exposed chorda tympani nerve. The sieve electrode and
Fig. 1. (A) Photomicrographs of the sieve electrode. Top: Micrograph of the recording sites and interconnecting traces. Bottom: Scanning electron micrograph of the nine holes, four with recording sites and five without (calibration bar = 20 Am). (B) Diagram showing position of implant in the chorda tympani nerve and the headcap as well as a detailed diagram of how the nerve stamps are connected to electrode with guide tubing. (C) Simultaneous recording from four channels. Lower panel: discriminated events of small single unit (S) and large single unit (L) in channel 2 and the single unit in channel 4. Bar graph: number of spikes elicited in first 5 s in response to four basic taste stimuli.
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cable were then threaded through this tunnel. The tubes containing the cut ends of the chorda tympani nerve were then inserted into the guide tubes and fixed in place using 110 nylon monofilament suture (Fig. 1B). The rats were housed individually on ad libitum food and water and were checked daily for any signs of postsurgical discomforts or stress. Surgical procedures were carried out under protocols approved by the National Institutes of Health and the University of Michigan Animal Care and Use Committee. 2.3. Recording After implanting, electrophysiological recordings were made twice a week until neural activity was detected. If any activity appeared, recordings were then made on a daily basis. Experiments were terminated either after 3 months if taste responses could not be evoked, or earlier if electrode impedance values were either too high or low indicating failure of the electrode. After termination, the animals were sacrificed and dissected to confirm that the nerve was still in place in the electrode. During recording rats were anesthetized with a short acting anesthetic consisting of an intramuscular injection of a 1:1 mixture of xylazine (2 mg/kg body weight) and ketamine (10 mg/kg body weight). This provided about 1 h of surgical level anesthesia. Once the animal was anesthetized, an amplifying equipment (Grass, West Warwick, RI, P511) was connected to the headcap plug. The anesthetized animal was laid on its side and the tongue gently retracted to expose the fungiform papillae and associated taste buds. Taste stimuli were solutions of 0.1 M NaCl, 0.5 M sucrose, 0.01 M HCl, and 0.02 M quinine HCl, applied twice in this order, and then a concentration series of NaCl (0.01 – 1.0 M) was applied. All solutions were made in distilled water and applied at room temperature. Solutions were gravity flowed over the tongue for f 20 s at 0.5 ml/s and the tongue was then rinsed with distilled water continually for 30 s before the next stimulus was applied. Recordings were made on four channels simultaneously. The amplified signals were connected to an A/D converter (CED, Cambridge, UK, 1401 Plus) interfaced to a computer. Data were acquired by the software program Spike2 (CED) and stored on the hard drive of the computer. 2.4. Data analysis Single unit analysis was possible for some records if the signal-to-noise (S/N) ratio was adequate and individual action potentials clearly distinguishable. Action potentials were discriminated by their amplitudes and waveforms using a template matching function of the Spike2 program. In longterm recording, the identities of individual units that were recorded on successive days were judged by the following criteria: (a) the units are recorded on the same channel; (b) the units are the only ones discriminated in the neural
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recording, and no other neural activity is present in that recording; and (c) there is a high similarity in the amplitude and the shape of action potentials for each unit across time, i.e., the unit of the subsequent day can be discriminated using the template created for that unit on the first day.
3. Results Sieve electrodes were implanted in 18 rats and recordings from regenerated axons were obtained in seven animals. Of the remaining 11 rats, 2 were terminated because of electrode failures and no response was recorded from nine rats despite testing for over 3 months. Neural activity was first detected 25 – 49 days after implanting. The earliest evoked responses were obtained 37 days after implanting, but in one animal, the first responses were recorded on the 88th day. Once the neural activity was detected on any channel, recordings were repeated daily. In most animals, when evoked recordings were obtained, the neural activity was stable for more than a week, but then disappeared within 2 weeks. However, in one animal, taste responses were recorded every day for more than 9 weeks. In two animals, taste responses were lost after 8 days but subsequently reappeared a few days later on the same channels. The number of single units contained in a record, and their S/N ratio varied from recording to recording. In some recordings, only one or two single units were identified and the S/N ratio was large enough to distinguish each action potential. 3.1. Simultaneous recording In three animals, neural activity was recorded on more than one channel, demonstrating that simultaneous recordings across the channels from different taste units were possible. Fig. 1C illustrates a simultaneous recording in which the data on channel 2 contains two single units of different amplitudes. The large unit responds with greatest frequency to 0.01 M HCl while the small unit responds with greatest frequency to 0.1 M NaCl. Only one single unit was recorded on channel 4 and it responded with the greatest frequency to NaCl. When the discharge patterns of action potentials on channels 2 (small amplitude unit) and 4 in response to NaCl are compared there are notable differences, indicating that these are two different fibers. 3.2. Long-term recording from single unit Long-term recordings from the same single unit are illustrated in Figs. 2 and 3. We were careful to ensure that the daily recordings were from the same single unit using the criteria described above. Long-term recordings over several days were made on five identified single units in five animals. Responses to four basic taste stimuli were examined
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Fig. 2. (A) Neural activity of the same single fiber in response to taste stimuli recorded on three successive days. (B) Long-term recordings from four fibers showing changes in taste response with time. Number of spikes in first 5 s are plotted for the first (closed circle) and second (open circle) stimulus application. Each bar is the average of the two applications. The first day of each graph is at 55 days (left upper), 52 days (right upper), 58 days (left lower), and 62 days (right lower) after implantation.
every day until the S/N ratio was not adequate to discriminate the single units. The frequency and firing pattern of action potentials evoked by taste stimuli changed over time. Furthermore, the change in response to each taste stimulus changed from day to day and therefore the response to the four basic taste stimuli was not constant. For example, the unit in Fig. 2A recorded from a single channel over 3 days responded best to 0.5 M sucrose solution on the first day. On the second day, the rhythmic discharge to sucrose (a characteristic response pattern of rat chorda tympani fibers to sucrose [10]) stimulation became more distinct and the number of action potentials in each burst of the rhythmic discharge increased. On the third day, the rhythmic response had disappeared. On the other hand, the responses evoked by NaCl, HCl, and quinine HCl decreased over the 3-day recording period.
Analysis of the mean frequency characteristics of the unit in Fig. 2A as well as three other units is shown in Fig. 2B. In all these recordings, the response magnitudes change from day to day. Fig. 3 presents data from a single unit over a period of 21 days. In this animal, taste responses were first recorded at 45 days after implanting and stable recordings from a single unit were maintained from the 77th to the 97th day after implantation. The superimposed trace of the daily averaged waveform of action potentials (Fig. 3B) demonstrates that the amplitude and waveform of the action potentials were constant during the 21-day recording period, indicating that recordings were made from the same single unit. Although the long-term recording was stopped because the S/N ratio deteriorated, responses to taste stimuli were still detectable for more than 2 weeks until the experiment was terminated.
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Fig. 3. Recording from the same single unit over 21 days. (A) Number of spikes elicited in first 5 s in response to four basic taste stimuli. Each bar is the average of two stimulus applications. The first day is 77 days after implantation. (B) Superimposed action potentials from all 21 recording days. Individual traces are the averaged action potential waveform of each day. (C) Discriminated events of the firing pattern of action potentials in response to NaCl and HCl. Note the marked change in firing pattern for NaCl, which is less evident for HCl.
As seen in Fig. 3A, the responses to taste stimuli, especially to NaCl and HCl changed gradually from day to day. The firing pattern of the action potentials in response to NaCl also changed (Fig. 3C). At first, this unit responded only with a transient burst of action potentials for the continuous flow of 0.1 M NaCl. Sustained firing of action potentials did not occur during stimulation even if the concentration of the NaCl was increased up to 1.0 M. Sustained responses appeared and increased gradually, reaching a peak at the 12th day. After that, the response frequency decreased, but the initial transient burst of action potentials remained. In contrast, the response to 0.01 M HCl was relatively stable for the first 12 days and gradually decreased over the rest of the recording period.
4. Discussion The major finding of this research is that the response characteristics of individual taste fibers change with time.
Since long-term recordings from the same whole chorda tympani nerve have demonstrated that response characteristics are stable for 3 months [15]; the variability of the single fiber responses was unexpected. It is possible that the response variability is a property of the regenerated nerve. The time needed for the cut chorda tympani nerve to regenerate as measured by taste bud number and ability to perform normal discriminatory responses was 7 weeks in rats [16]. As the first days of the recordings illustrated in Figs. 2 and 3 are all more than 7 weeks after surgery, we presume that the recordings were from nerves that have already achieved their fully regenerated response characteristics. However, regeneration through a sieve electrode may differ from regeneration following nerve crush or section. There was a wide variation in taste bud counts 90 days after nerve regeneration through a sieve electrode [3]. Moreover, regeneration of chorda tympani fibers may continue, which would not be reflected in taste bud counts or discriminative behavior measures. Thus, it is not possible to totally dismiss the role of regeneration in response variability over time.
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However, the source of the single fiber response variability may represent the normal dynamic properties of the taste receptor cells. The exact location of this variability is not easily determined because there are a number of confounding factors related to the final distribution of a single fiber. Single chorda tympani nerve fibers branch extensively not only to innervate several fungiform papillae but also as they ascend within a single fungiform papilla [2,8,9], and within a taste bud a single axon synaptically contacts up to five taste cells [5]. Therefore, the long-term recordings result from input from several taste receptor cells. As an added complication rat taste cells have a life span of 10.4 F 2 days [1], so that about 10% of the cells should be replaced in a day. Single chorda tympani nerve fibers may therefore be connected with taste cells in different taste buds at various stages in the cell cycle or possibly connected to cells of a specific cell cycle stage. On the other hand, detailed studies of chorda tympani nerve receptive fields in rat, cat, and sheep have demonstrated that a single fiber can innervate one fungiform papilla [6,8,11 – 14] and contact a single taste cell [5]. It is therefore conceivable that the long-term recordings could have originated from a single taste receptor cell and the cyclic variability in NaCl response of Fig. 3A could represent cellular life cycle changes accompanying turnover. In contrast, long-term recording variability from fibers innervating multiple taste receptors would result in more abrupt changes in response pattern as illustrated in Fig. 2. Because of technical difficulties, receptive fields were not mapped in the current study, but experiments are in progress in which the receptive field properties of the fibers will be examined over time. Conditions for recording neural activity and maintaining it may be very critical with this type of electrode. One possible assumption is that the recording is possible only when the node of Ranvier of a myelinated fiber is located on the annular metal of the recording site. This assumption could also explain why recordings of most single units are so short lived, because regenerative growth of the fibers conceivably shifts the position of the node. Increasing the number of recording sites would increase the chance of recording neural activity, but some modification of the recording sites such as lengthening the area of contact may be required to make the long-term recording more stable. The variability in the single fiber responses differs from the stability of the whole nerve responses [15]. Presumably this is due to averaging of the responses of all the fibers and perhaps indicates that across-fiber patterns are constant. Thus, afferent information arriving at the brain would also be constant. The variability in the single fiber responses is therefore a local phenomenon and potentially reflects shifts in the innervation of dynamically changing receptors. The results presented here represent the first successful long-term recordings from the same single afferent taste fibers. The results also demonstrate that it is possible to record from a sieve electrode array that relies on the proper-
ties of peripheral nerve regeneration. Although many investigators have tried to use sieve electrodes to record and stimulate peripheral nerves, this is the first time this technology has been used to record controlled evoked responses from a sensory nerve. Other investigators using sieve electrodes often only report recordings of spontaneous activity or do not systematically study evoked responses to carefully controlled stimulus conditions [7]. Long-term recordings from further fibers will hopefully confirm these initial findings presented here and provide new information on the cell biology of taste receptors.
Acknowledgements This work was funded by the National Institute on Deafness and Other Communication Disorders Grant DC04198 to R.M.B.
References [1] Beidler LM, Smallman RL. Renewal of cells within taste buds. J Cell Biol 1965;27:263 – 72. [2] Beidler LM. Innervation of rat fungiform papilla. In: Pfaffmann C, editor. Olfaction and taste III. New York: Rockefeller Univ. Press, 1969. pp. 352 – 69. [3] Bradley RM, Smoke RH, Akin T, Najafi K. Functional regeneration of glossopharyngeal nerve through micromachined sieve electrode arrays. Brain Res 1992;594:84 – 90. [4] Heiduschka P, Romann I, Stieglitz T, Thanos S. Perforated microelectrode arrays implanted in the regenerating adult central nervous system. Exp Neurol 2001;171:1 – 10. [5] Kinnamon JC, Sherman TA, Roper SD. Ultrastructure of mouse vallate taste buds: III. Patterns of synaptic connectivity. J Comp Neurol 1988;270:1 – 10. [6] Krimm RF, Hill DL. Innervation of single fungiform taste buds during development in rat. J Comp Neurol 1998;398:13 – 24. [7] Mensinger AF, Anderson DJ, Buchko CJ, Johnson MA, Martin DC, Tresco PA, et al. Chronic recordings of regenerating VIIIth nerve axons with a sieve electrode. J Neurophysiol 2000;83:611 – 5. [8] Miller Jr IJ. Peripheral interactions among single papilla inputs to gustatory nerve fibers. J Gen Physiol 1971;57:1 – 25. [9] Miller Jr IJ. Branched chorda tympani neurons and interactions among taste receptors. J Comp Neurol 1974;158:155 – 66. [10] Nagai T, Ueda K. Stochastic properties of gustatory impulse discharges in rat chorda tympani fibers. J Neurophysiol 1981;45:574 – 92. [11] Nagai T, Mistretta CM, Bradley RM. Developmental decrease in size of peripheral receptive fields of single chorda tympani nerve fibers and relation to increasing NaCl taste sensitivity. J Neurosci 1988;8: 64 – 72. [12] Oakley B. Receptive fields of cat taste fibers. Chem Senses Flavor 1975;1:431 – 42. [13] Pfaffmann C. Physiological and behavioral processes of the sense of taste. In: Wolstenholme GEW, Knight J, editors. Taste and smell in vertebrates. London: Churchill, 1970. p. 31 – 50. [14] Robinson PP. The characteristics and regional distribution of afferent fibres in the chorda tympani of the cat. J Physiol 1988;406: 345 – 59. [15] Shimatani Y, Grabauskiene S, Bradley RM. Long-term recording from the chorda tympani in rats. Physiol Behav 2002;76:143 – 9. [16] St John SJ, Markison S, Spector AC. Salt discriminability is related to
Y. Shimatani et al. / Physiology & Behavior 80 (2003) 309–315 number of regenerated taste buds after chorda tympani nerve section in rats. Am J Physiol Regul Integr Comp Physiol 1995;269:R141 – 53. [17] Takeda M, Suzuki Y, Obara N, Nagai Y. Apoptosis in mouse taste buds after denervation. Cell Tissue Res 1996;286:55 – 62. [18] Takeda M, Suzuki Y, Obara N, Nagai Y. Induction of apoptosis by
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colchicine in taste bud and epithelial cells of the mouse circumvallate papillae. Cell Tissue Res 2000;302:391 – 5. [19] Zeng Q, Oakley B. p53 and Bax: putative death factors in taste cell turnover. J Comp Neurol 1999;413:168 – 80.
Long-term recordings from afferent taste fibers Yuichi Shimatania, Stefan A. Niklesb, Khalil Najafib, Robert M. Bradleyc,d,* a
b
Department of Physiology, School of Medicine, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan Center for Integrated Sensors and Circuits, Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI 48109-2122, USA c Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA d Department of Molecular and Integrative Physiology, Medical School, University of Michigan, Ann Arbor, MI 48109-0622, USA Received 2 May 2003; received in revised form 7 July 2003; accepted 14 August 2003
Abstract The receptor cells of taste buds have a life span of about 10 days but it is not known if response characteristics of these receptors alter during the turnover cycle. To examine taste cell responses over time, a micromachined polyimide sieve electrode array was implanted between the cut ends of the rat chorda tympani nerve, which then regenerated through the electrode array. Long-term stable recordings from regenerated single afferent fibers innervating taste buds were possible using this technique for up to 21 days. Responses to taste stimuli recorded from the same fiber changed with time. The changes occurred in both the magnitude of response and the relative response profiles to four chemical stimuli, NaCl, sucrose, HCl, and quinine HCl. These changes in response characteristics were hypothesized to result from changes in the taste receptor cells as the receptor cells turnover in the taste buds. D 2003 Elsevier Inc. All rights reserved. Keywords: Taste; Chronic recording; Chorda tympani; Electrophysiology; Neural implant; Regeneration
1. Introduction Taste receptor cells are very dynamic structures that constantly turnover with a life span of about 10 days [1]. During turnover, the taste cells migrate from the periphery to the center of the taste buds and then die by apoptosis [17 – 19]. Taste cells are innervated by afferent sensory fibers travelling in the facial (VII), glossopharyngeal (IX), and vagus (X) nerves. Terminal fibers of these nerves branch as they approach the taste buds and synapse with the taste cells [2]. This anatomical arrangement of dynamically changing receptor cells and a relatively stable innervation poses an interesting series of questions [1]. What happens to the primary afferent nerve connections during turnover? Does an afferent fiber connect to a single taste receptor cell for its entire life span or do the cells get passed from fiber to fiber as the receptor cells migrate from the periphery to the center of
the taste bud? Do taste cells have the same response properties throughout their life cycle during the time they are connected to an afferent fiber? Does a single fiber change its response properties with time by disconnecting and reconnecting to cells of different response characteristics, or does it maintain the same properties by making connections repeatedly to cells of the same response properties? To answer these questions, we have taken advantage of modern microfabrication techniques combined with the regenerative properties of the peripheral taste system and implanted a sieve-like array of small holes surrounded by electrode sites between the cut ends of the chorda tympani nerve. The nerve then regenerates through the holes to reinnervate the taste buds [3]. The electrode sites are connected to external recording equipment via a cable and percutaneous headcap connector.
2. Methods * Corresponding author. Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Room 6228, Ann Arbor, MI 48109-1078, USA. Tel.: +1-734-763-1080; fax: +1-734-6472110. E-mail address: [email protected] (R.M. Bradley). 0031-9384/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2003.08.009
2.1. Electrode fabrication Sieve electrodes were microfabricated using a process similar to that described by Heiduschka et al. [4]. Briefly, a
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4-Am layer of polyimide (PI2611D-HD Microsystems) was cured on a silicon carrier wafer and metal layers evaporated onto the polyimide surface patterned to form the recording sites, interconnecting lines, and headcap contact pads. A second layer of polyimide completely insulated the metal layers. Selective etching was used to uncover the recording sites, holes, contact pads, and to define the electrode perimeter. The exposed recording sites and contact pads were then electroplated with 1 Am gold. The electrodes contained nine holes, 10 Am in diameter (Fig. 1A), four of which were electroplated gold electrode sites while the remaining five holes were included to increase the overall ‘‘transparency’’ of the electrode. The recording sites were connected to the contact pads on the headcap end via the metal interconnecting lines. Each electrode was then connected to a percutaneous headcap machined from stainless steel, with a base flange contoured to fit the skull curvature (Fig. 1B) and drilled for screws used to fasten the headcap to the skull. The Teflon connector, with four embedded gold pins was bonded to connector pads that terminate the interconnecting lines.
Small polyimide guide tubes (200 Am inner diameter and 200 Am long) were cemented to opposing faces of the electrode diaphragm. 2.2. Surgery Female Sprague – Dawley rats weighing 240– 260 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and laid on a heating pad to maintain body temperature. The skull was exposed by an incision through the skin and the periosteum carefully removed. Screw holes were made with a dental burr and the headcap fixed to the skull with titanium screws (1 mm diameter, 4 mm long; W. Lorenz, Jacksonville, FL). The chorda tympani nerve was exposed in the neck, carefully separated from surrounding tissue, and then cut. The cut ends of the chorda tympani were then inserted and sutured into short lengths of polyamide tubing concentric in diameter to the guide tubes. Via blunt dissection, a tunnel was made along the skull inside the zygomatic arch towards the exposed chorda tympani nerve. The sieve electrode and
Fig. 1. (A) Photomicrographs of the sieve electrode. Top: Micrograph of the recording sites and interconnecting traces. Bottom: Scanning electron micrograph of the nine holes, four with recording sites and five without (calibration bar = 20 Am). (B) Diagram showing position of implant in the chorda tympani nerve and the headcap as well as a detailed diagram of how the nerve stamps are connected to electrode with guide tubing. (C) Simultaneous recording from four channels. Lower panel: discriminated events of small single unit (S) and large single unit (L) in channel 2 and the single unit in channel 4. Bar graph: number of spikes elicited in first 5 s in response to four basic taste stimuli.
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cable were then threaded through this tunnel. The tubes containing the cut ends of the chorda tympani nerve were then inserted into the guide tubes and fixed in place using 110 nylon monofilament suture (Fig. 1B). The rats were housed individually on ad libitum food and water and were checked daily for any signs of postsurgical discomforts or stress. Surgical procedures were carried out under protocols approved by the National Institutes of Health and the University of Michigan Animal Care and Use Committee. 2.3. Recording After implanting, electrophysiological recordings were made twice a week until neural activity was detected. If any activity appeared, recordings were then made on a daily basis. Experiments were terminated either after 3 months if taste responses could not be evoked, or earlier if electrode impedance values were either too high or low indicating failure of the electrode. After termination, the animals were sacrificed and dissected to confirm that the nerve was still in place in the electrode. During recording rats were anesthetized with a short acting anesthetic consisting of an intramuscular injection of a 1:1 mixture of xylazine (2 mg/kg body weight) and ketamine (10 mg/kg body weight). This provided about 1 h of surgical level anesthesia. Once the animal was anesthetized, an amplifying equipment (Grass, West Warwick, RI, P511) was connected to the headcap plug. The anesthetized animal was laid on its side and the tongue gently retracted to expose the fungiform papillae and associated taste buds. Taste stimuli were solutions of 0.1 M NaCl, 0.5 M sucrose, 0.01 M HCl, and 0.02 M quinine HCl, applied twice in this order, and then a concentration series of NaCl (0.01 – 1.0 M) was applied. All solutions were made in distilled water and applied at room temperature. Solutions were gravity flowed over the tongue for f 20 s at 0.5 ml/s and the tongue was then rinsed with distilled water continually for 30 s before the next stimulus was applied. Recordings were made on four channels simultaneously. The amplified signals were connected to an A/D converter (CED, Cambridge, UK, 1401 Plus) interfaced to a computer. Data were acquired by the software program Spike2 (CED) and stored on the hard drive of the computer. 2.4. Data analysis Single unit analysis was possible for some records if the signal-to-noise (S/N) ratio was adequate and individual action potentials clearly distinguishable. Action potentials were discriminated by their amplitudes and waveforms using a template matching function of the Spike2 program. In longterm recording, the identities of individual units that were recorded on successive days were judged by the following criteria: (a) the units are recorded on the same channel; (b) the units are the only ones discriminated in the neural
311
recording, and no other neural activity is present in that recording; and (c) there is a high similarity in the amplitude and the shape of action potentials for each unit across time, i.e., the unit of the subsequent day can be discriminated using the template created for that unit on the first day.
3. Results Sieve electrodes were implanted in 18 rats and recordings from regenerated axons were obtained in seven animals. Of the remaining 11 rats, 2 were terminated because of electrode failures and no response was recorded from nine rats despite testing for over 3 months. Neural activity was first detected 25 – 49 days after implanting. The earliest evoked responses were obtained 37 days after implanting, but in one animal, the first responses were recorded on the 88th day. Once the neural activity was detected on any channel, recordings were repeated daily. In most animals, when evoked recordings were obtained, the neural activity was stable for more than a week, but then disappeared within 2 weeks. However, in one animal, taste responses were recorded every day for more than 9 weeks. In two animals, taste responses were lost after 8 days but subsequently reappeared a few days later on the same channels. The number of single units contained in a record, and their S/N ratio varied from recording to recording. In some recordings, only one or two single units were identified and the S/N ratio was large enough to distinguish each action potential. 3.1. Simultaneous recording In three animals, neural activity was recorded on more than one channel, demonstrating that simultaneous recordings across the channels from different taste units were possible. Fig. 1C illustrates a simultaneous recording in which the data on channel 2 contains two single units of different amplitudes. The large unit responds with greatest frequency to 0.01 M HCl while the small unit responds with greatest frequency to 0.1 M NaCl. Only one single unit was recorded on channel 4 and it responded with the greatest frequency to NaCl. When the discharge patterns of action potentials on channels 2 (small amplitude unit) and 4 in response to NaCl are compared there are notable differences, indicating that these are two different fibers. 3.2. Long-term recording from single unit Long-term recordings from the same single unit are illustrated in Figs. 2 and 3. We were careful to ensure that the daily recordings were from the same single unit using the criteria described above. Long-term recordings over several days were made on five identified single units in five animals. Responses to four basic taste stimuli were examined
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Fig. 2. (A) Neural activity of the same single fiber in response to taste stimuli recorded on three successive days. (B) Long-term recordings from four fibers showing changes in taste response with time. Number of spikes in first 5 s are plotted for the first (closed circle) and second (open circle) stimulus application. Each bar is the average of the two applications. The first day of each graph is at 55 days (left upper), 52 days (right upper), 58 days (left lower), and 62 days (right lower) after implantation.
every day until the S/N ratio was not adequate to discriminate the single units. The frequency and firing pattern of action potentials evoked by taste stimuli changed over time. Furthermore, the change in response to each taste stimulus changed from day to day and therefore the response to the four basic taste stimuli was not constant. For example, the unit in Fig. 2A recorded from a single channel over 3 days responded best to 0.5 M sucrose solution on the first day. On the second day, the rhythmic discharge to sucrose (a characteristic response pattern of rat chorda tympani fibers to sucrose [10]) stimulation became more distinct and the number of action potentials in each burst of the rhythmic discharge increased. On the third day, the rhythmic response had disappeared. On the other hand, the responses evoked by NaCl, HCl, and quinine HCl decreased over the 3-day recording period.
Analysis of the mean frequency characteristics of the unit in Fig. 2A as well as three other units is shown in Fig. 2B. In all these recordings, the response magnitudes change from day to day. Fig. 3 presents data from a single unit over a period of 21 days. In this animal, taste responses were first recorded at 45 days after implanting and stable recordings from a single unit were maintained from the 77th to the 97th day after implantation. The superimposed trace of the daily averaged waveform of action potentials (Fig. 3B) demonstrates that the amplitude and waveform of the action potentials were constant during the 21-day recording period, indicating that recordings were made from the same single unit. Although the long-term recording was stopped because the S/N ratio deteriorated, responses to taste stimuli were still detectable for more than 2 weeks until the experiment was terminated.
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Fig. 3. Recording from the same single unit over 21 days. (A) Number of spikes elicited in first 5 s in response to four basic taste stimuli. Each bar is the average of two stimulus applications. The first day is 77 days after implantation. (B) Superimposed action potentials from all 21 recording days. Individual traces are the averaged action potential waveform of each day. (C) Discriminated events of the firing pattern of action potentials in response to NaCl and HCl. Note the marked change in firing pattern for NaCl, which is less evident for HCl.
As seen in Fig. 3A, the responses to taste stimuli, especially to NaCl and HCl changed gradually from day to day. The firing pattern of the action potentials in response to NaCl also changed (Fig. 3C). At first, this unit responded only with a transient burst of action potentials for the continuous flow of 0.1 M NaCl. Sustained firing of action potentials did not occur during stimulation even if the concentration of the NaCl was increased up to 1.0 M. Sustained responses appeared and increased gradually, reaching a peak at the 12th day. After that, the response frequency decreased, but the initial transient burst of action potentials remained. In contrast, the response to 0.01 M HCl was relatively stable for the first 12 days and gradually decreased over the rest of the recording period.
4. Discussion The major finding of this research is that the response characteristics of individual taste fibers change with time.
Since long-term recordings from the same whole chorda tympani nerve have demonstrated that response characteristics are stable for 3 months [15]; the variability of the single fiber responses was unexpected. It is possible that the response variability is a property of the regenerated nerve. The time needed for the cut chorda tympani nerve to regenerate as measured by taste bud number and ability to perform normal discriminatory responses was 7 weeks in rats [16]. As the first days of the recordings illustrated in Figs. 2 and 3 are all more than 7 weeks after surgery, we presume that the recordings were from nerves that have already achieved their fully regenerated response characteristics. However, regeneration through a sieve electrode may differ from regeneration following nerve crush or section. There was a wide variation in taste bud counts 90 days after nerve regeneration through a sieve electrode [3]. Moreover, regeneration of chorda tympani fibers may continue, which would not be reflected in taste bud counts or discriminative behavior measures. Thus, it is not possible to totally dismiss the role of regeneration in response variability over time.
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However, the source of the single fiber response variability may represent the normal dynamic properties of the taste receptor cells. The exact location of this variability is not easily determined because there are a number of confounding factors related to the final distribution of a single fiber. Single chorda tympani nerve fibers branch extensively not only to innervate several fungiform papillae but also as they ascend within a single fungiform papilla [2,8,9], and within a taste bud a single axon synaptically contacts up to five taste cells [5]. Therefore, the long-term recordings result from input from several taste receptor cells. As an added complication rat taste cells have a life span of 10.4 F 2 days [1], so that about 10% of the cells should be replaced in a day. Single chorda tympani nerve fibers may therefore be connected with taste cells in different taste buds at various stages in the cell cycle or possibly connected to cells of a specific cell cycle stage. On the other hand, detailed studies of chorda tympani nerve receptive fields in rat, cat, and sheep have demonstrated that a single fiber can innervate one fungiform papilla [6,8,11 – 14] and contact a single taste cell [5]. It is therefore conceivable that the long-term recordings could have originated from a single taste receptor cell and the cyclic variability in NaCl response of Fig. 3A could represent cellular life cycle changes accompanying turnover. In contrast, long-term recording variability from fibers innervating multiple taste receptors would result in more abrupt changes in response pattern as illustrated in Fig. 2. Because of technical difficulties, receptive fields were not mapped in the current study, but experiments are in progress in which the receptive field properties of the fibers will be examined over time. Conditions for recording neural activity and maintaining it may be very critical with this type of electrode. One possible assumption is that the recording is possible only when the node of Ranvier of a myelinated fiber is located on the annular metal of the recording site. This assumption could also explain why recordings of most single units are so short lived, because regenerative growth of the fibers conceivably shifts the position of the node. Increasing the number of recording sites would increase the chance of recording neural activity, but some modification of the recording sites such as lengthening the area of contact may be required to make the long-term recording more stable. The variability in the single fiber responses differs from the stability of the whole nerve responses [15]. Presumably this is due to averaging of the responses of all the fibers and perhaps indicates that across-fiber patterns are constant. Thus, afferent information arriving at the brain would also be constant. The variability in the single fiber responses is therefore a local phenomenon and potentially reflects shifts in the innervation of dynamically changing receptors. The results presented here represent the first successful long-term recordings from the same single afferent taste fibers. The results also demonstrate that it is possible to record from a sieve electrode array that relies on the proper-
ties of peripheral nerve regeneration. Although many investigators have tried to use sieve electrodes to record and stimulate peripheral nerves, this is the first time this technology has been used to record controlled evoked responses from a sensory nerve. Other investigators using sieve electrodes often only report recordings of spontaneous activity or do not systematically study evoked responses to carefully controlled stimulus conditions [7]. Long-term recordings from further fibers will hopefully confirm these initial findings presented here and provide new information on the cell biology of taste receptors.
Acknowledgements This work was funded by the National Institute on Deafness and Other Communication Disorders Grant DC04198 to R.M.B.
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