Neuroseienee Letters, 142 (1992) 89 94 ,~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.

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NSL 08801

The cone electrode: ultrastructural studies following long-term recording in rat and monkey cortex
Philip R. K e n n e d y a, S u z a n n e S. M i r r a b a n d R o y A.E. B a k a y c
"Georgia Institute of" Technology and Yerkes Regional Primate Research Center, Emo O, Universio~, Atlanta, GA 30322 ( USA ), ~Department of Pathology and Laboratoo, Medicine. VA Medical Center and Emoo' UniversiO' School of Medicine, Atlanta, GA 30322 ( USA ) and'Department g[' Neurosurgeo', Emo O' University School of Medicine and VA Medical Center, Athmta, GA 30322 (US,4)
(Received 20 December 1991; Revised version received 14 April 1992: Accepted 30 April 1992)

Key wor&:

Cone electrode: Rat: Monkey; Electron microscopy; Neurite

The achievement of long-term recording of neural signals from the central nervous system has potential clinical and investigative application. To facilitate long-term recording, a novel cone electrode composed of an insulated gold wire within a hollow glass cone had been developed. Cone electrodes containing sciatic nerve or neurotrophic medium were implanted into cerebral cortex in rats and monkeys. Electrophysiologic recordings had been previously obtained from cone tissue for as long as 15 months following implantation and this tissue contained silver-positive processes. We now extend these observations to characterize the fine structural features of the tissue within these long-term implants. Electron microscopy revealed central myelinated axons, dendrites, synaptic profiles, blood vessels, and glia; peripheral nerve was not found in the cones in which sciatic nerve had been placed. These observations further suggest ingrowth of cortical neurites and elements into the hollow glass tip of the cone and support the feasibility of long-term recording using this electrode.

The cone electrode, a device developed to achieve long-term recording from neural tissue, consists of a 1-2 mm length of conical hollow glass tubing, 0.1 0.3 mm in diameter, glued to the end of a flexible recording wire. In a previous study, neurotrophic tissue or medium was inserted into the cone, which was then inserted into the superficial cerebral cortex of rats [9] and monkeys [10]. In the original study, connectivity of cone tissue and cortical neurons was suggested by the following observations: ( 1) Cone electrodes in which sciatic nerve segments were initially inserted contained silver positive fibers many months following implantation. (2) Retrograde tracing studies with fluorogold placed within the corticospinal tract at the upper cervical level revealed fluorescent dye in neuronal cell bodies as well as in adjacent cone tissue containing only processes. (3) Finally, electrical activity was recorded within the cones. These experiments were limited to sciatic nerve cone electrode insertions in rat and monkeys and did not provide direct confirmation of the tissue constituents within the cone. The current experiments extend the use of the
Correspondence: P.R. Kennedy, Neuroscience Laboratory, Georgia Institute of Technology, Room 325, CRB, 400 10th Street, Atlanta, GA 30322, USA.

cone electrode by substituting neurotrophic medium (Matrigel, Collaborative Research Inc., Bedford, MA [6]) for sciatic nerve inserts and characterize the fine structural features of the cone-contained and surrounding tissue in both species. Electrode construction, implantation, and method of recording of signals have been described [9]. Fig. 1 illustrates the electrode implanted below the surface of cortex. These electrodes were fabricated by gluing one or two recording wires, 2 or 3 mils in diameter, into a small conical piece of glass measuring 50-100/am at its deep end and 25(~400/lm at its superficial end where the wires enter. In the present experiments, however, glass cones were used without wires for two Matrigel-implanted rats. Implantations were made into the barrel cortex of rat for recording sensory responses to vibrissae deflections, and into the motor cortex of monkeys for monitoring signals related to arm and hand movements as determined perioperatively by surface microstimulation. For removal of the cone and its indwelling tissue, rats were perfused with 250 ml of normal saline, followed by 250 ml of 4% glutaraldehyde. In monkeys, the electrode tip, and about 1 cm of surrounding tissue, were removed en masse and immersed in 4% glutaraldehyde solution, In both rats and monkeys, the glass cone was immedi-

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ately removed by cracking it between forceps and returning the splinters piecemeal under a dissecting micro-scope. This procedure usually preserved tissue surrounding both ends of the cone. If the deep end was denuded of tissue, however, the indwelling tissue could be pulled gently from the glass cone. An overview of a cone removed from monkey cortex is shown in Fig. 2A. After cutting away the surrounding cortex in this example, a small amount of tissue surrounds the upper, wider end. and a minute amount remains at the lower end. A closer view of the tip of a different cone removed from rat cortex is shown in Fig. 2B, where tissue outside the cone is seen on the right, while the remainder of the tissue is encased within the glass. The diameter of the tissue narrows as it extends up the glass. In all cases the tissue was continuous inside the cone despite the narrowing. The unavoidable delay between removing the electrode from the brain and extracting the tissue from inside the glass cone often resulted in suboptimal fixation of tissue. For electron microscopy, tissue was fixed in 4% glutaraldehyde and treated with 0.2 M cacodylate buffer. The tissue was then postfixed in 2% osmium tetroxide, dehydrated in ascending concentrations of alcohol, treated with propylene oxide, and embedded in Epon. Ultrathin sections were examined on an RCA EMU-4 electron microscope following staining with 4% uranyl acetate and 0.1% lead citrate. Recordings were made from both rat and monkey central nervous systems as described [9]. In monkey CF63, electrical activity recorded at 15 months is shown in Fig. 3A. These waveshapes were recorded while the monkey reached for food with the hand contralateral to the primary m o t o r cortex that had been implanted with a sciatic nerve-filled electrode. Increased frequency of spike activity was associated with hand movements. Similar waveshapes were recorded three months after implantation of a Matrigel-filted electrode in barrel cortex of rat R34 as shown in Fig. 3B during sedation with Ketamine (30 mg/ kg, i.p.). This activity was evoked by deflecting the contralateral vibrissae on the rat's snout. Some waveshapes are large in amplitude, while others are small and blend together, becoming indistinct. In Fig. 3C, waveshapes are more distinct. These recordings were made 6 months after implantation while the monkey reached for food with the hand contralateral to the implanted electrode. Note the similarity of waveshapes 1, 2 and 3. The top trace also demonstrates a larger and two smaller waveshapes. These waveshapes may be the result of electrical activity in myelinated axons (as discussed below) that pass through the cone at varying distances from the recording wire. Virtually no neurons were found in the tissue in any cone. Electron microscopic observations were made in three

/Z/

Fig. 1. The glass cone is lm flanted into the superficial cortex in rats and monkeys (inset). The cartoon shows a neuron on the left and an astrocyte on the lower right adjacent to the implanted cone containing either sciatic nerve or trophic substrate.

adult rats and two adult monkeys that were implanted with cones containing either sciatic nerve or Matrigel. Sciatic nerve was used in monkey CF63 whose cone: electrode was removed at 15 months after recording semicontinuously from m o n t h three onward (example in Fig. 3A). Examination of tissue within the cone revealed central myelinated axons and capillaries (Fig. 4A). Astrocyte processes containing compact bundles of intermediate filaments were also seen as well as small cells with dark nuclei and cytoplasmic lipid vacuoles, presumably representing microglia or macrophages. No evidence of peripheral myelinated axons or Schwann cells was found. Tissue immediately adjacent to the cone also showed numerous central myelinated axons, some containing mitochondria. Sciatic nerve was used in rat IG32 whose cone electrode was removed after 12 months implantation. Virtually identical findings were encountered, although precise orientation of the intra- and extra-cone tissue was difficult to determine. Numerous central myelinated axons, synapses, swollen glial processes, and blood vessels were seen. One thinly myelinated axon suggesting remyelination was noted. Several neurites (dendrites and an axon) were filled with dense granules. Here, too, no evidence of residual peripheral nerve was noted. In addition, a fibrohistiocytic response with foreign body granulomata was observed. Matrigel was used as a neurotrophic substance placed in the glass cone before implantation in monkey CF49. Six months later, tissue removed from the cone revealed numerous central myelinated axons, Synaptic profiles were seen, some with swollen dendritic processes. Rare degenerating axons were noted. Interspersed glial processes contained abundant filaments. R a t WIG-2 was implanted in the cortical leg area with a Matrigel-filled cone. Four months later, analysis showed numerous cen-

91 tral myelinated axons and glial processes packed with filaments (Fig. 4B). The adjacent superficial cortex showed densely packed astrocytic processes filled with filaments and an overlying pial membrane. The leg area of rat W I G - 4 was implanted with two cones removed after one month. Examination of tissue from the cones (shown grossly in Fig. 2B) revealed similar findings in both specimens: synaptic profiles were observed along with a few central myelinated axons (Fig. 4C,D). In addition, rare neuronal cell bodies, dendrites, and capillaries were present (Fig. 4E). These results illustrate a number of important points. The presence of central myelinated axons, synaptic profiles, dendrites, blood vessels and supporting cells such as glia suggests that neural tissue grew into the glass cone and remained viable for many months. Alternatively, some of the tissue may have been mechanically introduced into the cone during implantation or with subsequent movements. However, the finding in previous experiments that neither tissue ingrowth nor electrophysiological activity occurs in empty control cones, n=5 [9] militates against this possibility. The electrical activity recorded through the wires is likely due to action potentials travelling along the myelinated axons. Since multiple myelinated fibers are found inside the cone, it comes as no surprise that multiple action potentials are recorded. These have different amplitudes, with the largest presumably being closest to the recording wire, or optimally juxtaposed between two wires as shown in Fig. 1. These different sized potentials are then separated using waveshape analysis techniques. The time required for ingrowth of axons would also explain the delay of three weeks or longer before activity can be recorded [9]. Dendrites, too, were occasionally found inside the cone tissue, suggesting that they can grow in and form synaptic complexes (small arrows in Fig. 4C E). The other tissues found inside the cone consisted of supporting stroma such as blood vessels and glia. This suggests that near-normal neuropil may take up residence inside the cone even after just four weeks {Fig. 4C E). Reactive glial changes, of course, might be detrimental to recording electrical events, since gliosis might increase the distance between the recording wire tip and the axons. However, the recordings do not appear to be affected by these fibrillary glial processes since activity persisted t\~r over a year in rats (1G21, IG32) and monkey CF63 (Fig. 3B) or half a year in numerous rats and in monkey CF49 (sciatic nerve insert prior to the Matrigel implant), and monkey R C Z I (Fig. 3C). No evidence was found for remnants of the original insert of sciatic nerve. It was probably phagocytosed by macrophages or microglia that were observed in some

2A

Monkey CF63

2B

Rat Wig4

Fig. 2. A: a glass cone and its tissue after removal from monkey CF63 is shown lying on gridlines that are I mm apart. The smooth conical shape of the center piece is due to the glass and is almost 2 mm in length. Some small remnants of tissue extrude from this end be[\)re folding backward. B: a close-up view of tissue extruding from the narrow end of another glass cone. Bubbles can be seen under the glass and on the tissue. The tissue narrows as it extends up the glass (to the left), but in no case was it absent from inside the glass. It always extended from one end to the other, Bar - 100 j4m.

specimens. Presumably, it acted as an attractant for the neurites. It contains various factors, such as nerve growth factor, fibronectin, laminin and others that may have contributed to the observed neurite ingrowth [5, ll]. There was evidence of Jbreign body reaction only in rat IG32. We had expected a possible reaction to the cyanoacrylate glue holding the wires to the glass, and, to a lesser extent, the gold wires, their Teflon insulation, and the glass. Therefore, any problems encountered with recording neural activity are unlikely to be related to foreign body reaction. When the recordings ceased, it was always because the cone was removed for histological examination, or because the subject damaged the implant. Even when this occurred (as in some of the monkeys), the cone sometimes was retrieved for tissue analysis.

~2

3A

Monkey CF63

3C

Monkey RCZ
1 2 3

3B

Rat R 3 4

Fig. 3. A: examples of recordings from monkey CF63 15 months after implantation. The spikes are transient changes in amplitude reflecting the electrical events adjacent to the recording wires. These transients originate most likely from the myelinated axons, and on that basis are classified as action potentials. More details of recording methods and results are described elsewhere [9]. B: a similar example from rat R34 3 months after implantation. Calibration bars = 20 mV and 10 ms for both A and B. C: a further example from monkey RCZ showing 125 ms of recordings (top trace), with individual spikes 1,2, and 3 displayed on a shorter time base of 20 ms. Horizontal calibration bar = 20 ms for the upper trace, 3.3 ms for the lower three traces. Vertical calibration bar = 20 mV for all traces.

This study does not demonstrate any obvious histological advantage to using sciatic nerve or a substrate such as Matrigel. However, the ease with which Matrigel can be drawn into the glass cone compared to the hours needed to obtain a fresh nerve specimen and insert it into the cone [9] would strongly favor using Matrigel whenever possible. Thus, we recommend using Matrigel in rats. The growth of neurites into the sciatic nerve is not surprising. There is precedence for this observation in the work of Aguayo and colleagues who implanted autologous sciatic nerve in the central nervous system of rats and showed that neurites grow into the length of nerve for a few centimeters [2, 3, 7, 8, 12, 15]. In these studies, however, no functionally useful tissue bridge formed due to the overabundance of connections. The cone electrode is conceptually based upon these

findings of Aguayo and colleagues. It was designed specifically for long-term recordings as a prosthetic neural controller requiring a small number of signals, not necessarily the same all the time, that the patient can voluntarily control for years. With these signals, it is possible that paralyzed patients will control muscle stimulators for restoration of movement. The cone electrode is less well suited for experimental paradigms if very precise histological localization of the recorded neurons is a requisite. In these instances, it would be difficult to determine the cells of origin of the myelinated axons. Other electrodes designed specifically for this purpose [1, 13] are built as arrays of etched silicon shafts bearing multiple recording sites pushed into the cortex. So far, recorded activity has not persisted beyond a few weeks, but the expectation is that these arrays will record over years. Inherent movement problems that lead to gliosis around the tip of the

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Fig. 4. Tissue within cone. All bars - 1/am. A: monkey CF63 15 months alter implantation of sciatic nerve-filled cone. Despite poor preservation of tissue, central myelinated axons (arrows) and a capillary (*) are noted, x3920. B: rat WIG 2 following 4 months implantation oJ"Matrigel-filled cone. A glial process (G) filled with intermediate filaments is seen along with central myelinated axons, x3640. (',l): rat WIG 4 following I month implantation of Matrigcl-filled cone. Neuropil is notcd with synapses {small arrm~s), cemral myelinated axon> (large arro\~s), neurites, and a few dilated processes. (7. 6720; D, 8580. E: rat WIG 4. In another region, a capillar 5 (*) is noted within neuropil containing rgtrc neuronal perikarya (N), dendrites (large arrow), and synapses (small arrows). 5880.

shafts m a y s e p a r a t e the r e c o r d i n g s u r f a c e f r o m the recorded neurons and reduce the s i g n a l - t o - n o i s e lexel. S u c h p r o b l e m s w e r e p r e v i o u s l y e n c o u n t e r e d w i t h a hat pin e l e c t r o d e ; w a x i n g a n d w a n i n g o f signals o c c u r r e d as the e l e c t r o d e m o v e d [14]. N e v e r t h e l e s s , o n e hat pin elect r o d e did r e c o r d for t h r e e years. T h e c o n e e l e c t r o d e , on t h e o t h e r h a n d , c o n t a i n s the tissue b e i n g r e c o r d e d , so

t h a t as the b r a i n m o v e s d u r i n g activity, the r e c o r d e d tissue m o v e s w i t h the e l e c t r o d e . T h i s m a y e x p l a i n the persistcnce a n d stability o f the r e c o r d e d a c t i v i t y in the c o n e electrode. T h e s e e x p e r i m e n t s w e r e c a r r i c d o u t with the p e r m i s sion o f the I n s t i t u t i o n a l A n i m a l C a r e a n d Use C o m m i t -

~4 tee o f both Yerkes Research Center o f E m o r y University a n d the G e o r g i a Institute o f Technology. M a r g a r e t L. Miles provided excellent technical assistance with the electron microscopic studies. D o n n J o h n s o n provided the illustrations in Fig. 1. This investigation was supported in part by N I H G r a n t RR-00165 from the National C e n t e r for Research Resources to the Yerkes Regional P r i m a t e Research Center. The Yerkes C e n t e r is fully accredited by the A m e r i c a n A s s o c i a t i o n of L a b o r a tory A n i m a l Care. The investigation was also supported in part by a g r a n t from the A m e r i c a n Paralysis Association to Dr. K e n n e d y . 1 BeMent, S.L., Wise, K.D., Anderson, D.J., Najafi, K. and Drake, K.L., Solid-state electrodes for multichannel multiplexed intracortical neuronal recording, IEEE Trans. Biomed. Eng., 33 (1986) 230241. 2 Benfey,M, and Aguayo, A.-J., Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature, 296 (1982) 150-152. 3 David, S. and Aguayo, A.-J., Axonal elongation into peripheral nerve system 'bridges' after central nervous system injury in adult rats, Science,214 (1981) 931 033. 4 David, S. and Aguayo, A.-J., Axonal regeneration after crush injury of rat central nervous system fibers innervating peripheral nerve grafts, J. Neurocytol., 14 (1985) l- 12. 5 DiStefano, ES. and Johnson, E.M., Induction of nerve growth factor (NGF) receptors on cultured rat Schwann cells, Soc. Neurosci. Abstr., 12 (1986) 392. 6 Gasser, U.E. and Hatten, M.E., Neuron-gila interactions of rat hippocampal cells In vitro: glial-guided neuronal migration and neuronal regulation of glial differentiation, J. Neurosci., 4 (1984) 1276 1285. 7 Gauthier, E and Rasminsky, M., Activity of medullary respiratory neurons regenerating axons into peripheral nerve grafts in adult rat. Brain Res., 438 (t988) 225 236. 8 Keirstead, S.A., Rasminsky, M., Fukuda, Y., Carter, D.A., Aguayo, A.-J. and Vidal-Sanz, M., Electrophysiological responses in hamster superior colliculus evoked by regenerating retinal axons. Science, 246 (1989) 255- 257. 9 Kennedy, ER., The cone electrode: a long-term electrode that ~ecords from neurites grown onto its recording surface. J. Neurosci. Methods, 29 (1989) 181 193. I0 Kennedy, ER., Bakay, R.A.E., Oyesiku, N. and Banks, D.M., Long-term recording of cortical units using the Cone Electrode in monkeys, Soc. Neurosci. Abstr., 16 (1990) 1134. 11 Letourneau, E, Pech, I., McCarthy, J., Palm, S., Skubitz, A., Charonis, A. and Furcht, L.T., Interactions of nerve fibers with fibronectin and laminin, Int. Symp. Neural Regen. Abstr., Asilomar, 1987, 16. 12 Munz, M., Rasminsky, M., Aguayo, A.-J., Vidal-Sanz, M. and Devor, M.G.~ Functional activity of rat brainstem neurons regenerating axons along peripheral nerve grafts, Brain Res., 340 (1985) 115 125. 13 Najafi, K. and Wise, K.D., An implantable multielectrode array with on-chip signal processing, IEEE J. Solid-State Circuits, SC-21 (1986) 1035-1044. t4 Schmidt, E.M., McIntosh, J.S. and Bak, M.J., Long-term implants of Parylene-C coated microelectrodes, Med, Biol. Eng. Comp., 26 (1988) 96 101. 15 So, K.-F. and Aguayo, A.-J., Lengthy regrowth of cut axons from ganglion cells after peripheral nerve transplantation into the retinae of adult rats, Brain Res.. 238 (1985) 349-354.