- Email: [email protected]
A method for determining the position of chronically implanted platinum microwire electrodes
,lournal of Neuroscience Method~'. 5 (1982) 7- I 1 Elsevier Biomcdical Prcss
7
Research Papers
A method for determining the position of chronically implanted platinum microwire electrodes A.J.S. Summerlee, A.C. Paisley and C.L. Goodall Department of A naton~v, The Medical School, UniversiO' Walk, Bristol BS8 1 TD ( U.K.) (Received April 27th, 1981 ) (Revised version received June 20th, 1981 ) (Accepted June 22nd, 1981 ) K
Introduction
There has been an increasing interest in microwire recording techniques ever since they were first introduced by Strumwasser in 1958. A variety of different metals or metal alloys have been implanted into nervous tissue to record the activity of single neurones in several different anatomical sites, for example, the cerebral cortex (Burns et al., 1974), the reticular formation (Trulson and Jacobs, 1979), cerebellum (Palmer, 1978) and hypothalamus (Paisley and Summerlee, 1980). Platinum, which is chemically and biologically inert, is often used for construction of microwire electrodes due to its excellent properties for recording neuronal activity. The drawback with using platinum for recording electrodes stems from its inertness. There is no stain that is specific for platinum ions deposited in nervous tissue, which is analogous to the technique for staining iron deposits by the Prussian blue method. To determine the position of the tip of a platinum recording electrode it has been necessary to serially reconstruct histological sections of brain tissue to follow the electrode tracks surrounded by a collagen scar (Burns et al. 1974; Summerlee and Lincoln, 1981). Our aim was to produce a simple and readily applied technique that could be used to determine reliably the position of the tip of a particular platinum microwire implanted into the brain. 0165-0270/82/0000-0000/$02.75
t.' 1982 Elsevier Biomedical Press
Materials and methods
Adult female Californian rabbits were chronically implanted with platinum microwire electrodes (30/~m in diameter) insulated along their length and square cut across the tip. The procedure for implanting the microwires is described elsewhere (Summerlee and Lincoln, 1981). At the end of the experimental period, usually greater than 6 months, specific electrodes were chosen for the marking technique. The rabbit was sedated with xylazine ( 2 m g - k g - I : Rompun, Bayer) and the selected electrode was connected to the positive output of a DC source. The negative pole was connected to the reference electrode of the recording system, a stainless-steel stud screwed through the skull. A current of 100 /~A was passed through the platinum microwire for 1 rain. The animal was allowed to recover from the sedative and returned to the free-range run. An astrocytosis developed around the tip of the electrode in response to the electrical lesion. The animal was premedicated 7 10 days later with xylazine and anaesthetized (pentobarbitone 40 m g . kg t: Sagatal, May and Baker). The brain was perfused with f o r m a l i n - a m m o n i u m bromide solution (Carleton and Drury, 1957) administered through the left ventricle. The brain was removed and left to fix in f o r m a l i n - a m m o n i u m bromide for 14 days, then transferred to a solution of formalin and 9% sucrose for a further 7 days. Forty-eight hours before sectioning the brain was transferred to a solution of 30% sucrose. Serial sections (20 /~m thick) were then cut on a freezing microtome. Alternate sections were stained with Cajal's gold chloride sublimate (Carleton and Drury, 1957) and cresyl violet. Results
A discrete lesion (60-100 t~m in diameter) was produced (Fig. 1) using a current strength of 100 # A for 1 min. The area of the astrocytosis which occurred in response to the electrical lesion varied with the intensity of the current passed through the electrode. A current of 200 #A produced a lesion that covered an area greater than 500 Cm, whereas 50 CA did not produce an astrocytosis that could be detected by the stain. Astrocytes were stained black by the Cajal's gold chloride sublimate method and the background tissue stained reddish-purple. Fibre tracts stained clearly dark red. The positive staining was simply not a reaction to the electro-coagulated tissue, as tissue fixed and stained immediately after lesioning only occasionally showed a slight darkening of the affected area. There was no astrocytosis that could be seen by the staining method along the shafts of the electrodes (see Discussion). As alternate sections were stained with cresyl violet, it was possible to determine accurately the position of the tip of the recording electrode with respect to the cell bodies in the nervous tissue. Fig. I. Two plates of a histological section (20 t~m thick) cut from a rabbit brain. The sections are stained with Cajal's gold chloride sublimate which is specific for astrocytes. The upper part of the figure is a complete frontal section to show the position of the astrocytosis (?) that developed in response to a pin-point electrolytic lesion. The lower part of the figure is an enlargement of the area around the astrocytosis. The astrocytes stain black against a reddish-purple background.
"1
IO
Discussion It is important to verify the position of the tip of electrodes used to record neuronal activity, particularly for chronic recording techniques where several microwire electrodes are implanted into one animal. Until now the methods used were simply to follow the tracks of scarfing left by the electrodes in the brain tissue by a serial reconstruction of histological sections, e.g. Masson's green trichrome (see Burns et al., 1974; Summerlee and Lincoln, 1981). These tracks are present for all the electrodes implanted into the brain, yet some of the electrodes may not record neuronal activity, so a stain which highlights generalized gliotic tissue is of limited value. It is therefore necessary to have a technique that can demonstrate histologically the site of recording for any chosen electrode. Cajal's gold chloride sublimate method of staining has been used previously to examine nervous tissue for the presence of astrocytes (Penfield, 1927; Ramon Y Cajal, 1959). The present technique makes use of the fact that an astrocytosis will develop in response to damage to the nervous system, providing that sufficient time is left for the scarring to occur. No gliosis however, could be detected around the shafts of all the implanted electrodes using the Cajal stain. The difference compared with other stains for gliosis (Masson's green) in other studies (see Burns et al., 1974) is possibly due to the aseptic approach for implantation of our microwires. The technique is not suitable for the rapid localization of electrodes placed acutely in the nervous tissue as a prolonged fixation time is required before the brain can be sectioned. However, as these chronically implanted electrodes can be used for recording neuronal activity in excess of 6 months, this fixation time is negligible compared with the value of positive histological identification of the site of recording.
Acknowledgements The authors are grateful to D.W. Lincoln for making facilities available to them. The work was supported by a project grant to D.W. Lincoln from the Medical Research Council.
References Burns, B.D., Stean, J.P.B. and Webb, A.C. (1974) Recording for several days from single cortical neurons in completely unrestrained cats, Electroenceph. clin. Neurophysiol.. 36:314-318. Carleton, H.M. and Drury, R.A.B. (1957) Histological techniques for normal and pathological tissue and the identification of parasites, 3rd. Edn., Oxford University Press. London, pp. 245-246. Paisley, A.C. and Summerlee, A.J.S. (1980) A reappraisal of the effect of sleep on the spontaneous activity of hypothalamic neurones, Neurosci. Len., Suppl. 5: SI29. Palmer, C. (1978) A microwire technique for recording single neurons in unrestrained animals. Br. Res. B.C., 3: 001-005. Penfield, W. (1927) The mechanism of cicatrical contraction in the brain. Brain. 50:499-517
Ramon Y Cajal, S. (1959) Degeneration and regeneration of the nervous system, R.M. May (Ed.) Hafner, New York, Vol. II, pp. 714-733. Strumwasser, F. (1958) Long-term recording from single neurons in brain of unrestrained mammals, Science, 127:469-470. Summerlee, A.J.S. and Lincoln, D.W. (1981) Electrophysiological recordings from oxytocinergic neurones in the unanaesthetized, lactating rat, J. Endocr., in press. Trulson and Jacobs (1979) Raphe unit activity in freely-moving cats: correlation with level of behavioural arousal, Brain Res., 163: 135-150.
7
Research Papers
A method for determining the position of chronically implanted platinum microwire electrodes A.J.S. Summerlee, A.C. Paisley and C.L. Goodall Department of A naton~v, The Medical School, UniversiO' Walk, Bristol BS8 1 TD ( U.K.) (Received April 27th, 1981 ) (Revised version received June 20th, 1981 ) (Accepted June 22nd, 1981 ) K
Introduction
There has been an increasing interest in microwire recording techniques ever since they were first introduced by Strumwasser in 1958. A variety of different metals or metal alloys have been implanted into nervous tissue to record the activity of single neurones in several different anatomical sites, for example, the cerebral cortex (Burns et al., 1974), the reticular formation (Trulson and Jacobs, 1979), cerebellum (Palmer, 1978) and hypothalamus (Paisley and Summerlee, 1980). Platinum, which is chemically and biologically inert, is often used for construction of microwire electrodes due to its excellent properties for recording neuronal activity. The drawback with using platinum for recording electrodes stems from its inertness. There is no stain that is specific for platinum ions deposited in nervous tissue, which is analogous to the technique for staining iron deposits by the Prussian blue method. To determine the position of the tip of a platinum recording electrode it has been necessary to serially reconstruct histological sections of brain tissue to follow the electrode tracks surrounded by a collagen scar (Burns et al. 1974; Summerlee and Lincoln, 1981). Our aim was to produce a simple and readily applied technique that could be used to determine reliably the position of the tip of a particular platinum microwire implanted into the brain. 0165-0270/82/0000-0000/$02.75
t.' 1982 Elsevier Biomedical Press
Materials and methods
Adult female Californian rabbits were chronically implanted with platinum microwire electrodes (30/~m in diameter) insulated along their length and square cut across the tip. The procedure for implanting the microwires is described elsewhere (Summerlee and Lincoln, 1981). At the end of the experimental period, usually greater than 6 months, specific electrodes were chosen for the marking technique. The rabbit was sedated with xylazine ( 2 m g - k g - I : Rompun, Bayer) and the selected electrode was connected to the positive output of a DC source. The negative pole was connected to the reference electrode of the recording system, a stainless-steel stud screwed through the skull. A current of 100 /~A was passed through the platinum microwire for 1 rain. The animal was allowed to recover from the sedative and returned to the free-range run. An astrocytosis developed around the tip of the electrode in response to the electrical lesion. The animal was premedicated 7 10 days later with xylazine and anaesthetized (pentobarbitone 40 m g . kg t: Sagatal, May and Baker). The brain was perfused with f o r m a l i n - a m m o n i u m bromide solution (Carleton and Drury, 1957) administered through the left ventricle. The brain was removed and left to fix in f o r m a l i n - a m m o n i u m bromide for 14 days, then transferred to a solution of formalin and 9% sucrose for a further 7 days. Forty-eight hours before sectioning the brain was transferred to a solution of 30% sucrose. Serial sections (20 /~m thick) were then cut on a freezing microtome. Alternate sections were stained with Cajal's gold chloride sublimate (Carleton and Drury, 1957) and cresyl violet. Results
A discrete lesion (60-100 t~m in diameter) was produced (Fig. 1) using a current strength of 100 # A for 1 min. The area of the astrocytosis which occurred in response to the electrical lesion varied with the intensity of the current passed through the electrode. A current of 200 #A produced a lesion that covered an area greater than 500 Cm, whereas 50 CA did not produce an astrocytosis that could be detected by the stain. Astrocytes were stained black by the Cajal's gold chloride sublimate method and the background tissue stained reddish-purple. Fibre tracts stained clearly dark red. The positive staining was simply not a reaction to the electro-coagulated tissue, as tissue fixed and stained immediately after lesioning only occasionally showed a slight darkening of the affected area. There was no astrocytosis that could be seen by the staining method along the shafts of the electrodes (see Discussion). As alternate sections were stained with cresyl violet, it was possible to determine accurately the position of the tip of the recording electrode with respect to the cell bodies in the nervous tissue. Fig. I. Two plates of a histological section (20 t~m thick) cut from a rabbit brain. The sections are stained with Cajal's gold chloride sublimate which is specific for astrocytes. The upper part of the figure is a complete frontal section to show the position of the astrocytosis (?) that developed in response to a pin-point electrolytic lesion. The lower part of the figure is an enlargement of the area around the astrocytosis. The astrocytes stain black against a reddish-purple background.
"1
IO
Discussion It is important to verify the position of the tip of electrodes used to record neuronal activity, particularly for chronic recording techniques where several microwire electrodes are implanted into one animal. Until now the methods used were simply to follow the tracks of scarfing left by the electrodes in the brain tissue by a serial reconstruction of histological sections, e.g. Masson's green trichrome (see Burns et al., 1974; Summerlee and Lincoln, 1981). These tracks are present for all the electrodes implanted into the brain, yet some of the electrodes may not record neuronal activity, so a stain which highlights generalized gliotic tissue is of limited value. It is therefore necessary to have a technique that can demonstrate histologically the site of recording for any chosen electrode. Cajal's gold chloride sublimate method of staining has been used previously to examine nervous tissue for the presence of astrocytes (Penfield, 1927; Ramon Y Cajal, 1959). The present technique makes use of the fact that an astrocytosis will develop in response to damage to the nervous system, providing that sufficient time is left for the scarring to occur. No gliosis however, could be detected around the shafts of all the implanted electrodes using the Cajal stain. The difference compared with other stains for gliosis (Masson's green) in other studies (see Burns et al., 1974) is possibly due to the aseptic approach for implantation of our microwires. The technique is not suitable for the rapid localization of electrodes placed acutely in the nervous tissue as a prolonged fixation time is required before the brain can be sectioned. However, as these chronically implanted electrodes can be used for recording neuronal activity in excess of 6 months, this fixation time is negligible compared with the value of positive histological identification of the site of recording.
Acknowledgements The authors are grateful to D.W. Lincoln for making facilities available to them. The work was supported by a project grant to D.W. Lincoln from the Medical Research Council.
References Burns, B.D., Stean, J.P.B. and Webb, A.C. (1974) Recording for several days from single cortical neurons in completely unrestrained cats, Electroenceph. clin. Neurophysiol.. 36:314-318. Carleton, H.M. and Drury, R.A.B. (1957) Histological techniques for normal and pathological tissue and the identification of parasites, 3rd. Edn., Oxford University Press. London, pp. 245-246. Paisley, A.C. and Summerlee, A.J.S. (1980) A reappraisal of the effect of sleep on the spontaneous activity of hypothalamic neurones, Neurosci. Len., Suppl. 5: SI29. Palmer, C. (1978) A microwire technique for recording single neurons in unrestrained animals. Br. Res. B.C., 3: 001-005. Penfield, W. (1927) The mechanism of cicatrical contraction in the brain. Brain. 50:499-517
Ramon Y Cajal, S. (1959) Degeneration and regeneration of the nervous system, R.M. May (Ed.) Hafner, New York, Vol. II, pp. 714-733. Strumwasser, F. (1958) Long-term recording from single neurons in brain of unrestrained mammals, Science, 127:469-470. Summerlee, A.J.S. and Lincoln, D.W. (1981) Electrophysiological recordings from oxytocinergic neurones in the unanaesthetized, lactating rat, J. Endocr., in press. Trulson and Jacobs (1979) Raphe unit activity in freely-moving cats: correlation with level of behavioural arousal, Brain Res., 163: 135-150.