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A chronic, moveable nonrotating electrode for brain stimulation in the rat
Physiology & Behavior, Vol. 26, pp. 891-894. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
A Chronic, Moveable Nonrotating Electrode for Brain Stimulation in the Rat ELEFTHERIOS
MILIARESSIS 1AND ALAIN GRATTON
School o f Psychology, University of Ottawa, Ottawa, Canada, K I N 6N5 R e c e i v e d 13 N o v e m b e r 1980 MILIARESSIS, E. AND A. GRATTON. A chronic, moveable nonrotating electrode for brain stimulation in the rat. PHYSIOL. BEHAV. 26(5) 891-894, 1981.--An easy to construct, moveable nonrotating electrode of small dimensions is described. Its reliability and usefulness are demonstrated in circling behavior in mesencephalic areas of the rat. Moveable electrode
Circling
Rat
COMPARISON of the behavioral effects of brain stimulation between sites belonging to different animals is subject to two main limitations: As shown by Wise [8] and Valenstein et al. [7], stimulation of a discrete hypothalamic region may result in different behaviors depending on the animal under investigation. Second, when mapping a circumscribed brain area, there is an appreciable uncertainty as to the precise spatial relationship between the location of the electrode and the surrounding structures across different brains. The above limitations can be partially circumvented by testing several sites in the same animal with the use of a dorsoventrally moveable electrode. Moveable electrodes for brain stimulation in the rat have been constructed by Wise [9] and by Mathis and Schmitt [3] and have been found to be very useful for precise behavioral mapping of the lateral hypothalamus, mesencepbalon, periaqueductal grey and locus coeruleus [1, 2, 6, 8]. The main limitation in Mathis and Schmitt's electrode lies in the complexity of the design and the relatively large dimensions of the device (approximately 1.2 cm large and 3.0 cm high). Wise's electrode is significantly smaller. However, its main limitation is that the stimulating wire rotates with each movement. As stressed by the author of this electrode, the wire has to be soldered perfectly concentric in order to avoid lesioning the brain at each rotation. Following our experience, perfect concentricity is very difficult to achieve, so that only a small percentage of the constructed electrodes can be used. In the present report we describe an easy to make, small, nonrotating electrode for brain stimulation in the rat. In addition the reliability and usefulness of our device are supported by behavioral data obtained in the mesencephalon. MATERIAL AND CONSTRUCTION
Construction of a complete electrode requires the following commercially available materials: One 13 mm piece of 0.25 in. dia. nylon bar to be used as a pedestal; one 16 mm piece of 2-56 threaded rod obtained from a screw to be used
as an excursion screw; one 3 mm piece of 0.125 in. dia. brass bar to be used as a moveable slug; one 3 mm long number 0-80 screw to b e used as retaining screw; 250 /~dia. stainless-steel insulated stimulating wire. The three fu'st materials are machined on a jeweller's lathe as follows: (the letters presently used correspond to the different steps or parts in Fig. 1A). The piece of nylon (a) is bored out for 9 mm of its length (b) using a 1/8 in. reamer. The hole should be started using a N ° 31 drill. The remaining 4 mm (c) is then bored out with a N ° 51 drill and the resulting hole is threaded with a 2-56 tap. Two mm from the bottom of the plastic pedestal thus obtained, a 1 mm thick ridge (d) is cut and two vertical notches are made to insure anchoring of the pedestal in cement. Finally, a N ° 56 drill is used to make a hole (e), 2 mm from the top of the pedestal. The hole is then threaded using a 0-80 tap to fit the retaining screw. The 3 mm slug (f) obtained from 1/8 in. dia. brass bar stock is bored out to a depth of 1 ram (g) using a 1/16 in. end mill and its opposite end is drilled at a 1 mm depth using a N ° 72 drill (h). The upper end of the slug is rounded using sanding paper to facilitate entry of the slug into the pedestal. The upper 4 mm of the excusion screw (i) is trimmed down to fit an amphenol female plug (part No. 202-S02) while 1.5 mm from its lower end (k) is trimmed down to fit the milled end of the brass slug. Finally 1.5 mm of thread at the upper part (l) of the screw are flattened with a file to provide grip when the screw is rotated. A complete electrode weighs about 1 g. Figure 1B shows the above components as assembled and fixed on a rat's cranium. The electrode wire is soldered into the hole made at the lower part of the brass slug. Care must be taken to cover the soldered joint as well as the entire lower surface of the slug with insulating material to avoid current loss caused by seeping cerebrospinal fluid (CSF). Seepage of CSF can be minimized and contact between the stimulating wire and the cement can be prevented by packing the unimplanted part of the wire with petroleum gel. One rotation of the excursion screw lowers the electrode by 0.45 ram. Using the above described lengths of pedestal and slug,
1To whom reprint requests should be addressed.
C o p y r i g h t © 1981 Brain R e s e a r c h Publications Inc.--0031-9384/81/050891-04502.00/0
892
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FIG. 1. A: Schematic representation of the 3 main components as machined. From left tO right: plastic pedestal, brass slug and brass excursion screw~ For explanation see text. B: The complete electrode as assembled and fixed on a rat's cranium. (a) retaining screw; (b) stimulating wire (c) anchoring screws; (d) dental cement; (e) amphenot, male plug used as indifferent electrode; (f) petroleum gel.
MOVEABLE ELECTRODE
893
V
I / ~
iI iI /~ //////
RAT 622
/I iie i i i i i I / I / I / iI / / /
~..~
SG
/"'- PCS
C~rcling
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FIG. 2. Changes in pulse frequency required at different brain sties in rat 622 in order to reach a performance of I rotation per sec in circling behavior. The column at the ordinates represents a qualitative expression of the strength of behavior in each site, i.e., the most heavily shaded area indicating the lowest pulse frequency required to obtain the criterion performance. AC: aqueduct; FLM: fasciculus longitudinal& medialis; FOR: reticular formation; PCS: superior cerebeUar peduncle; SG: periaqueductal grey; TTS: tractus tectospinalis; tp: nucleus tegmenti pontis; vt: nucleus ventralis tegmenti.
the maximum excursion of the wire is 6.0 mm. This excursion range was chosen for studies on rats. Of course, its extent can be adapted to fit larger-brained animals. Accidental rotation of the excursion screw is prevented by tightening the retaining screw as shown. APPLICATION
Movement of the electrode is accomplished by grasping the flattened part of the excursion screw with a pair of small forceps and by turning it a predetermined portion of a revolution. With obedient rats this operation can be accomplished easily, without anesthesia. With the use of the above described moveable nonrotating electrode, mesencephalic regions were tested for circling behavior. The electrode was implanted 3.0 mm below the surface of the skull and subsequently lowered down by steps of 227 /.t. The stimulation consisted of cathodal pulses (0.1 msec in duration) of constant intensity (250/~A), and variable frequency. The efficacy of the stimulation was inferred by the pulse frequency required in order for the animal to reach a criterion performance of one rotation per sec. This procedure allows one to keep the size of the stimulated field constant and to avoid distortion of the measurements by behavioral floor and ceil-
ing effects (for detailed exposition of this problem see [10]). This procedure seems therefore essential in the establishment of discrete behavioral brain mapping. Circling behavior was studied in a cylinder of 20 cm in dia. The animal received continuous stimulation until 3 consecutive rotations were completed (for procedural details see [4]). Data for each stimulated site were obtained from several speed-frequency functions. At the end of the experiments the animals were sacrificed and their brains were sliced (28 ~ thick) in a cryostat microtome and subsequently stained with thionine for the purpose of macroscopic examination of the electrode tracks. Figure 2 shows the required pulse frequency changes in order to obtain the criterion performance of circling in 20 dorsoventral locations in a single animal. It can be observed that circling is elicited from two independent and relatively large brain regions. These regions are respectively located within the periaqueductal grey and lateral to the median raphe nucleus. Negative sites were found within and immediately above the superior cerebellar peduncle (PCS). Especially in the region below the PCS, the U-shaped progressive changes in the required pulse frequency provides a fairly detailed picture of the relative importance of each site in circling behavior. The present observations related to the
894
MILIARESSIS AND GRATTON
raphe region confirm our previous f'mdings obtained from several animals with the use of a single fixed electrode [4,5]. Extensive examination of this region for circling falls outside the scope of this presentation. However we would like to stress that the present data obtained from a single animal provide a more complete anatomicalpicture when compared to our previous studies in which several animals with fixed electrodes were used [4,5]. Since the placement of the stimulated sites is inferred by the location of the tip of the electrode, some adjustment must be made in order to compensate for possible dilatation or shrinkage of the brain due to histological procedure. This factor which was not considered in the present report, is presently under investigation. RELIABILITYOF THE ELECTRODE The main disadvantage of our electrode lies in the fact that the brass slug and excursion screw constitute two independent pieces, therefore leaving the possibility for contact interruption. As a consequence we thought that a tiny drop
of mercury in the hole of insertion of the screw into the slug would be essential in order to insure uninterrupted electrical conductivity. However, routine subsequent testing revealed that mercury is not necessary. Electrical conductivity was continuously monitored during stimulation by looking at the voltage drop across the rat's active and indifferent electrodes. In addition, loss can be revealed by sudden changes in the intensity of the recorded behavior. Data for circling behavior obtained from a single site of an animal tested throughout a period of one month showed variations which do not exceed the expected changes due to behavioral variability (mean pulse frequency required to reach the behavioral criterion and standard error for 9 sessions =45.6 _+ 0.8). In the present report we described an easy to make, small nonrotating electrode for brain stimulation in the rat. In addition we showed that this electrode is reliable. Its main advantage in comparison to other moveable electrodes is that the stimulation wire is nonrotating but still the electrode is of small dimensions and easy to make.
REFERENCES 1. Corbett, D. and R. Wise. Intracranial self-stimulation in relation to the ascending noradrenergic fiber systems of the pontine tegmentum and caudal midbrain: A moveable electrode mapping study. Brain Res. 177: 423-436, 1975. 2. Corbett, D. and R. Wise. Intracranial self-stimulation in relation to the ascending dopaminergic systems of the midbrain: A moveable electrode mapping study. Brain Res. 185: 1-15, 1980. 3. Mathis, G. and P. Schrnitt. Dispositif h demeure pour la descente progressive d'une 61eetrode dans le cerveau du rat. Physiol. Behav. 12: 281-283, 1974. 4. Miliaressis, E. Refractoriness of neurons subserving circling following stimulation of the median raphe region in the rat. Physiol. Behav., 1981, in press. 5. Miliaressis, E. and P. P. Romprr. Self-stimulation and circling: Differentiation of the neural substrata by behavioral measurement by the use of the double pulse technique. Physiol. Beha v. 25: 939--943, 1980.
6. Schmitt, P., F. Eclancher and P. Carli. Etude des systrmes de renforcement nrgatif et de renforcement positif au niveau de la substance grise centrale chez le rat. Physiol. Behav. 12: 271279, 1974. 7. Valenstein, E. S., V. C. Cox and J. W. Kakolewski. Reexamination of the role of the hypothalamus in motivation. Psychol. Rev. 77: 16--31, 1970. 8. Wise, R. A. Individual differences in effects of hypothalamic stimulation: The role of stimulation locus. Physiol. Behav. 6: 569--572, 1971. 9. Wise, R. A. Moveable electrode for chronic brain stimulation in the rat. Physiol. Behav. 16: 105-106, 1976. 10. Yeomans, J. S. Quantitative measurement of neural poststimulation excitability with behavioral methods. Physiol. Behay. 15: 593--602, 1975.
A Chronic, Moveable Nonrotating Electrode for Brain Stimulation in the Rat ELEFTHERIOS
MILIARESSIS 1AND ALAIN GRATTON
School o f Psychology, University of Ottawa, Ottawa, Canada, K I N 6N5 R e c e i v e d 13 N o v e m b e r 1980 MILIARESSIS, E. AND A. GRATTON. A chronic, moveable nonrotating electrode for brain stimulation in the rat. PHYSIOL. BEHAV. 26(5) 891-894, 1981.--An easy to construct, moveable nonrotating electrode of small dimensions is described. Its reliability and usefulness are demonstrated in circling behavior in mesencephalic areas of the rat. Moveable electrode
Circling
Rat
COMPARISON of the behavioral effects of brain stimulation between sites belonging to different animals is subject to two main limitations: As shown by Wise [8] and Valenstein et al. [7], stimulation of a discrete hypothalamic region may result in different behaviors depending on the animal under investigation. Second, when mapping a circumscribed brain area, there is an appreciable uncertainty as to the precise spatial relationship between the location of the electrode and the surrounding structures across different brains. The above limitations can be partially circumvented by testing several sites in the same animal with the use of a dorsoventrally moveable electrode. Moveable electrodes for brain stimulation in the rat have been constructed by Wise [9] and by Mathis and Schmitt [3] and have been found to be very useful for precise behavioral mapping of the lateral hypothalamus, mesencepbalon, periaqueductal grey and locus coeruleus [1, 2, 6, 8]. The main limitation in Mathis and Schmitt's electrode lies in the complexity of the design and the relatively large dimensions of the device (approximately 1.2 cm large and 3.0 cm high). Wise's electrode is significantly smaller. However, its main limitation is that the stimulating wire rotates with each movement. As stressed by the author of this electrode, the wire has to be soldered perfectly concentric in order to avoid lesioning the brain at each rotation. Following our experience, perfect concentricity is very difficult to achieve, so that only a small percentage of the constructed electrodes can be used. In the present report we describe an easy to make, small, nonrotating electrode for brain stimulation in the rat. In addition the reliability and usefulness of our device are supported by behavioral data obtained in the mesencephalon. MATERIAL AND CONSTRUCTION
Construction of a complete electrode requires the following commercially available materials: One 13 mm piece of 0.25 in. dia. nylon bar to be used as a pedestal; one 16 mm piece of 2-56 threaded rod obtained from a screw to be used
as an excursion screw; one 3 mm piece of 0.125 in. dia. brass bar to be used as a moveable slug; one 3 mm long number 0-80 screw to b e used as retaining screw; 250 /~dia. stainless-steel insulated stimulating wire. The three fu'st materials are machined on a jeweller's lathe as follows: (the letters presently used correspond to the different steps or parts in Fig. 1A). The piece of nylon (a) is bored out for 9 mm of its length (b) using a 1/8 in. reamer. The hole should be started using a N ° 31 drill. The remaining 4 mm (c) is then bored out with a N ° 51 drill and the resulting hole is threaded with a 2-56 tap. Two mm from the bottom of the plastic pedestal thus obtained, a 1 mm thick ridge (d) is cut and two vertical notches are made to insure anchoring of the pedestal in cement. Finally, a N ° 56 drill is used to make a hole (e), 2 mm from the top of the pedestal. The hole is then threaded using a 0-80 tap to fit the retaining screw. The 3 mm slug (f) obtained from 1/8 in. dia. brass bar stock is bored out to a depth of 1 ram (g) using a 1/16 in. end mill and its opposite end is drilled at a 1 mm depth using a N ° 72 drill (h). The upper end of the slug is rounded using sanding paper to facilitate entry of the slug into the pedestal. The upper 4 mm of the excusion screw (i) is trimmed down to fit an amphenol female plug (part No. 202-S02) while 1.5 mm from its lower end (k) is trimmed down to fit the milled end of the brass slug. Finally 1.5 mm of thread at the upper part (l) of the screw are flattened with a file to provide grip when the screw is rotated. A complete electrode weighs about 1 g. Figure 1B shows the above components as assembled and fixed on a rat's cranium. The electrode wire is soldered into the hole made at the lower part of the brass slug. Care must be taken to cover the soldered joint as well as the entire lower surface of the slug with insulating material to avoid current loss caused by seeping cerebrospinal fluid (CSF). Seepage of CSF can be minimized and contact between the stimulating wire and the cement can be prevented by packing the unimplanted part of the wire with petroleum gel. One rotation of the excursion screw lowers the electrode by 0.45 ram. Using the above described lengths of pedestal and slug,
1To whom reprint requests should be addressed.
C o p y r i g h t © 1981 Brain R e s e a r c h Publications Inc.--0031-9384/81/050891-04502.00/0
892
M I L I A R E S S I S AN D G R A T T O N
l"~
A-
measurements in m m ....= I m m
®
---N
6.4
4
2 ®i~
x-j
®
.5
®i ~ !
J
< ~
16
@ 3
®
1.5 ,r
.5
•
i~ 1.7 =i =
1.7 ~i
3
FIG.la
FIGlb
®
**©oo¢>
0.
@
L2o i~o ~ . . . .
/
"~
/
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
FIG. 1. A: Schematic representation of the 3 main components as machined. From left tO right: plastic pedestal, brass slug and brass excursion screw~ For explanation see text. B: The complete electrode as assembled and fixed on a rat's cranium. (a) retaining screw; (b) stimulating wire (c) anchoring screws; (d) dental cement; (e) amphenot, male plug used as indifferent electrode; (f) petroleum gel.
MOVEABLE ELECTRODE
893
V
I / ~
iI iI /~ //////
RAT 622
/I iie i i i i i I / I / I / iI / / /
~..~
SG
/"'- PCS
C~rcling
' '" " " "'""::::~
~FLM
] .,;/" .-"
P~
I' FOR TTS--~ ' i
i b
=
" 0
--//
i
40
i
i
I00
T
,
~
i
i
,
J
i
,
200 Frequency of pulses
FIG. 2. Changes in pulse frequency required at different brain sties in rat 622 in order to reach a performance of I rotation per sec in circling behavior. The column at the ordinates represents a qualitative expression of the strength of behavior in each site, i.e., the most heavily shaded area indicating the lowest pulse frequency required to obtain the criterion performance. AC: aqueduct; FLM: fasciculus longitudinal& medialis; FOR: reticular formation; PCS: superior cerebeUar peduncle; SG: periaqueductal grey; TTS: tractus tectospinalis; tp: nucleus tegmenti pontis; vt: nucleus ventralis tegmenti.
the maximum excursion of the wire is 6.0 mm. This excursion range was chosen for studies on rats. Of course, its extent can be adapted to fit larger-brained animals. Accidental rotation of the excursion screw is prevented by tightening the retaining screw as shown. APPLICATION
Movement of the electrode is accomplished by grasping the flattened part of the excursion screw with a pair of small forceps and by turning it a predetermined portion of a revolution. With obedient rats this operation can be accomplished easily, without anesthesia. With the use of the above described moveable nonrotating electrode, mesencephalic regions were tested for circling behavior. The electrode was implanted 3.0 mm below the surface of the skull and subsequently lowered down by steps of 227 /.t. The stimulation consisted of cathodal pulses (0.1 msec in duration) of constant intensity (250/~A), and variable frequency. The efficacy of the stimulation was inferred by the pulse frequency required in order for the animal to reach a criterion performance of one rotation per sec. This procedure allows one to keep the size of the stimulated field constant and to avoid distortion of the measurements by behavioral floor and ceil-
ing effects (for detailed exposition of this problem see [10]). This procedure seems therefore essential in the establishment of discrete behavioral brain mapping. Circling behavior was studied in a cylinder of 20 cm in dia. The animal received continuous stimulation until 3 consecutive rotations were completed (for procedural details see [4]). Data for each stimulated site were obtained from several speed-frequency functions. At the end of the experiments the animals were sacrificed and their brains were sliced (28 ~ thick) in a cryostat microtome and subsequently stained with thionine for the purpose of macroscopic examination of the electrode tracks. Figure 2 shows the required pulse frequency changes in order to obtain the criterion performance of circling in 20 dorsoventral locations in a single animal. It can be observed that circling is elicited from two independent and relatively large brain regions. These regions are respectively located within the periaqueductal grey and lateral to the median raphe nucleus. Negative sites were found within and immediately above the superior cerebellar peduncle (PCS). Especially in the region below the PCS, the U-shaped progressive changes in the required pulse frequency provides a fairly detailed picture of the relative importance of each site in circling behavior. The present observations related to the
894
MILIARESSIS AND GRATTON
raphe region confirm our previous f'mdings obtained from several animals with the use of a single fixed electrode [4,5]. Extensive examination of this region for circling falls outside the scope of this presentation. However we would like to stress that the present data obtained from a single animal provide a more complete anatomicalpicture when compared to our previous studies in which several animals with fixed electrodes were used [4,5]. Since the placement of the stimulated sites is inferred by the location of the tip of the electrode, some adjustment must be made in order to compensate for possible dilatation or shrinkage of the brain due to histological procedure. This factor which was not considered in the present report, is presently under investigation. RELIABILITYOF THE ELECTRODE The main disadvantage of our electrode lies in the fact that the brass slug and excursion screw constitute two independent pieces, therefore leaving the possibility for contact interruption. As a consequence we thought that a tiny drop
of mercury in the hole of insertion of the screw into the slug would be essential in order to insure uninterrupted electrical conductivity. However, routine subsequent testing revealed that mercury is not necessary. Electrical conductivity was continuously monitored during stimulation by looking at the voltage drop across the rat's active and indifferent electrodes. In addition, loss can be revealed by sudden changes in the intensity of the recorded behavior. Data for circling behavior obtained from a single site of an animal tested throughout a period of one month showed variations which do not exceed the expected changes due to behavioral variability (mean pulse frequency required to reach the behavioral criterion and standard error for 9 sessions =45.6 _+ 0.8). In the present report we described an easy to make, small nonrotating electrode for brain stimulation in the rat. In addition we showed that this electrode is reliable. Its main advantage in comparison to other moveable electrodes is that the stimulation wire is nonrotating but still the electrode is of small dimensions and easy to make.
REFERENCES 1. Corbett, D. and R. Wise. Intracranial self-stimulation in relation to the ascending noradrenergic fiber systems of the pontine tegmentum and caudal midbrain: A moveable electrode mapping study. Brain Res. 177: 423-436, 1975. 2. Corbett, D. and R. Wise. Intracranial self-stimulation in relation to the ascending dopaminergic systems of the midbrain: A moveable electrode mapping study. Brain Res. 185: 1-15, 1980. 3. Mathis, G. and P. Schrnitt. Dispositif h demeure pour la descente progressive d'une 61eetrode dans le cerveau du rat. Physiol. Behav. 12: 281-283, 1974. 4. Miliaressis, E. Refractoriness of neurons subserving circling following stimulation of the median raphe region in the rat. Physiol. Behav., 1981, in press. 5. Miliaressis, E. and P. P. Romprr. Self-stimulation and circling: Differentiation of the neural substrata by behavioral measurement by the use of the double pulse technique. Physiol. Beha v. 25: 939--943, 1980.
6. Schmitt, P., F. Eclancher and P. Carli. Etude des systrmes de renforcement nrgatif et de renforcement positif au niveau de la substance grise centrale chez le rat. Physiol. Behav. 12: 271279, 1974. 7. Valenstein, E. S., V. C. Cox and J. W. Kakolewski. Reexamination of the role of the hypothalamus in motivation. Psychol. Rev. 77: 16--31, 1970. 8. Wise, R. A. Individual differences in effects of hypothalamic stimulation: The role of stimulation locus. Physiol. Behav. 6: 569--572, 1971. 9. Wise, R. A. Moveable electrode for chronic brain stimulation in the rat. Physiol. Behav. 16: 105-106, 1976. 10. Yeomans, J. S. Quantitative measurement of neural poststimulation excitability with behavioral methods. Physiol. Behay. 15: 593--602, 1975.