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A simple multiple-cannula headpiece for the rat
Physiology& Behavior,Vol. 26, pp. 897-900. PergamonPress and Brain Research Publ., 1981.Printed in the U.S.A.
A Simple Multiple-Cannula Headpiece for the Rat G E O R G E G. D O U G H E R T Y , JR. 1 A N D E V E R E T r H. E L L I N W O O D , JR.
Behavioral Neuropharmacology Laboratory, Department o f Psychiatry Duke University Medical Center, Durham, N C 27710 Received 1 D e c e m b e r 1980 DOUGHERTY, G. G., JR. AND E. H. ELLINWOOD, JR. A simple multiple-cannula headpiecefor the rat. PHYSIOL. BEHAV. 26(5) 897-900, 1981.--The construction of a muRiple-cannula headpiece, its implantation into the rat brain, and its use in intracranial injections is described. The headpiece is rapidly and cheaply produced from common hospital and laboratory supplies once several machined metal parts have been made, and its construction is easily adapted to changes in injection technique, e.g., infusion by osmotic minipump. Cannula
Stereotaxictechnique
Rat
Osmoticminipump
headpiece, covering the tops of the guide cannulae and stylets between injections. The machined metal parts used to mass-produce these headpieces are described below. The premise used in the design of this headpiece was that the precise relative positioning of several guide cannulae within the headpiece would allow their simultaneous implantation into intended sites in the rat brain. To this end, holes were drilled in the surface of a small block of aluminum (the "base block") to have the same relative antero-posterior (AP) and lateral (L) stereotaxic positions as would the guide cannulae intended for implantation. Great accuracy was used in positioning these holes. The hole diameter was chosen to allow a snug fit with inserted 26 gauge tubing. The relative hole depths were determined from the relative stereotaxic depth coordinates for the cannulae. The cylindrical headpiece was made by placing guide cannulae into the holes of the base block after inner surfaces were coated with stopcock grease. Freshly mixed dental acrylic cement was then poured into a mold placed about the protruding guides on the base block. The mold was made from two oblong pieces of aluminum, each with a half a cylinder cut from one side. The walls of the mold were tapped with threads so that the molded piece produced would have threads on its outer surface. Fine-pitch 3/8 in. threading was used. The base block had been constructed with a 2 mm ridge on one side, parallel to the AP axis. When one edge of the mold was flush with this ridge, the cylindrical opening was centered over the drilled holes. Twenty-six gauge, 1/2 in. long hypodermic needles were used for the guide cannulae. The needles were denuded of their plastic adapters, leaving a thin layer of ceramic about the shaft, before they were placed point-down into the holes in the base block. The stylets were constructed from 32 gauge stainless steel tubing (Small Parts, Inc.), with a short "handle" of larger
THE STIMULATION of specific brain sites by administration of small amounts of drugs or neurotransmitters has come to be regarded as a useful technique in the study of brain and behavior [7]. Our work has often involved the assessment of brain changes following chronic exposure to a drug, by means of studying the response to subsequent administration of "pharmacologic probes" into brain nuclei. A practical advantage is gained if multiple brain sites can be tested in a single chronically-treated animal. However, the stereotaxic implantation of multiple cannulae for such a study can be very time-consuming, especially when a large number of subjects is required. The technical awkwardness of the successive positioning and cementing of cannulae commercially available or described by others [2, 3, 4, 5] is also an undesirable feature of the procedure. We have confronted these difficulties by constructing a headpiece conraining several guide cannulae, which can then be accurately implanted with a single positioning of the stereotaxic arm. After an initial modest outlay of funds for a number of machined metal parts, the headpieces and the remainder of the cannula system can be rapidly and cheaply produced from common hospital supplies and available metal and plastic tubing. The system is useful in the implantation of several cannulae only when all cannulae are to have the same angle with the vertical. CONSTRUCTION The finished cannula system (see Fig. l) consists of (1) a cylindrical headpiece of molded dental acrylic cement 9 mm in diameter and 7 mm in height; (2) 26 gauge guide cannulae embedded in the headpiece parallel to the cylindrical axis and extending above and below it; (3) 32 gauge stylets for the guides; (4) 32 gauge internal cannulae attached to polyethylene tubing; and (5) a plastic cap which screws onto the
1Send reprint requests to G. G. Dougherty, Jr., M.D., Department of Psychiatry, Duke University Medical Center, Box 3870, Durham, NC 27710.
897
898
DOUGHERTY AND E L L I N W O O D
FIG. 1. A base block is pictured on the stereotaxic zeroing stand (left); the headpiece has been grasped by the stereotaxic arm in proper orientation. The mold for headpiece construction is shown on the right.
tubing crimped on one end so that they extended 0.5 mm beyond the end of the guide cannulae when placed in them. The internal cannulae were also made from 32 gauge stainless steel tubing, A 3 mm piece of 26 gauge tubing was fastened about the smaller tubing with epoxy glue at a position carefully measured so that when this rim was flush with the top of the guide the internal cannula tip extended 2 mm beyond the guide tip. Polyethylene tubing (PE-20, Becton, Dickinson, and Co.) was fitted over the 26 gauge rim and fastened with epoxy glue to achieve a water-tight junction. The plastic caps for the headpieces were made from pieces of 3 cc plastic syringes (Monoject, Sherwood Medical Products) tapped on the inside with 3/8 in. fine-pitched thread. IMPLANTATION
Once the headpiece was made, stereotaxic measurements could be done to position the guide cannula intended for the deepest brain site; all the other cannulae would then be correctly placed---provided the headpiece was oriented properly. To ensure the proper orientation of the headpiece in the stereotaxic apparatus, we built a stereotaxic arm consisting of a spring-powered clip designed to grasp the headpiece fh-mly, and a small ball and socket device with the ball attached to the
headpiece clip. The ball could be easily rotated about any axis and then fixed fuanly in a given orientation. To achieve the correct orientation of the finished headpiece in the clip, we used a stereotaxic zeroing stand. The base block was positioned parallel to the stereotaxic coordinate directions. With the headpiece replaced in the holes of the base block, the stereotaxic arm was lowered to grasp the headpiece, and that orientation was preserved by tightening of the ball and socket. This last device was a luxury as a simple clip on a stereotaxic arm has also been satisfactory in holding the headpiece. Locations for skull holes were marked individually for drilling by standard micromanipulator measurements; the oriented headpiece was aligned over these holes by making similar measurements for the longest cannula in the headpiece. However, clips grasping the headpiece from the side permit simultaneous marking of skull hole sites by 32 gauge tubing passed through the guides of a headpiece positioned over the skull, and we are altering our ball and socket clip accordingly; holes will still be drilled individually. The remainder of the implantation procedure involved standard use of skull screws and acrylic cement to VLXthe headpiece to the skull. Examination of histologic sections of operated rat brains has confirmed that the above system can produce accurate
MULTIPLE-CANNULA HEADPIECE
899
FIG. 2. An operated rat is injected via two of four intracerebral cannula guides, after removal of protective cap.
placements of multiple cannulae. For rats housed individually, the headpieces have remained in place for several months when careful technique was used to fix the headpiece to the skull. INJECTION The animal was injected by removal of cap and stylets and insertion of the internal cannulae. If the animal was held during injection, the friction between the internal and guide cannulae was nearly always sufficient to hold the internal cannulae in place, even during large amounts of head movement (Fig. 2). Chronic infusions in behaving animals have required that the internal cannulae be more permanently fixed in place in the guides. For each headpiece a single plug containing the multiple internal cannulae was made. Dental acrylic cement was poured into a mold composed of an open-topped cap screwed onto the headpiece, with internal cannulae in place. The cap had been cut lengthwise and rejoined with tape for easy removal, and all inner surfaces coated with stopcock grease. Polyethylene tubing from internal cannulae was coated with epoxy glue, as acrylic solvent dissolves the former. The plug then fit snugly under a headpiece cap with a hole in the top for emerging tubing. Most rats required brief ether anesthesia for the insertion of plugs with several can-
,.,;/,.,/,;,.,.~,/'~~ "
_ .....
=~.,.~.
FIG. 3. Cross-sectional view of the headpiece adapted for use with the osmotic minipump.
900
D O U G H E R T Y AND ELLINWOOI3
nulae. However, it should be noted that the twisting of two or more tubes from conventional infusion pumps by the animal's turning can severely limit the period of behavioral observation. USE WITH OSMOTIC MINIPUMP The control over headpiece construction available to the experimenter with this system allowed for easy adaptation of the headpiece to alterations in infusion technique. F o r example, one or more Alzet osmotic minipumps [1] implanted subcutaneously in the rat's interscapular region could be used as pumping sources for chronic intracerebral infusion. We simply incorporated one or more extra connect-
ing tubes into the headpiece during its construction (Fig. 3J. These connecting tubes allowed the infusion solution to flow from the minipump up through the headpiece, around a loop of tubing under the headpiece cap, back down through the headpiece and into the brain via the internal cannula, In this case, the flow rate of the pump was measured by introducing a small bubble into the loop above the headpiece and monitoring its movement. The adaptability that this headpiece construction method offers the investigator may also prove useful for other interventions involving multiple implantations such as cannula injections combined with electrode recording or stimulation, and concurrent infusions of more than one brain site to study the dynamic interaction of different regions of the brain.
REFERENCES 1. Alzet Osmotic Minipump Technical Bulletin 2(3): 1978. 2. Brakkee, J. H., V. M. Wiegant and W. H. Gispen. A simple technique for rapid implantation of a permanent cannula into the rat brain ventrieular system. Lab. AnOn. Sci. 29: 78--81, 1979. 3. Harvey, E. A. and R. J. Stephens. Construction and implantation of a cannula system for repeated injections into localized regions of the rat brain. J. Pharm. Pharmac. 27: 43--45, 1975. 4. Kokkinidis, L., L. Raffler and H. Anisman. Simple and compact cannula system for mice. Pharmac. Biochem. Behav. 6: 595-597, 1977.
5. Lavenhar, S. R. and A. L. Palanker. Cannula system for local stimulation of the rat brain. Pharmac. Biochem. Behav. 4: 351352, 1976. 6. Myers, R. D. and F. Mora. In vivo neurochemical analysis~ by push-pull perfusion, of the mesocortical dopaminergic system of the rat during self-stimulation. Brain Res. Bull. 2: 105-I 121 1977. 7. Myers, R. D. Handbook o f Drug and Chemical Stimulation o f the Brain. New York: Van Nostrand Reinhold, 1974, pp. 37-39.
A Simple Multiple-Cannula Headpiece for the Rat G E O R G E G. D O U G H E R T Y , JR. 1 A N D E V E R E T r H. E L L I N W O O D , JR.
Behavioral Neuropharmacology Laboratory, Department o f Psychiatry Duke University Medical Center, Durham, N C 27710 Received 1 D e c e m b e r 1980 DOUGHERTY, G. G., JR. AND E. H. ELLINWOOD, JR. A simple multiple-cannula headpiecefor the rat. PHYSIOL. BEHAV. 26(5) 897-900, 1981.--The construction of a muRiple-cannula headpiece, its implantation into the rat brain, and its use in intracranial injections is described. The headpiece is rapidly and cheaply produced from common hospital and laboratory supplies once several machined metal parts have been made, and its construction is easily adapted to changes in injection technique, e.g., infusion by osmotic minipump. Cannula
Stereotaxictechnique
Rat
Osmoticminipump
headpiece, covering the tops of the guide cannulae and stylets between injections. The machined metal parts used to mass-produce these headpieces are described below. The premise used in the design of this headpiece was that the precise relative positioning of several guide cannulae within the headpiece would allow their simultaneous implantation into intended sites in the rat brain. To this end, holes were drilled in the surface of a small block of aluminum (the "base block") to have the same relative antero-posterior (AP) and lateral (L) stereotaxic positions as would the guide cannulae intended for implantation. Great accuracy was used in positioning these holes. The hole diameter was chosen to allow a snug fit with inserted 26 gauge tubing. The relative hole depths were determined from the relative stereotaxic depth coordinates for the cannulae. The cylindrical headpiece was made by placing guide cannulae into the holes of the base block after inner surfaces were coated with stopcock grease. Freshly mixed dental acrylic cement was then poured into a mold placed about the protruding guides on the base block. The mold was made from two oblong pieces of aluminum, each with a half a cylinder cut from one side. The walls of the mold were tapped with threads so that the molded piece produced would have threads on its outer surface. Fine-pitch 3/8 in. threading was used. The base block had been constructed with a 2 mm ridge on one side, parallel to the AP axis. When one edge of the mold was flush with this ridge, the cylindrical opening was centered over the drilled holes. Twenty-six gauge, 1/2 in. long hypodermic needles were used for the guide cannulae. The needles were denuded of their plastic adapters, leaving a thin layer of ceramic about the shaft, before they were placed point-down into the holes in the base block. The stylets were constructed from 32 gauge stainless steel tubing (Small Parts, Inc.), with a short "handle" of larger
THE STIMULATION of specific brain sites by administration of small amounts of drugs or neurotransmitters has come to be regarded as a useful technique in the study of brain and behavior [7]. Our work has often involved the assessment of brain changes following chronic exposure to a drug, by means of studying the response to subsequent administration of "pharmacologic probes" into brain nuclei. A practical advantage is gained if multiple brain sites can be tested in a single chronically-treated animal. However, the stereotaxic implantation of multiple cannulae for such a study can be very time-consuming, especially when a large number of subjects is required. The technical awkwardness of the successive positioning and cementing of cannulae commercially available or described by others [2, 3, 4, 5] is also an undesirable feature of the procedure. We have confronted these difficulties by constructing a headpiece conraining several guide cannulae, which can then be accurately implanted with a single positioning of the stereotaxic arm. After an initial modest outlay of funds for a number of machined metal parts, the headpieces and the remainder of the cannula system can be rapidly and cheaply produced from common hospital supplies and available metal and plastic tubing. The system is useful in the implantation of several cannulae only when all cannulae are to have the same angle with the vertical. CONSTRUCTION The finished cannula system (see Fig. l) consists of (1) a cylindrical headpiece of molded dental acrylic cement 9 mm in diameter and 7 mm in height; (2) 26 gauge guide cannulae embedded in the headpiece parallel to the cylindrical axis and extending above and below it; (3) 32 gauge stylets for the guides; (4) 32 gauge internal cannulae attached to polyethylene tubing; and (5) a plastic cap which screws onto the
1Send reprint requests to G. G. Dougherty, Jr., M.D., Department of Psychiatry, Duke University Medical Center, Box 3870, Durham, NC 27710.
897
898
DOUGHERTY AND E L L I N W O O D
FIG. 1. A base block is pictured on the stereotaxic zeroing stand (left); the headpiece has been grasped by the stereotaxic arm in proper orientation. The mold for headpiece construction is shown on the right.
tubing crimped on one end so that they extended 0.5 mm beyond the end of the guide cannulae when placed in them. The internal cannulae were also made from 32 gauge stainless steel tubing, A 3 mm piece of 26 gauge tubing was fastened about the smaller tubing with epoxy glue at a position carefully measured so that when this rim was flush with the top of the guide the internal cannula tip extended 2 mm beyond the guide tip. Polyethylene tubing (PE-20, Becton, Dickinson, and Co.) was fitted over the 26 gauge rim and fastened with epoxy glue to achieve a water-tight junction. The plastic caps for the headpieces were made from pieces of 3 cc plastic syringes (Monoject, Sherwood Medical Products) tapped on the inside with 3/8 in. fine-pitched thread. IMPLANTATION
Once the headpiece was made, stereotaxic measurements could be done to position the guide cannula intended for the deepest brain site; all the other cannulae would then be correctly placed---provided the headpiece was oriented properly. To ensure the proper orientation of the headpiece in the stereotaxic apparatus, we built a stereotaxic arm consisting of a spring-powered clip designed to grasp the headpiece fh-mly, and a small ball and socket device with the ball attached to the
headpiece clip. The ball could be easily rotated about any axis and then fixed fuanly in a given orientation. To achieve the correct orientation of the finished headpiece in the clip, we used a stereotaxic zeroing stand. The base block was positioned parallel to the stereotaxic coordinate directions. With the headpiece replaced in the holes of the base block, the stereotaxic arm was lowered to grasp the headpiece, and that orientation was preserved by tightening of the ball and socket. This last device was a luxury as a simple clip on a stereotaxic arm has also been satisfactory in holding the headpiece. Locations for skull holes were marked individually for drilling by standard micromanipulator measurements; the oriented headpiece was aligned over these holes by making similar measurements for the longest cannula in the headpiece. However, clips grasping the headpiece from the side permit simultaneous marking of skull hole sites by 32 gauge tubing passed through the guides of a headpiece positioned over the skull, and we are altering our ball and socket clip accordingly; holes will still be drilled individually. The remainder of the implantation procedure involved standard use of skull screws and acrylic cement to VLXthe headpiece to the skull. Examination of histologic sections of operated rat brains has confirmed that the above system can produce accurate
MULTIPLE-CANNULA HEADPIECE
899
FIG. 2. An operated rat is injected via two of four intracerebral cannula guides, after removal of protective cap.
placements of multiple cannulae. For rats housed individually, the headpieces have remained in place for several months when careful technique was used to fix the headpiece to the skull. INJECTION The animal was injected by removal of cap and stylets and insertion of the internal cannulae. If the animal was held during injection, the friction between the internal and guide cannulae was nearly always sufficient to hold the internal cannulae in place, even during large amounts of head movement (Fig. 2). Chronic infusions in behaving animals have required that the internal cannulae be more permanently fixed in place in the guides. For each headpiece a single plug containing the multiple internal cannulae was made. Dental acrylic cement was poured into a mold composed of an open-topped cap screwed onto the headpiece, with internal cannulae in place. The cap had been cut lengthwise and rejoined with tape for easy removal, and all inner surfaces coated with stopcock grease. Polyethylene tubing from internal cannulae was coated with epoxy glue, as acrylic solvent dissolves the former. The plug then fit snugly under a headpiece cap with a hole in the top for emerging tubing. Most rats required brief ether anesthesia for the insertion of plugs with several can-
,.,;/,.,/,;,.,.~,/'~~ "
_ .....
=~.,.~.
FIG. 3. Cross-sectional view of the headpiece adapted for use with the osmotic minipump.
900
D O U G H E R T Y AND ELLINWOOI3
nulae. However, it should be noted that the twisting of two or more tubes from conventional infusion pumps by the animal's turning can severely limit the period of behavioral observation. USE WITH OSMOTIC MINIPUMP The control over headpiece construction available to the experimenter with this system allowed for easy adaptation of the headpiece to alterations in infusion technique. F o r example, one or more Alzet osmotic minipumps [1] implanted subcutaneously in the rat's interscapular region could be used as pumping sources for chronic intracerebral infusion. We simply incorporated one or more extra connect-
ing tubes into the headpiece during its construction (Fig. 3J. These connecting tubes allowed the infusion solution to flow from the minipump up through the headpiece, around a loop of tubing under the headpiece cap, back down through the headpiece and into the brain via the internal cannula, In this case, the flow rate of the pump was measured by introducing a small bubble into the loop above the headpiece and monitoring its movement. The adaptability that this headpiece construction method offers the investigator may also prove useful for other interventions involving multiple implantations such as cannula injections combined with electrode recording or stimulation, and concurrent infusions of more than one brain site to study the dynamic interaction of different regions of the brain.
REFERENCES 1. Alzet Osmotic Minipump Technical Bulletin 2(3): 1978. 2. Brakkee, J. H., V. M. Wiegant and W. H. Gispen. A simple technique for rapid implantation of a permanent cannula into the rat brain ventrieular system. Lab. AnOn. Sci. 29: 78--81, 1979. 3. Harvey, E. A. and R. J. Stephens. Construction and implantation of a cannula system for repeated injections into localized regions of the rat brain. J. Pharm. Pharmac. 27: 43--45, 1975. 4. Kokkinidis, L., L. Raffler and H. Anisman. Simple and compact cannula system for mice. Pharmac. Biochem. Behav. 6: 595-597, 1977.
5. Lavenhar, S. R. and A. L. Palanker. Cannula system for local stimulation of the rat brain. Pharmac. Biochem. Behav. 4: 351352, 1976. 6. Myers, R. D. and F. Mora. In vivo neurochemical analysis~ by push-pull perfusion, of the mesocortical dopaminergic system of the rat during self-stimulation. Brain Res. Bull. 2: 105-I 121 1977. 7. Myers, R. D. Handbook o f Drug and Chemical Stimulation o f the Brain. New York: Van Nostrand Reinhold, 1974, pp. 37-39.