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Application of a new intraventricular injection technique in rat brain norepinephrine studies
Life Sciences Dol . 18, pp . 197-204 Printed in the U.S .A .
Pergamon Press
APPLICATION OF A NEW INTRAD~ITRICOIAR INJECTIOIQ TßCHNIQUE IN RAT BRAIN NORßPINSPHRINß STUDIßS Pred R. Popick Research Department, Pharmaceuticals Division CIBA-GSIGY CORPORATION, Summt, Nev .Teraey 07901 (Received is final form December 22, 1975)
Summ A sew technique has been devised for iajectiag substances directly into the right lateral ventricle of the rat . Injections are made with the aid of s stainless steel bead designed to conform to the rat's skull and equipped with a rigid sleeve . When properly aligned with the caudal edge of the optic orbit, the sleeve is positioned over the right lateral ventricle. The sleeve prevents beading of the injection needle and controls its penetration to the desired depth. Histological ezamination and H3-norepinephrine disposition studies have damcnstrated the validity and reproducibility of the method. Ia contrant to previous methods, as anesthetic is not required and surgical trauma is avoided. Furthermore, the efficiency of the new technique is such that one rat can be injected per minute . Many recent studlae of the affects of drugs oa the physiological disposition of intraventricu].arly injected H3-norepinephriae employed the injection method described by Noble (1), or som modification of that method . Anew bead injection method has been devised which has three distinct advantages : the rats are not anesthetized ; they are sot subjected to surgical trauma ; the efficiency has been improved to the eztant that one rat can be injected per minute . Three NFrmetaboli~m studies and hiatological az~{~tione ware used in assessing the efficacy of thn injection method, sad in all cases the results indicate that the norepiaephriae brain stores ware effectively labeled . Materials and Methods sana Design A stainless steel band, 56 mm long and 12 mm wide, wan shaped to conform to the skull of a 200 gram rat (Fig . 1) . A hole was drilled 2 s~ to the right of the center and 7 .5 mm back from the forward edge to accommodate a 21-gauge (i .d .) stainless steel tube 9 .5 mm in length . The tube was soldered Abbreviations NB ~ Norepiaephriae NM ~ Normetanephrine DMA ~ Daaillylnaadelic acid MAO ~ Monoamina azidase
DHMA DHPG MHPG COMT
~ ~ ~ ~
3,4 Dihydrozymaadelic acid 3,4 Dihydroxyphenylglycol 3-Methozy-4-I~ydrozy-phealylglycol Catechol-O-Methyltraasferase
197
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Intraventricular Injection Technique
Vol. 18, No . 2
into place perpendicularly to the surface and eztendiag 1 mm belw and 8 >® above the band so that it could serve as a rigid sleeve .
FIQ 1 Injection _Technique Male Sprague-Davley rats vniQh no 190 - 210 grams vets used . The rat vas held so that the ventral surface of the head vas pressed against a flat surface . The skin covering the skull vas made taut and the bead vas than slipped back onto the skull tortil the forward edge vas in line tirith the caudal edge of the optic orbit ; the sleeve vas then over the right half of the brain (Fig, 2) .
FIQ 2 Once ahP d the band t+ras held in place with firm pressure to prevent its roving and to insure that the sleeve vas in contact with the skull. A 100 a Hamilton syringe equipped with a 27-gauge, 1/2-inch stainless steel needle vas inserted into the eleeva . A 26-gauge, 1/2-inch stainless steel needle easy also be used . Slight but firm pressure vas applied to enable the needle to penetrate the skull and descend 3.2 mm into the lateral ventricle. The needle vas able to penetrate
Vol . 18, No . 2
Iatravantricular Injection Technique
199
the bone without bending, as a result of the added strength provided by the sleeve . When the foraiard edge of the band was is proper alignment, the needle penetrated the skull 1.5 - 2.0 mm lateral to the crossing of the eagittal and coronal sutures (Fig . 3) . 11!!DL! ~llTRAT10N
Fre~w
Caarl
FIQ S After injection of 10 a of NE-H 3, the needle was allowed to remain in place for about 10 seconds and then withdrawn ; the solution remaining is the needle was After sash injeotion a resw needle was requtiredl pulled back into the syringe . Preparation of NE-H 3 1-[7-H3 ] Noradreaalina (Amersham/Seeds), specific activity of 8.8 Ci /mmole and a radioactive concentration of 0 .9 aCi/ml, was evaporated to one-third original volume under reduced pressure . Thie yielded a radioactive concentration of 2.7 mCi/al . Tissue PreQaratiaa At various times after iatraveatricular injection rata were sacrificed by decapitation . Brains were removed, rinsed with distilled water, blotted dry and weighed. Each was placed is oas-half volume (0 .5 ml/g) of cold 0 .3 N perchloric acid solution, containing pargyline (1 mM), pyrogallol (1 mM), and disodium ethylea~ai~+++etetraacetate (1 mt~ . The braise were sonicated separately for 30 seconds with a Heat Systems Model 350 W saaifier (60x duty cycle, output dial at 6) . Samples were then centrifuged for ]5 minutes in a Mistral 4L centrifuge net at 5 °C sad s speed of 3,000 B.P .M. Chromatography Labeled NE sad its metabolites were separated as Whataaa P-81 cellulose phosphate paper by ascending chromatography . The solvent system was 0 .2 M ammonium acetate-iaopropaaol (2 :1) used previously by itovacsics and Saelens (2) . The carrier solution consisted of 0.3 N perchloric acid containinô 1 .5 mß/ml N8 bitartrata, 2 m8/nl NM HC1, 1 mg/ml D~lA, 1.5 mg/ml VMA, 1.5 mg/nl DHPG sad 2 .0 n8/al MHPG . Ten J1 of the carrier and thirty a,of .the brain supernatant were spotted sad chromatographed for 18 hours. The compounds were then visualized with I2 vapor . Each sample strip was s~~++++pd on the Actigraph III, Nuclear Chicago at 1 R c/a, 60 cm/hr, 10 second sasq:ling period, 1 minute printout, with
200
Intraventricular Injection Technique
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a slit width of 6 mm . This chramatograpt~y method leads to as avarlap of the VMA and the MBPG spots . This can give rise to as overestimate of the VMA and an underestimate of the MHPG . However, the eca~er separates the two peaks and by dividing the overlap is proportion to the respective peaks, a reasonable estimate of each metabolite can be obtained . Statistics Brain levels of NS-H3 and its metabolites were calculated as percent of the total CPM recovered from the strip . Mean and standard error were computed for each metabolite . The statistical significance between drug and control was determined by Student's t-test when variances were homogeneous and by Behren's t-test (3) when variances were heterogeneous . Results Split-Brain Studies To determine whether the bead injection method was actually delivering the NFrH 3 into the lateral ventricle, the left half of the brain was assayed for total NSrH3 . Counts is the left half demonstrate that the NS-H 3 has circulated throughout the ventricular system . Four rate were injected with 10 a of N&-H3 into the right lateral ventricle according to the procedure above . 7hey were sacri~icad two hours later, and the brains ware divided by a mid-sagittal cut . The mean CPM for the left half was 30,719 ± 736 . According to Noble (1), if the injection had not delivered the NS-H3 into the lateral ventricle, the counts would have remained localised at the site of injection without circulating into the left half of the brain . Histologic Studies Ten rats ware given a 5 a injection of a 1X solution of bramcresol green by the band injection technique . Lateral eectious of the brains were taken and in each case a direct needle tract could be seen entering the right lateral ventricle . The entire ventricular system was stained a dark blue, indicating that the injection did indeed deliver the dye into the lateral ventricle . NFrH3 Metabolism :
Time Curve
A 10 ul injection of NB-H3 was made with the aid of the band into the lateral ventricle of twenty-four rats . The rats were sacrificed at various tine intervals (5, 10, 15, 60, 120, 240 minutes) and curves for each metabolite ware plotted (Fig . ~+) . During the initial phase NB disappeared rapidly from the brain and platesued by 15 minutes . NM peaked at 10 mimites and platesued at 60 minutes . DHlfA and DHPG plateaued at 15 giautas and regained approzimately constant for 4 hours . VMA rose rapidly, peaked at 60 minutes, then rapidly declined sad plateaued between 120 and 240 minutes . MHPG showed a slight rise to about 3X of total counts in the first 15 minutes, rose to about 13i of total counts between 60 and 120 minutes, and then platesued . The total CPM showed a decline of 76X fran as initial peak of 3,439 CPM . Effect of Pyrogallol on N&-H?Metabolism Pyrogallol, a COMT inhibitor, was edwiniA tared i .p . ..at a dose of 200 mg/kg 30 minutas prior to the ïntraveatriculsr injection of NSrH3 by the band injection technique . One group (N ~ 4) was sacrificed at 5 minutes and the other at 60 minutes post NSrH3 . The results are shown is Fig . 5 . At 5 minutes the
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Iatraventricular Ic~jection Technique
201
i p~A " ~IlC . . ~ .
T" 1! ~I~IITY FIG. 4 RATE OF DISAPPEARANCE OF INTRAVENTRICULARLY INJECTED H3-NOREPINEPHRINE AND THE FORMATION OF SOME OF ITS METABOLITES.
PYROGALLOL COMT INHIBITOR
i
6 MIN . CONTROL
!i OF TOTAL COUNTS
PYROGALLOL ' 30 20 10
NE
NM
DHMA
VMA MHPG
NE F1ß . 6
EFFECTS OF PYROGALLOL ON THE METABOLITE PATTERN RATS WERE INJECTED LP. 30 MINUTES PRE NE-H2 INJECTION. DOSE WAS 200 nN./ko . ONE GROUP SACRIFH~D AT 6 MINUTES POST NE-li3 AND THE OTHER AT BO MINUTES POST NE-H3.
NM
DHMA VMA MHPß
202
Intraveatricular Injection Technique
Vol . 18, No . 2
effect on the metabolite pattern (changes in X of total counts) was as follows : NM, a decrease of 10 .5X (p < 0 .01) ; DHIlA aad DHPG, an increase of 11 .1X (p < 0 .01) ; VMA, a decrease of 7 .6X (p < 0 .01) . At 60 minutes the effect was as follows : NM, a decrease of 4 .3X (p < 0 .03) ; DHMA and DHPG, ea increase of 28 .6X (p < 0 .01) ; VMA, a decrease of 19 .3X (p < 0 .01) . Sffeçt of Parg~line oa NFrS3 Metabolism Pargyline, an MAO inhibitor, was administered i .p . at a dose of 100 mg/,kg 3O minutes prior to the intraventricular injection of the N&-H3 . One group (N - 4) was sacrificed at 10 minutas aad the other at 120 minutes post NE-H3 . The results are shown in Fig . 6 . At 10 minutes the effect an the metabolite pattern (changes in X of total counts) was as follows : NS, an increase of 11 .1X (p < 0 .02) ; NM, an increase of 15 .4X (p < 0 .01) ; DHMA and DHPG, a decrease of 5 .7X (p < 0 .01) ; VMA, a decrease of 20 .OX (p < 0 .01) . At 120 minutes effect was as follows : NM, an increase of 53 .OX (p < 0 .01) ; D1~fA sad DHPG, a decrease of 1 .1X (p < 0 .01) ; VMA, a decrease of 23 .7X (p < 0 .01) ; MSPG, a decrease of 11 .OX (p < 0 .01) .
Flß. 6 EFFECTf OF PAROYLINE ON THE METABOLITE PATTERN RATt MIERE IN.IECTED LP. 80 MINUTES PRE NE"Ila INJECTION. DO:E WAi 100 nK./k} ONE ßROUP iACRIFICED AT 10 MINUTE! PORT NE-H9 AND THE OTHER AT 1Z0 MINUTEÔ POiT NE-H8.
Discussion The results demonstrate that the band injection technique ie .very satisfactory . Ia the split-brain studies the counts circulated throughout the ventricular system and did not remain localized at the injection site . The histological studies ,again showed the circulation of the dye throughout the ventricular system. Finally, in testing the effect of a CO!!T inhibitor and an MAO inhibitor the expected results were obtained . All previous intraveatricular injection techniques for acute studies required as anesthetic, usually diethyl ether . Anesthetics are not without
Vol . 18, No . 2
Intraveatricular Iajection Technique
203
effect on neuronal transmission, electrical activity, and biogenic amines . The band Iajection technique enables the study of the metabolism sad function of intravemtricularly injected substances is the rat brain without the limitations imposed by the use of an anesthetic . A surgical procedure was also previously required which 1s a stress factor . The only stress to which the rat is subjected when using the bead is the brief restraint required during the injection procedure. No adverse reactions were observed Ia any of the animals following the injection procedure . The band Iajection technique is not limited to injections into the lateral ventricle . Modifications of the bead design would enable investigators to reach other areas of the brain, either for injection purposes or lesloaing . Nobel (1) was able to inject 15 - 20 rats per hour, using ether and a surgical procedure; with the bead it 3s feasible to inject one rat per minute or aizty rats per hour . Acknowledgment The author would like to thank Mr . Robert Poempner for ezecuting the bead design and tir. Bernard Miller for his illustrations, sad wishes to e~rese hie appreciation to Dr . William D . Cash, Ms . Patricia Loo, Dr . Aaron Feldstein and Dr . Gertrude P . gaina for their most helpful guidance . References 1.
E. P . NOBLE, R. J . WITRTMAN and J. ABELROD, Life Sciences , 6, 281-291 (1967) .
2.
G. B. KOVACSICS aad J . ~. SAELEDiS, Arçh . Iat . Pharmacodyn., 174, 481 (1968) .
3.
R. STEEL and J . TORBIE, Principles and Procedures of Statistics , (p . 81) Magraw Hill, N. Y. (1960) .
Pergamon Press
APPLICATION OF A NEW INTRAD~ITRICOIAR INJECTIOIQ TßCHNIQUE IN RAT BRAIN NORßPINSPHRINß STUDIßS Pred R. Popick Research Department, Pharmaceuticals Division CIBA-GSIGY CORPORATION, Summt, Nev .Teraey 07901 (Received is final form December 22, 1975)
Summ A sew technique has been devised for iajectiag substances directly into the right lateral ventricle of the rat . Injections are made with the aid of s stainless steel bead designed to conform to the rat's skull and equipped with a rigid sleeve . When properly aligned with the caudal edge of the optic orbit, the sleeve is positioned over the right lateral ventricle. The sleeve prevents beading of the injection needle and controls its penetration to the desired depth. Histological ezamination and H3-norepinephrine disposition studies have damcnstrated the validity and reproducibility of the method. Ia contrant to previous methods, as anesthetic is not required and surgical trauma is avoided. Furthermore, the efficiency of the new technique is such that one rat can be injected per minute . Many recent studlae of the affects of drugs oa the physiological disposition of intraventricu].arly injected H3-norepinephriae employed the injection method described by Noble (1), or som modification of that method . Anew bead injection method has been devised which has three distinct advantages : the rats are not anesthetized ; they are sot subjected to surgical trauma ; the efficiency has been improved to the eztant that one rat can be injected per minute . Three NFrmetaboli~m studies and hiatological az~{~tione ware used in assessing the efficacy of thn injection method, sad in all cases the results indicate that the norepiaephriae brain stores ware effectively labeled . Materials and Methods sana Design A stainless steel band, 56 mm long and 12 mm wide, wan shaped to conform to the skull of a 200 gram rat (Fig . 1) . A hole was drilled 2 s~ to the right of the center and 7 .5 mm back from the forward edge to accommodate a 21-gauge (i .d .) stainless steel tube 9 .5 mm in length . The tube was soldered Abbreviations NB ~ Norepiaephriae NM ~ Normetanephrine DMA ~ Daaillylnaadelic acid MAO ~ Monoamina azidase
DHMA DHPG MHPG COMT
~ ~ ~ ~
3,4 Dihydrozymaadelic acid 3,4 Dihydroxyphenylglycol 3-Methozy-4-I~ydrozy-phealylglycol Catechol-O-Methyltraasferase
197
lgg
Intraventricular Injection Technique
Vol. 18, No . 2
into place perpendicularly to the surface and eztendiag 1 mm belw and 8 >® above the band so that it could serve as a rigid sleeve .
FIQ 1 Injection _Technique Male Sprague-Davley rats vniQh no 190 - 210 grams vets used . The rat vas held so that the ventral surface of the head vas pressed against a flat surface . The skin covering the skull vas made taut and the bead vas than slipped back onto the skull tortil the forward edge vas in line tirith the caudal edge of the optic orbit ; the sleeve vas then over the right half of the brain (Fig, 2) .
FIQ 2 Once ahP d the band t+ras held in place with firm pressure to prevent its roving and to insure that the sleeve vas in contact with the skull. A 100 a Hamilton syringe equipped with a 27-gauge, 1/2-inch stainless steel needle vas inserted into the eleeva . A 26-gauge, 1/2-inch stainless steel needle easy also be used . Slight but firm pressure vas applied to enable the needle to penetrate the skull and descend 3.2 mm into the lateral ventricle. The needle vas able to penetrate
Vol . 18, No . 2
Iatravantricular Injection Technique
199
the bone without bending, as a result of the added strength provided by the sleeve . When the foraiard edge of the band was is proper alignment, the needle penetrated the skull 1.5 - 2.0 mm lateral to the crossing of the eagittal and coronal sutures (Fig . 3) . 11!!DL! ~llTRAT10N
Fre~w
Caarl
FIQ S After injection of 10 a of NE-H 3, the needle was allowed to remain in place for about 10 seconds and then withdrawn ; the solution remaining is the needle was After sash injeotion a resw needle was requtiredl pulled back into the syringe . Preparation of NE-H 3 1-[7-H3 ] Noradreaalina (Amersham/Seeds), specific activity of 8.8 Ci /mmole and a radioactive concentration of 0 .9 aCi/ml, was evaporated to one-third original volume under reduced pressure . Thie yielded a radioactive concentration of 2.7 mCi/al . Tissue PreQaratiaa At various times after iatraveatricular injection rata were sacrificed by decapitation . Brains were removed, rinsed with distilled water, blotted dry and weighed. Each was placed is oas-half volume (0 .5 ml/g) of cold 0 .3 N perchloric acid solution, containing pargyline (1 mM), pyrogallol (1 mM), and disodium ethylea~ai~+++etetraacetate (1 mt~ . The braise were sonicated separately for 30 seconds with a Heat Systems Model 350 W saaifier (60x duty cycle, output dial at 6) . Samples were then centrifuged for ]5 minutes in a Mistral 4L centrifuge net at 5 °C sad s speed of 3,000 B.P .M. Chromatography Labeled NE sad its metabolites were separated as Whataaa P-81 cellulose phosphate paper by ascending chromatography . The solvent system was 0 .2 M ammonium acetate-iaopropaaol (2 :1) used previously by itovacsics and Saelens (2) . The carrier solution consisted of 0.3 N perchloric acid containinô 1 .5 mß/ml N8 bitartrata, 2 m8/nl NM HC1, 1 mg/ml D~lA, 1.5 mg/ml VMA, 1.5 mg/nl DHPG sad 2 .0 n8/al MHPG . Ten J1 of the carrier and thirty a,of .the brain supernatant were spotted sad chromatographed for 18 hours. The compounds were then visualized with I2 vapor . Each sample strip was s~~++++pd on the Actigraph III, Nuclear Chicago at 1 R c/a, 60 cm/hr, 10 second sasq:ling period, 1 minute printout, with
200
Intraventricular Injection Technique
Vol . 18, No . 2
a slit width of 6 mm . This chramatograpt~y method leads to as avarlap of the VMA and the MBPG spots . This can give rise to as overestimate of the VMA and an underestimate of the MHPG . However, the eca~er separates the two peaks and by dividing the overlap is proportion to the respective peaks, a reasonable estimate of each metabolite can be obtained . Statistics Brain levels of NS-H3 and its metabolites were calculated as percent of the total CPM recovered from the strip . Mean and standard error were computed for each metabolite . The statistical significance between drug and control was determined by Student's t-test when variances were homogeneous and by Behren's t-test (3) when variances were heterogeneous . Results Split-Brain Studies To determine whether the bead injection method was actually delivering the NFrH 3 into the lateral ventricle, the left half of the brain was assayed for total NSrH3 . Counts is the left half demonstrate that the NS-H 3 has circulated throughout the ventricular system . Four rate were injected with 10 a of N&-H3 into the right lateral ventricle according to the procedure above . 7hey were sacri~icad two hours later, and the brains ware divided by a mid-sagittal cut . The mean CPM for the left half was 30,719 ± 736 . According to Noble (1), if the injection had not delivered the NS-H3 into the lateral ventricle, the counts would have remained localised at the site of injection without circulating into the left half of the brain . Histologic Studies Ten rats ware given a 5 a injection of a 1X solution of bramcresol green by the band injection technique . Lateral eectious of the brains were taken and in each case a direct needle tract could be seen entering the right lateral ventricle . The entire ventricular system was stained a dark blue, indicating that the injection did indeed deliver the dye into the lateral ventricle . NFrH3 Metabolism :
Time Curve
A 10 ul injection of NB-H3 was made with the aid of the band into the lateral ventricle of twenty-four rats . The rats were sacrificed at various tine intervals (5, 10, 15, 60, 120, 240 minutes) and curves for each metabolite ware plotted (Fig . ~+) . During the initial phase NB disappeared rapidly from the brain and platesued by 15 minutes . NM peaked at 10 mimites and platesued at 60 minutes . DHlfA and DHPG plateaued at 15 giautas and regained approzimately constant for 4 hours . VMA rose rapidly, peaked at 60 minutes, then rapidly declined sad plateaued between 120 and 240 minutes . MHPG showed a slight rise to about 3X of total counts in the first 15 minutes, rose to about 13i of total counts between 60 and 120 minutes, and then platesued . The total CPM showed a decline of 76X fran as initial peak of 3,439 CPM . Effect of Pyrogallol on N&-H?Metabolism Pyrogallol, a COMT inhibitor, was edwiniA tared i .p . ..at a dose of 200 mg/kg 30 minutas prior to the ïntraveatriculsr injection of NSrH3 by the band injection technique . One group (N ~ 4) was sacrificed at 5 minutes and the other at 60 minutes post NSrH3 . The results are shown is Fig . 5 . At 5 minutes the
Vol . 18, No . 2
Iatraventricular Ic~jection Technique
201
i p~A " ~IlC . . ~ .
T" 1! ~I~IITY FIG. 4 RATE OF DISAPPEARANCE OF INTRAVENTRICULARLY INJECTED H3-NOREPINEPHRINE AND THE FORMATION OF SOME OF ITS METABOLITES.
PYROGALLOL COMT INHIBITOR
i
6 MIN . CONTROL
!i OF TOTAL COUNTS
PYROGALLOL ' 30 20 10
NE
NM
DHMA
VMA MHPG
NE F1ß . 6
EFFECTS OF PYROGALLOL ON THE METABOLITE PATTERN RATS WERE INJECTED LP. 30 MINUTES PRE NE-H2 INJECTION. DOSE WAS 200 nN./ko . ONE GROUP SACRIFH~D AT 6 MINUTES POST NE-li3 AND THE OTHER AT BO MINUTES POST NE-H3.
NM
DHMA VMA MHPß
202
Intraveatricular Injection Technique
Vol . 18, No . 2
effect on the metabolite pattern (changes in X of total counts) was as follows : NM, a decrease of 10 .5X (p < 0 .01) ; DHIlA aad DHPG, an increase of 11 .1X (p < 0 .01) ; VMA, a decrease of 7 .6X (p < 0 .01) . At 60 minutes the effect was as follows : NM, a decrease of 4 .3X (p < 0 .03) ; DHMA and DHPG, ea increase of 28 .6X (p < 0 .01) ; VMA, a decrease of 19 .3X (p < 0 .01) . Sffeçt of Parg~line oa NFrS3 Metabolism Pargyline, an MAO inhibitor, was administered i .p . at a dose of 100 mg/,kg 3O minutes prior to the intraventricular injection of the N&-H3 . One group (N - 4) was sacrificed at 10 minutas aad the other at 120 minutes post NE-H3 . The results are shown in Fig . 6 . At 10 minutes the effect an the metabolite pattern (changes in X of total counts) was as follows : NS, an increase of 11 .1X (p < 0 .02) ; NM, an increase of 15 .4X (p < 0 .01) ; DHMA and DHPG, a decrease of 5 .7X (p < 0 .01) ; VMA, a decrease of 20 .OX (p < 0 .01) . At 120 minutes effect was as follows : NM, an increase of 53 .OX (p < 0 .01) ; D1~fA sad DHPG, a decrease of 1 .1X (p < 0 .01) ; VMA, a decrease of 23 .7X (p < 0 .01) ; MSPG, a decrease of 11 .OX (p < 0 .01) .
Flß. 6 EFFECTf OF PAROYLINE ON THE METABOLITE PATTERN RATt MIERE IN.IECTED LP. 80 MINUTES PRE NE"Ila INJECTION. DO:E WAi 100 nK./k} ONE ßROUP iACRIFICED AT 10 MINUTE! PORT NE-H9 AND THE OTHER AT 1Z0 MINUTEÔ POiT NE-H8.
Discussion The results demonstrate that the band injection technique ie .very satisfactory . Ia the split-brain studies the counts circulated throughout the ventricular system and did not remain localized at the injection site . The histological studies ,again showed the circulation of the dye throughout the ventricular system. Finally, in testing the effect of a CO!!T inhibitor and an MAO inhibitor the expected results were obtained . All previous intraveatricular injection techniques for acute studies required as anesthetic, usually diethyl ether . Anesthetics are not without
Vol . 18, No . 2
Intraveatricular Iajection Technique
203
effect on neuronal transmission, electrical activity, and biogenic amines . The band Iajection technique enables the study of the metabolism sad function of intravemtricularly injected substances is the rat brain without the limitations imposed by the use of an anesthetic . A surgical procedure was also previously required which 1s a stress factor . The only stress to which the rat is subjected when using the bead is the brief restraint required during the injection procedure. No adverse reactions were observed Ia any of the animals following the injection procedure . The band Iajection technique is not limited to injections into the lateral ventricle . Modifications of the bead design would enable investigators to reach other areas of the brain, either for injection purposes or lesloaing . Nobel (1) was able to inject 15 - 20 rats per hour, using ether and a surgical procedure; with the bead it 3s feasible to inject one rat per minute or aizty rats per hour . Acknowledgment The author would like to thank Mr . Robert Poempner for ezecuting the bead design and tir. Bernard Miller for his illustrations, sad wishes to e~rese hie appreciation to Dr . William D . Cash, Ms . Patricia Loo, Dr . Aaron Feldstein and Dr . Gertrude P . gaina for their most helpful guidance . References 1.
E. P . NOBLE, R. J . WITRTMAN and J. ABELROD, Life Sciences , 6, 281-291 (1967) .
2.
G. B. KOVACSICS aad J . ~. SAELEDiS, Arçh . Iat . Pharmacodyn., 174, 481 (1968) .
3.
R. STEEL and J . TORBIE, Principles and Procedures of Statistics , (p . 81) Magraw Hill, N. Y. (1960) .