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Reduction of GABA synthesis following lesions of inhibitory vestibulotrochlear pathway
326
Brain Research, 91 (1975) 326-330 O Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Reduction of GABA synthesis following lesions of inhibitory vestibulotrochlear pathway
SUZANNE ROFFLER-TARLOV ANDEDWARD TARLOV Department of Neurology, The Children's Hosp#al Medical Center, Departments of Neurology and Pharmacology, Harvard Medical School, and the Neurosurgical Service, Massachusetts General Hospital, Boston, Mass. 02115 (U.S.A.)
(Accepted March llth, 1975)
We have studied the synthesis of suspected neurotransmitter compounds including gamma-aminobutyric acid (GABA) by the normal cat trochlear nucleus (TN) and following lesions of the inhibitory ipsilateral pathway from the superior vestibular nucleus (SVN) to the TN. In addition to its motor neurons, which innervate the contralateral superior oblique, the trochlear nucleus contains the nerve terminals of a dense ipsilateral projection from the SVN s. The SVN receives afferent projections from the ganglia of the semicircular canals 1. Electrophysiological experiments show that in cats, SVN neurons exert an inhibitory influence on the ipsilateral T N neurons via this pathway; stimulation of the ipsilateral vestibular (VIII) nerve or direct stimulation of the SVN produces short latency inhibitory postsynaptic potentials (IPSP's) in T N neurons6; this response is blocked by bicuculline and picrotoxinL During the course of our studies, Precht et al. 7 reported that lesions of the ipsilateral medial longitudinal fasciculus (MLF) reduced endogenous GABA in a midbrain area which included the T N and oculomotor nuclei. This finding is of interest because axons passing from the SVN to the T N and oculomotor nuclei pass through the M L F as do other fibers passing rostrally and caudally in the brain stem. To screen a number of substances as possible neurotransmitters specificially in the inhibitory S V N - T N pathway, we have compared the capacity of normal T N and T N deafferented of SVN connections to synthesize suspected transmitter compounds. We have used a radiochemical method to measure the ability of fresh isolated T N tissue in vitro to synthesize suspected neurotransmitters from their radioactive precursors 2. The usefulness of this technique as a screening procedure for transmitter compounds is based upon a neuron's ability to synthesize and accumulate its own transmitter substance 3. Among the compounds tested, acetylcholine, dopamine, tyramine and GABA, only the synthesis of GABA was dependent on intact SVN nerve terminals in TN, and therefore our data concerning this compound only will be described here. Studies of other suspected transmitters related to the vestibulo-ocular pathways are in progress. To produce SVN axon terminal degeneration in ipsilateral TN, discrete stereo-
327 taxic lesions were made in the SVN or its efferent pathways on the left side in 5 adult cats. There were 7 control cats; 2 in which the electrode was passed through the cerebellum without producing a vestibular nuclear lesion and 5 in which lesions were made in vestibular nuclei other than the SVN. The lesions were made with a 0.5 mm or a 0.25 mm steel electrode using cathodal direct current. Twelve to 14 days later the animals were guillotined and the brains quickly removed with sterile precautions. After the brains were chilled in the freezer for 3-5 min to render the tissue firmer, cubes of tissue were taken under x 16 magnification from the ventral portion of the central gray matter just dorsal to the M L F on each side, at the level of the decussation of the trochlear nerves. The remainder of the block was embedded, serially sectioned, and stained with Toluidine blue. Absence of the trochlear nucleus in serial sections through the midbrain confirmed the nucleus had been removed for incubation. The block containing the vestibular nuclei was fixed and sectioned for histological confirmation of the location of the lesion. Each TN was pre-incubated for 30 rain at 37 °C in non-radioactive sterile tissue culture medium (L-15 from Microbiological Associates) which contained no glutamate or glutamine. The tissue was then incubated for 2 h at 37 °C in 50-100/~1 of tissue culture medium containing 20 #M [3-all]glutamate (16.2 Ci/mmole from New England Nuclear, Boston). When necessary the [3H]glutamate was purified by high voltage electrophoresis at pH 2, see ref. 4. The [3H]glutamate was usually boiled just before use in 2N HCI for 1 h, see ref. 4, to remove a contaminant which can be converted to GABA in the absence of glutamic acid decarboxylase, the enzyme which catalyzes the conversion of glutamate to GABA ~. After incubation, the tissues were washed for 5-7 rain in three changes of nonradioactive medium. The radioactivity was extracted from the tissue by freezing and thawing in 90 #1 pH 2 buffer (0.47 M formic acid, 1.4 M acetic acid) and 15 #1 of 1 sodium dodecyl sulfate in pH 2 buffer. A 45 #1 sample of the extract was applied to Whatman 3 MM chromatography paper. A 10 #1 sample of the radioactive medium which had bathed the tissue and a sample of radioactive medium which had been incubated in the absence of tissue were applied to separate lanes of the paper. To visualize the positions of the precursor and product, standard non-radioactive solutions of glutamate and GABA were also applied to each of the lanes. Subsequently, the precursor and its product were separated at pH 2 by high voltage electrophoresis z and their positions were visualized by dipping the strip into a solution of ninhydrin and cadmium acetate. The lanes were then cut into 1-cm strips which were placed in 1-dram vials with 0.5 ml of 0.1 N HC1 to elute the radioactivity 4. Four milliliters of scintillation fluid (Aquasol) were added after the vials had been gently shaken for 30 min. The radioactivity was counted in a Packard liquid scintillation counter. The identity of GABA formed was confirmed by high voltage electrophoresis, followed by ascending chromatography in N-butanol-acetic acid-water (60:15:25) 4. The distribution and relative size of lesions made in the vestibular nuclei are represented in Fig. 1. Two large lesions (nos. 9 and 15) ablate nearly the entire SVN. There is one small lesion in SVN, no. 13. Two of the lesions, 1l and 14, transect the efferent pathway from the SVN, which passes rostromedially into the MLF 8. Lesions
328
5~
/3//50.
Fig. 1. Diagrammatic representation, in horizontal sections at 3 levels, of lesions in the left vestibular complex. Numbers refer to animal in which lesion was made. Most dorsal section is at right next to the genu of the facial (VII) nerve, most medial is at left next to brachium conjunctivum (B.C.). Nuclei in the vestibular complex are the superior (S), the medial (M), the lateral (L), and the descending (D). Their positions are shown on the inset diagram. Lesions indicated with open circles had no effect on synthesis of gamma-aminobutyric acid (GABA) in the ipsilateral trochlear nucleus (TN). Lesions marked with hatches, lying in the superior nucleus or its efferent pathway, produced marked depression of TN GABA synthesis.
4, 6, 8, 10 and 12 involve other parts of the vestibular complex. The TN from all 7 control cats in which the S V N - T N pathway was intact formed and stored [3H], G A B A from [3H]glutamate. The amount of [aH]GABA formed on each side varied no more than 15 ~ between the left and the right TN in any of the animals with intact S V N - T N pathways (Table I). in contrast, all lesions which involved the SVN or its efferent pathways produced a marked decrease in the ability of excised T N tissue to synthesize and store GABA. The left T N from cats 9 and 15 in which large lesions of the left SVN had been made 2 weeks earlier were able to make only 36 ~ and 31 O//o respectively of the G A B A formed by the right TN. In animals 11 and 14, in which the efferent pathway from the left SVN to the ipsilateral T N was interrupted, the left T N made 45 ~ and 55 ~ of the GABA made by the right. In all of the control cats and in cats 9, II, 14 and 15 with lesions of the SVN or its efferent pathway, histological examination of the midbrains showed that the T N had been completely removed for studies. In cat 13, in which a small lesion had been placed successfully in the left SVN, G A B A synthesis by the left TN was 130 of that synthesized by the right (Table l). Serial histological sections of the midbrain of this cat provide an explanation for this discrepant result, since the sections showed that removal of T N was incomplete. On the left side, an estimated two-thirds of the volume of the TN was removed whereas on the right, a smaller volume of the TN, estimated at one-third was removed for incubation. Thus the volume of TN incubated and tested for GABA synthesizing capacity was smaller on the right than on the left: furthermore, a very small lesion such as this would be suspected from our previous studies to produce minimal axon degeneration in the TN. These facts account for the greater GABA synthesizing activity of the left T N compared to the right in this experiment.
329 TABLE I EFFECT OF LEFT-SIDED LESIONS IN CAT VFSTIBULAR NUCLEI ON FORMATION OF GAMMA-AMINOBUTYRIC ACID
(GABA)
IN IPSILATERAL TROCHLEAR NUCLEUS ( T N )
See Fig. 1 for exact site and size of lesions. All lesions were made stereotaxically on the left side in adult cats, with steel electrode using cathodal direct current. Cats were killed 12-14 days postoperatively. In animals 2 and 3 which had no lesions in the vestibular complex, the electrode was passed through the cerebellum at the angle of approach to the vestibular target. GABA formation by trochlear nuclei from animals in control group varied from 314,559 disint./min/mg to 1,080,823 disint./min/mg. Superior-trochlear pathway intact Cat Site of lesion in vestibular complex
2 3 6 4 10 8 12
Superior-trochh'ar path way interrupted
GABA Cat Site of lesion in vestibular formation: camplex disint./ min/ mg left T N divided by disint.~rain~ mg right T N
No lesion 1.09 No lesion 0.93 Medial nucleus 0.88 Lateral nucleus 0.85 Lateral nucleus 0.91 Lateraland descending 1.01 Descending and medial 0.94
13 9 15 11 14
Superior (SVN) (small lesion) SVN (large lesion) SVN (large lesion) SVN-TN pathway SVN-TN pathway
GABA Jbrmation: disbtt./ m in./ n :g h'Jt T N divided by disint./min/ mg right T N
1.30" 0.36 0.31 0.45 0.55
* TN tissue incubated in cat 13 with a small lesion consisted of approximately two-thirds of the TN on the left and one-third on the right, accounting for the greater GABA formation on the side of a superior vestibular nucleus lesion (see text). In all other cats removal of the TN was complete.
T a b l e I expresses the d a t a in terms o f [ a H ] G A B A f o r m e d / m g tissue removed. The weights o f the T N tissue r e m o v e d were fairly consistent on the two sides, m e a n weight for the left T N f r o m 12 cats being 3.35 ~ 0.25 mg a n d those f r o m the r i g h t side 2.95 :k 0.18 mg. W i t h the exception o f cat 13, serial histological sections showed t h a t the entire T N was removed. Thus, v a r i a t i o n in weight o f tissue r e m o v e d reflects v a r y i n g a m o u n t s o f s u r r o u n d i n g tissue included in the sample. H o w e v e r , the findings r e p o r t e d are the s a m e w h e t h e r expressed as [ a H ] G A B A f o r m e d / t r o c h l e a r nucleus or [ a H ] G A B A f o r m e d / m g tissue. T h a t is, lesions which ablate a significant p o r t i o n o f the S V N or its efferent p a t h w a y are a s s o c i a t e d with a m a r k e d decrease in the ability o f the ipsilateral T N to synthesize G A B A , whereas lesions l o c a t e d outside the S V N T N p a t h w a y d o n o t p r o d u c e this specific effect. T h e m o s t likely i n t e r p r e t a t i o n o f o u r results is t h a t the m a r k e d decrease in G A B A synthesis b y ipsilateral T N tissue following lesions o f the S V N - T N p a t h w a y is a consequence o f d e g e n e r a t i o n o f the s y n a p t i c t e r m i n a l s o f axons which arise in the S V N a n d n o r m a l l y synapse in the T N , These t e r m i n a l b o u t o n s o f S V N n e u r o n s a p p e a r to a c c o u n t for a large p o r t i o n o f the G A B A synthesis which occurs in the T N o f a n i m a l s in which the S V N - T N p a t h w a y is intact. The decrease o f G A B A synthesizing c a p a b i l i t y b y T N deafferented f r o m SVN correlates with the time at
330 which o u r previous studies s have shown extensive d e g e n e r a t i o n o f SVN axon terminals. Other d a t a also p o i n t to G A B A as t r a n s m i t t e r in this pathway. Bicuculline and picrotoxin, which block the IPSPs generated in tile T N after s t i m u l a t i o n o f the VIII nerve or o f SVN, act by b l o c k i n g G A B A transmission 6,7. Precht's d e m o n s t r a t i o n o f r e d u c t i o n o f e n d o g e n o u s G A B A in the m i d b r a i n area o f the T N following destruction o f the ipsilateral M L F 7 is o f interest in relation to our findings, as the M L F contains, a m o n g other fibers, the axons passing from the SVN to the T N which we have destroyed. O u r experiments indicate the p r o b a b l e source o f these g a b a m i n e r g i c fibers to be the SVN, and localize their t e r m i n a t i o n s to the T N . The m a r k e d depression o f G A B A synthesis in T N coincident with d e g e n e r a t i o n o f SVN n e u r o n terminal.~ in T N indicates G A B A ' s likely t r a n s m i t t e r function in the inhibitory S V N - T N pathway. This w o r k was s u p p o r t e d by N I N D S G r a n t s 05172 a n d The C h i l d r e n ' s H o s p i t a l M e d i c a l Center M e n t a l R e t a r d a t i o n a n d H u m a n D e v e l o p m e n t a l R e s e a r c h P r o g r a m G r a n t H D 03773. W e t h a n k Peter Sargent a n d Dr. E. M e r l e r for their assistance with the transm i t t e r screening m e t h o d a n d high voltage electrophoresis.
1 GACEK, R., The course and central termination of first order neurons supplying vestibular end organs in the cat, Acta oto-laryng. (Stockh.), Suppl. 254 (1969) 1-66. 2 HILDEBRAND,J. G., BARKER,D. L., HERBERT, E., AND KRAVlrZ, E. A., Screening for neurotransmitters : a rapid radiochemical procedure, J. Neurobiol., 2 (1971) 231-246. 3 KRAVlTZ, E. A., Acetylcholine, gamma-aminobutyric acid and glutamic acid: physiological and chemical studies related to their roles as neurotransmitter agents. In G. QUARTON,T. MELN~CHUK AND F. SCHMITT(Eds.), The Neurosciences, A Study Program, Rockefeller University Press, New York, 1967, pp. 433-444. 4 MAINS, R. E., AND PATTERSON, P. H., Primary cultures of dissociated sympathetic neurons. I. Establishment of long-term growth in culture and studies of differentiated properties, J. Cell Biol., 59 (1973) 329-345. 5 MOLINOEE,P. B., AND KRAVlTZ, E. A., The metabolism of gamma-aminobutyric acid (GABA) in the lobster nervous system: glutamic decarboxylase, J. Neurochem., 15 (1968) 391-409. 6 PRECHT, W., AND BAKER, R., Synaptic organization of the vestibulo-trochlear pathway, Exp. Brain Res., 14 (1972) 158-184. 7 PRECHT, W., BAKER, R., AND OKADA,Y., Evidence for GABA as the synaptic transmitter of the inhibitory vestibulo-ocular pathway, Exp. Brain Res., 18 (1973) 415-428. 8 TARLOV, E., Organization of vestibulo-oculomotor projections in the cat, Brain Research, 20 (1970) 159-179.
Brain Research, 91 (1975) 326-330 O Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Reduction of GABA synthesis following lesions of inhibitory vestibulotrochlear pathway
SUZANNE ROFFLER-TARLOV ANDEDWARD TARLOV Department of Neurology, The Children's Hosp#al Medical Center, Departments of Neurology and Pharmacology, Harvard Medical School, and the Neurosurgical Service, Massachusetts General Hospital, Boston, Mass. 02115 (U.S.A.)
(Accepted March llth, 1975)
We have studied the synthesis of suspected neurotransmitter compounds including gamma-aminobutyric acid (GABA) by the normal cat trochlear nucleus (TN) and following lesions of the inhibitory ipsilateral pathway from the superior vestibular nucleus (SVN) to the TN. In addition to its motor neurons, which innervate the contralateral superior oblique, the trochlear nucleus contains the nerve terminals of a dense ipsilateral projection from the SVN s. The SVN receives afferent projections from the ganglia of the semicircular canals 1. Electrophysiological experiments show that in cats, SVN neurons exert an inhibitory influence on the ipsilateral T N neurons via this pathway; stimulation of the ipsilateral vestibular (VIII) nerve or direct stimulation of the SVN produces short latency inhibitory postsynaptic potentials (IPSP's) in T N neurons6; this response is blocked by bicuculline and picrotoxinL During the course of our studies, Precht et al. 7 reported that lesions of the ipsilateral medial longitudinal fasciculus (MLF) reduced endogenous GABA in a midbrain area which included the T N and oculomotor nuclei. This finding is of interest because axons passing from the SVN to the T N and oculomotor nuclei pass through the M L F as do other fibers passing rostrally and caudally in the brain stem. To screen a number of substances as possible neurotransmitters specificially in the inhibitory S V N - T N pathway, we have compared the capacity of normal T N and T N deafferented of SVN connections to synthesize suspected transmitter compounds. We have used a radiochemical method to measure the ability of fresh isolated T N tissue in vitro to synthesize suspected neurotransmitters from their radioactive precursors 2. The usefulness of this technique as a screening procedure for transmitter compounds is based upon a neuron's ability to synthesize and accumulate its own transmitter substance 3. Among the compounds tested, acetylcholine, dopamine, tyramine and GABA, only the synthesis of GABA was dependent on intact SVN nerve terminals in TN, and therefore our data concerning this compound only will be described here. Studies of other suspected transmitters related to the vestibulo-ocular pathways are in progress. To produce SVN axon terminal degeneration in ipsilateral TN, discrete stereo-
327 taxic lesions were made in the SVN or its efferent pathways on the left side in 5 adult cats. There were 7 control cats; 2 in which the electrode was passed through the cerebellum without producing a vestibular nuclear lesion and 5 in which lesions were made in vestibular nuclei other than the SVN. The lesions were made with a 0.5 mm or a 0.25 mm steel electrode using cathodal direct current. Twelve to 14 days later the animals were guillotined and the brains quickly removed with sterile precautions. After the brains were chilled in the freezer for 3-5 min to render the tissue firmer, cubes of tissue were taken under x 16 magnification from the ventral portion of the central gray matter just dorsal to the M L F on each side, at the level of the decussation of the trochlear nerves. The remainder of the block was embedded, serially sectioned, and stained with Toluidine blue. Absence of the trochlear nucleus in serial sections through the midbrain confirmed the nucleus had been removed for incubation. The block containing the vestibular nuclei was fixed and sectioned for histological confirmation of the location of the lesion. Each TN was pre-incubated for 30 rain at 37 °C in non-radioactive sterile tissue culture medium (L-15 from Microbiological Associates) which contained no glutamate or glutamine. The tissue was then incubated for 2 h at 37 °C in 50-100/~1 of tissue culture medium containing 20 #M [3-all]glutamate (16.2 Ci/mmole from New England Nuclear, Boston). When necessary the [3H]glutamate was purified by high voltage electrophoresis at pH 2, see ref. 4. The [3H]glutamate was usually boiled just before use in 2N HCI for 1 h, see ref. 4, to remove a contaminant which can be converted to GABA in the absence of glutamic acid decarboxylase, the enzyme which catalyzes the conversion of glutamate to GABA ~. After incubation, the tissues were washed for 5-7 rain in three changes of nonradioactive medium. The radioactivity was extracted from the tissue by freezing and thawing in 90 #1 pH 2 buffer (0.47 M formic acid, 1.4 M acetic acid) and 15 #1 of 1 sodium dodecyl sulfate in pH 2 buffer. A 45 #1 sample of the extract was applied to Whatman 3 MM chromatography paper. A 10 #1 sample of the radioactive medium which had bathed the tissue and a sample of radioactive medium which had been incubated in the absence of tissue were applied to separate lanes of the paper. To visualize the positions of the precursor and product, standard non-radioactive solutions of glutamate and GABA were also applied to each of the lanes. Subsequently, the precursor and its product were separated at pH 2 by high voltage electrophoresis z and their positions were visualized by dipping the strip into a solution of ninhydrin and cadmium acetate. The lanes were then cut into 1-cm strips which were placed in 1-dram vials with 0.5 ml of 0.1 N HC1 to elute the radioactivity 4. Four milliliters of scintillation fluid (Aquasol) were added after the vials had been gently shaken for 30 min. The radioactivity was counted in a Packard liquid scintillation counter. The identity of GABA formed was confirmed by high voltage electrophoresis, followed by ascending chromatography in N-butanol-acetic acid-water (60:15:25) 4. The distribution and relative size of lesions made in the vestibular nuclei are represented in Fig. 1. Two large lesions (nos. 9 and 15) ablate nearly the entire SVN. There is one small lesion in SVN, no. 13. Two of the lesions, 1l and 14, transect the efferent pathway from the SVN, which passes rostromedially into the MLF 8. Lesions
328
5~
/3//50.
Fig. 1. Diagrammatic representation, in horizontal sections at 3 levels, of lesions in the left vestibular complex. Numbers refer to animal in which lesion was made. Most dorsal section is at right next to the genu of the facial (VII) nerve, most medial is at left next to brachium conjunctivum (B.C.). Nuclei in the vestibular complex are the superior (S), the medial (M), the lateral (L), and the descending (D). Their positions are shown on the inset diagram. Lesions indicated with open circles had no effect on synthesis of gamma-aminobutyric acid (GABA) in the ipsilateral trochlear nucleus (TN). Lesions marked with hatches, lying in the superior nucleus or its efferent pathway, produced marked depression of TN GABA synthesis.
4, 6, 8, 10 and 12 involve other parts of the vestibular complex. The TN from all 7 control cats in which the S V N - T N pathway was intact formed and stored [3H], G A B A from [3H]glutamate. The amount of [aH]GABA formed on each side varied no more than 15 ~ between the left and the right TN in any of the animals with intact S V N - T N pathways (Table I). in contrast, all lesions which involved the SVN or its efferent pathways produced a marked decrease in the ability of excised T N tissue to synthesize and store GABA. The left T N from cats 9 and 15 in which large lesions of the left SVN had been made 2 weeks earlier were able to make only 36 ~ and 31 O//o respectively of the G A B A formed by the right TN. In animals 11 and 14, in which the efferent pathway from the left SVN to the ipsilateral T N was interrupted, the left T N made 45 ~ and 55 ~ of the GABA made by the right. In all of the control cats and in cats 9, II, 14 and 15 with lesions of the SVN or its efferent pathway, histological examination of the midbrains showed that the T N had been completely removed for studies. In cat 13, in which a small lesion had been placed successfully in the left SVN, G A B A synthesis by the left TN was 130 of that synthesized by the right (Table l). Serial histological sections of the midbrain of this cat provide an explanation for this discrepant result, since the sections showed that removal of T N was incomplete. On the left side, an estimated two-thirds of the volume of the TN was removed whereas on the right, a smaller volume of the TN, estimated at one-third was removed for incubation. Thus the volume of TN incubated and tested for GABA synthesizing capacity was smaller on the right than on the left: furthermore, a very small lesion such as this would be suspected from our previous studies to produce minimal axon degeneration in the TN. These facts account for the greater GABA synthesizing activity of the left T N compared to the right in this experiment.
329 TABLE I EFFECT OF LEFT-SIDED LESIONS IN CAT VFSTIBULAR NUCLEI ON FORMATION OF GAMMA-AMINOBUTYRIC ACID
(GABA)
IN IPSILATERAL TROCHLEAR NUCLEUS ( T N )
See Fig. 1 for exact site and size of lesions. All lesions were made stereotaxically on the left side in adult cats, with steel electrode using cathodal direct current. Cats were killed 12-14 days postoperatively. In animals 2 and 3 which had no lesions in the vestibular complex, the electrode was passed through the cerebellum at the angle of approach to the vestibular target. GABA formation by trochlear nuclei from animals in control group varied from 314,559 disint./min/mg to 1,080,823 disint./min/mg. Superior-trochlear pathway intact Cat Site of lesion in vestibular complex
2 3 6 4 10 8 12
Superior-trochh'ar path way interrupted
GABA Cat Site of lesion in vestibular formation: camplex disint./ min/ mg left T N divided by disint.~rain~ mg right T N
No lesion 1.09 No lesion 0.93 Medial nucleus 0.88 Lateral nucleus 0.85 Lateral nucleus 0.91 Lateraland descending 1.01 Descending and medial 0.94
13 9 15 11 14
Superior (SVN) (small lesion) SVN (large lesion) SVN (large lesion) SVN-TN pathway SVN-TN pathway
GABA Jbrmation: disbtt./ m in./ n :g h'Jt T N divided by disint./min/ mg right T N
1.30" 0.36 0.31 0.45 0.55
* TN tissue incubated in cat 13 with a small lesion consisted of approximately two-thirds of the TN on the left and one-third on the right, accounting for the greater GABA formation on the side of a superior vestibular nucleus lesion (see text). In all other cats removal of the TN was complete.
T a b l e I expresses the d a t a in terms o f [ a H ] G A B A f o r m e d / m g tissue removed. The weights o f the T N tissue r e m o v e d were fairly consistent on the two sides, m e a n weight for the left T N f r o m 12 cats being 3.35 ~ 0.25 mg a n d those f r o m the r i g h t side 2.95 :k 0.18 mg. W i t h the exception o f cat 13, serial histological sections showed t h a t the entire T N was removed. Thus, v a r i a t i o n in weight o f tissue r e m o v e d reflects v a r y i n g a m o u n t s o f s u r r o u n d i n g tissue included in the sample. H o w e v e r , the findings r e p o r t e d are the s a m e w h e t h e r expressed as [ a H ] G A B A f o r m e d / t r o c h l e a r nucleus or [ a H ] G A B A f o r m e d / m g tissue. T h a t is, lesions which ablate a significant p o r t i o n o f the S V N or its efferent p a t h w a y are a s s o c i a t e d with a m a r k e d decrease in the ability o f the ipsilateral T N to synthesize G A B A , whereas lesions l o c a t e d outside the S V N T N p a t h w a y d o n o t p r o d u c e this specific effect. T h e m o s t likely i n t e r p r e t a t i o n o f o u r results is t h a t the m a r k e d decrease in G A B A synthesis b y ipsilateral T N tissue following lesions o f the S V N - T N p a t h w a y is a consequence o f d e g e n e r a t i o n o f the s y n a p t i c t e r m i n a l s o f axons which arise in the S V N a n d n o r m a l l y synapse in the T N , These t e r m i n a l b o u t o n s o f S V N n e u r o n s a p p e a r to a c c o u n t for a large p o r t i o n o f the G A B A synthesis which occurs in the T N o f a n i m a l s in which the S V N - T N p a t h w a y is intact. The decrease o f G A B A synthesizing c a p a b i l i t y b y T N deafferented f r o m SVN correlates with the time at
330 which o u r previous studies s have shown extensive d e g e n e r a t i o n o f SVN axon terminals. Other d a t a also p o i n t to G A B A as t r a n s m i t t e r in this pathway. Bicuculline and picrotoxin, which block the IPSPs generated in tile T N after s t i m u l a t i o n o f the VIII nerve or o f SVN, act by b l o c k i n g G A B A transmission 6,7. Precht's d e m o n s t r a t i o n o f r e d u c t i o n o f e n d o g e n o u s G A B A in the m i d b r a i n area o f the T N following destruction o f the ipsilateral M L F 7 is o f interest in relation to our findings, as the M L F contains, a m o n g other fibers, the axons passing from the SVN to the T N which we have destroyed. O u r experiments indicate the p r o b a b l e source o f these g a b a m i n e r g i c fibers to be the SVN, and localize their t e r m i n a t i o n s to the T N . The m a r k e d depression o f G A B A synthesis in T N coincident with d e g e n e r a t i o n o f SVN n e u r o n terminal.~ in T N indicates G A B A ' s likely t r a n s m i t t e r function in the inhibitory S V N - T N pathway. This w o r k was s u p p o r t e d by N I N D S G r a n t s 05172 a n d The C h i l d r e n ' s H o s p i t a l M e d i c a l Center M e n t a l R e t a r d a t i o n a n d H u m a n D e v e l o p m e n t a l R e s e a r c h P r o g r a m G r a n t H D 03773. W e t h a n k Peter Sargent a n d Dr. E. M e r l e r for their assistance with the transm i t t e r screening m e t h o d a n d high voltage electrophoresis.
1 GACEK, R., The course and central termination of first order neurons supplying vestibular end organs in the cat, Acta oto-laryng. (Stockh.), Suppl. 254 (1969) 1-66. 2 HILDEBRAND,J. G., BARKER,D. L., HERBERT, E., AND KRAVlrZ, E. A., Screening for neurotransmitters : a rapid radiochemical procedure, J. Neurobiol., 2 (1971) 231-246. 3 KRAVlTZ, E. A., Acetylcholine, gamma-aminobutyric acid and glutamic acid: physiological and chemical studies related to their roles as neurotransmitter agents. In G. QUARTON,T. MELN~CHUK AND F. SCHMITT(Eds.), The Neurosciences, A Study Program, Rockefeller University Press, New York, 1967, pp. 433-444. 4 MAINS, R. E., AND PATTERSON, P. H., Primary cultures of dissociated sympathetic neurons. I. Establishment of long-term growth in culture and studies of differentiated properties, J. Cell Biol., 59 (1973) 329-345. 5 MOLINOEE,P. B., AND KRAVlTZ, E. A., The metabolism of gamma-aminobutyric acid (GABA) in the lobster nervous system: glutamic decarboxylase, J. Neurochem., 15 (1968) 391-409. 6 PRECHT, W., AND BAKER, R., Synaptic organization of the vestibulo-trochlear pathway, Exp. Brain Res., 14 (1972) 158-184. 7 PRECHT, W., BAKER, R., AND OKADA,Y., Evidence for GABA as the synaptic transmitter of the inhibitory vestibulo-ocular pathway, Exp. Brain Res., 18 (1973) 415-428. 8 TARLOV, E., Organization of vestibulo-oculomotor projections in the cat, Brain Research, 20 (1970) 159-179.