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Further evidence for GABAergic afferents to the lateral habenula
Brain Research, 152 (1978) 609-613 ~© Elsevier/North-Holland Biomedical Press
609
Further evidence for GABAergic afferents to the lateral habenula
ZEHAVA GOTTESFELD and DAVID M. JACOBOWITZ Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.)
(Accepted March 30th, 1978)
The stria medullaris (SM) is a massive fiber system which interconnects a variety of brain regions with the habenular nuclei (for review see refs. 3, 7 and 17). The identity of neurotransmitters associated with habenular afferents which pass via the SM is not known. In a recent work it has been demonstrated that the activity of glutamate decarboxylase (GAD), a marker for GA BAergic neurons, was decreased in the lateral habenula (LHb) as a result of SM lesion4,L This suggested that GABAergic fibers in the SM underwent anterograde degeneration with a subsequent loss of G A D at the projection site, the LHb. The most direct approach to identifying GABA-containing neurons and their pathways is to see them. The recent development of an immunocytochemical technique for G A D 1,e2 calls for a highly purified enzyme, and has been used to a limited extent only. A more widely used approach to visualizeS, 9 and biochemically trace GABAergic cells has been the high-affinity GABA uptake (ref. 21 for review). Whether the accumulation of GABA is by net uptake or by exchange diffusion has been questioned by several workers15, z0. Nevertheless, this process has been shown to be highly specific 10, and has been used as a tool to label neurotransmitter-specific neurons 2t. In the present study the GABA uptake process is used as an additional approach to tracing GABAergic pathways in the aM 4,5. If G A D is associated with GABAergic terminals, then its decreased activity in the L H b after SM lesion 4,5 should be correlated with a reduced high-affinity uptake of GABA. The results, indeed, demonstrate a decreased GABA accumulation in the LHb. Bilateral SM lesions were placed in male Sprague-Dawley rats (300-350 g) as described previously 4. Sham animals received similar treatments, except that the electrode was placed 0.2 mm above the SM and current was not delivered. The accuracy of the lesions was verified microscopically4,5. The experiment was designed to compare in parallel sham-treated and lesioned rats of identical survival periods. The rats were decapitated in alternate sequence and the brain was placed in a Vibratome (Oxford Laboratories, Foster City, Calif.) containing 0.32 M sucrose solution at 4 °C, and 300 #m sections were cut 1~. The habenular * Visiting Scientist to the National Institute of Mental Health.
610 TABLE 1 Kinetics and high-affTnity uptake of GABA and glutamate in the habenular nuclei Results are expressed as mean z~z S.E.M. For high-affinity uptake (8-10 experiments) the habenular nuclei were homogenized in 0.32 M sucrose and centrifuged at t000 × g for 10 min. Ten ixl aliquots (in triplicates) of the supernatant were incubated at 25 °C for 4 rain in the presence of GABA or glutamate (5 × 10-7 Mfinal concentration). The results were corrected after subtracting the blank values (see text). For the kinetics studies (3 separate experiments) similar aliquots of the 1000 g supernatant were incubated in the presence of 0.3-1 ltM of [aH]GABA or Jail]glutamate. Double reciprocal plots gave straight lines. Km (IzM)
GABA Lateral habenula (LHb) Medial habenula (MHb) Glutamate Lateral habenula (LHb) Medial habenula (MHb)
l/.mx
High-affTnity uptake
nmole/mg prot. ~rain
L Hb: MHb
dpm/llg prot./rain
LHb :MHb
5.0 ± 0.5
962 ± 110
1.51
1673 ± 128
3.6
3.3 ± 0.3
637 ± 72
2.5 ~- 0.3
909 ± 101
1.5 ~ 0.2
294 ± 50
470 ± 34
3.09
2318 ~ 255
3.8
603 i 47
TABLE II High-affinity GABA uptake and its inhibition by structural analogues Results are expressed as mean dz S.E.M. The number of experiments in parentheses. For further details see Table I and text. dpmlttg prot.lmin
% inhibition
Lateral habenula Control 2,4-Diaminobutyric acid (1 mM) fl-Alanine (0.5 mM)
1673 _k 128 (10) 467 :~- 56 (4) 1154 5- 107 (4)
72 31
Medial habenula Control 2.4-Diaminobutyric acid (1 mM) fl-Alanine (0.5 mM)
470 :~ 34 (10) 266 ± 30 (4) 204 _L 31 (4)
43 57
nuclei, lateral ( L H b ) a n d medial ( M H b ) , were dissected u n d e r the stereomicroscope, each nucleus was pooled f r o m 3 sections per rat and h o m o g e n i z e d in 0.32 M sucrose s o l u t i o n (final volume 75 #1). The h o m o g e n a t e s were centrifuged at 1000 × g for 10 m i n a n d 10 #1 s u p e r n a t a n t aliquots (in triplicates) c o n t a i n i n g 3-6 # g proteins were used. High-affinity uptake of G A B A or, for comparison, glutamate was measured in oxygenated Krebs phosphate buffer, p H 7.4 (140 m M NaC1, 5 m M KCI, 1.2 m M CaC12, 1.2 m M MgC12, 15 m M sodium phosphate a n d 5 m M glucose) in a final volume of
611 TABLE Ill Effect of bilateral stria medullaris lesions on GA BA and glutamate uptake by habenular nuelei
Results are expressed as mean ± S.E.M. of groups of 4-8 rats. Sham and lesioned rats werecompared in parallel for each survival period. For further details see text. Survival (days)
Uptake (dpm/l~gprot./min)
% Change
P*
Sham
Lesion
4 35 35
1994 ± 239 1199 =t=356 424 ± 46
1178 ± 264 452 ± 141 473 ± 76
41 --62
< 0.05 < 0.001
7 7
2318 ± 255 603 =~ 47
2864 ± 517 299 ± 54
24 --50
NS < 0.005
GA BA
Lateral habenula Medial habenula Glutamate
Lateral habenula Medial habenula
* Significant compared to sham using Student's t-test, two-tailed. 0.25 ml. The samples were preincubated in a shaking water bath for 5 min at 25 °C. Uptake was initiated by adding 50/~1 of 5 × 10 7 M (final concentration) of [3H]GA BA (spec. act. 43 Ci/mmole) or [3H]L-glutamate (spec. act. 40 Ci/mmole) with continued incubation for 4 rain. Uptake was stopped by excessive dilution with cold buffer. The radioactivity-containing particles ('crude synaptosomal fraction') were collected by filtration on Mill±pore filters (0.42/~m pore size), extracted with 2 ml cellosolve and counted in a liquid scintillation counter. Blanks (in triplicates) were treated identically but kept on ice, and their counts amounted to about one-fifth of the sample counts. The results were corrected by subtracting the blank values and expressed in dpm//~g prot./min. Protein was assayed in 5 #l aliquots of the 1000 g supernatant 1(~. Under the present experimental conditions the metabolic degradation of GABA is apparently insignificant, since l0 -s M aminooxyacetic acid, a GABA-transaminase inhibitor, did not affect the uptake. More than 90 ~o of the counts were in either GABA or glutamate (unpublished observations). GABA and glutamate accumulation were completely abolished by hypotonicity or in the presence of 0.2 % Triton X-100. In a Na+-free medium or in the presence of 10 7 M ouabain, uptake was greatly inhibited (by more than 88 ~i), as also shown by others2,1L The uptake of GABA was higher in the LHb compared to MHb (Table 1). This is consistent with previous observations of a markedly higher G A D activity in the LHb 4. Glutamate accumulation was also higher in the LHb. This differential uptake could be attributed, at least in part, to a higher Vm~x in the LHb (Table I). Both neurons and glial cells possess a high-affinity uptake mechanism for both GABA and glutamate6,S,lt,ls, 19. The uptake of GABA by neurons and gliae can be pharmacologically distinguished by 2,4-diaminobutyric acid and fl-alanine, respectively (see ref. 11 for review). Table II demonstrates that in the LHb a larger proportion of GABA uptake (72 %) was into nerve terminals. On the other hand, the accumulation of GA BA in neurons and gliae of the MHb did not markedly differ. As a result of SM lesions (Table Ill) the capacity of the LHb to take up GABA
612 was lost by 41 ~o a n d 62 ~ after survival o f 4 and 35 days, respectively. This coincides with a similar loss o f G A D activity, 4 4 % a n d 5 7 ~ after 4 a n d 14 days, respectively, following S M lesions 5. The possibility o f glial p r o l i f e r a t i o n in the L H b is unlikely, since the lesion was placed r e m o t e l y from the nucleus, and gliosis would have resulted in a n increase in b o t h G A D activity a n d G A B A uptake. In contrast, G A B A uptake in the M H b was n o t affected by the lesion in a c c o r d with lack o f change in G A D activity in this region 5. It is o f interest to note t h a t the u p t a k e o f g l u t a m a t e did not significantly change in the L H b after S M lesion (Table l l l ) , whereas a m a r k e d loss (50 ~ ) was f o u n d in the M H b . This could suggest a g l u t a m a t e r g i c i n p u t to the M H b t h r o u g h the SM, but a m o r e direct e x p e r i m e n t a l a p p r o a c h has yet to verify it. In conclusion, evidence based on G A B A high-affinity u p t a k e f u r t h e r s u p p o r t s the suggestion that G A B A e r g i c fibers within the stria medullaris project to the lateral habenula. The expert technical assistance o f Mr. R o b e r t P. M c D e v i t t a n d the excellent secretarial w o r k o f Ms. J u d y G. Blumenthal is gratefully a c k n o w l e d g e d .
1 Barber, R. and Saito, K., Light microscopic visualization of GAD and GABA-T in immunocytochemical preparations of rodent CNS. In E. Roberts, T. N. Chase and D. B. Tower (Eds.), GABA in Nervous System Function, Vol. 5, Kroc Foundation Series, Raven Press, New York, 1974, pp. 113-132. 2 Bennett, J. P., Jr., Logan, W. J. and Snyder, S. H., Amino acids as central nervous transmitters : the influence of ions, amino acid analogues, and ontogeny on transport systems for L-glutamic and L-aspartic acids and glycine into central nervous synaptosomes of the rat, J. Neurochem., 21 (1973) 1533-1550. 3 Cragg, B. G., The connections of the habenula in the rabbit, Exp. Neurol., 3 (1961) 388-409. 4 Gottesfeld, Z. and Jacobowitz, D. M., Neurochemical and anatomical studies of GABAergic neurons. In S. Garattini, J. F. Pujol and R. Samanin (Eds.), Interactions Between Putative Neurotransmitters in the Brain, Raven Press, New York, 1978, pp. I09-126. 5 Gottesfeld, Z., Massari, J. V., Muth, E. A., and Jacobowitz, D. M., Stria medullaris: a possible pathway containing GABAergic afferents to the lateral habenula, Brain Research, 130 (1977) 184-189. 6 Henn, F. A., Goldstein, M. N. and Hamberger, A., Uptake of the neurotransmitter candidate glutamate by glia, Nature (Lond.), 249 (1974) 663-664. 7 Herkenham, M. and Nauta, W. J. H., Afferent connections of the habenular nuclei in the rat. A horseradish peroxidase study, with a note on the fiber-of-passage problem, J. comp. Neurol., 173 (1977) 123-146. 8 H6kfelt, T. and Ljungdahl, A., Application of cytochemical techniques to the study of suspected transmitter substances in the nervous system. In E. Costa, L. L. Iversen and R. Paoletti (Eds.), Studies of Neurotransmitters at the Synaptic Level, Vol. 6, Advance. Biochem. Psychopharmacol.,
Raven Press, New York, 1972, pp. 1-36. 9 Iversen, L. L. and Bloom, F. E., Studies on the uptake of [ZH]GABA and [3H]glycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 10 Iversen, L. L. and Johnston, G. A. R., GABA uptake in rat central nervous system : comparison of uptake in slices and homogenates and the effects of some inhibitors, J. Neurochem., 18 (1971) 1939-1950. 11 Iversen, L. L. and Kelly, J. S., Uptake and metabolism of ~-aminobutyric acid by neurons and glial cells, Biochem. PharmacoL, 24 (1975) 933--938.
613 12 lversen, L. L. and Neal, M. J., The uptake of [aH]GABA by slices of rat cerebral cortex, J. Neurochem., 15 (1968) 1141 1149. 13 Jacobowitz, D. M., Removal of discrete fresh regions of the rat brain, Brain Research, 80 (1974) 111-115. 14 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Parts o f the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 15 Levi, G. and Raiteri, M., Exchange of neurotransmitter amino acid at nerve endings can simulate high affinity uptake, Nature (Lond.), 250 (1974) 735-737. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 17 Marburg, O., The structure and fiber connections of the human habenula, J. comp. Neurol., 80 (1944) 211 233. 18 Schon, F. and Kelly, J. S., Autoradiographic localization of [Z3H]GABA and ['~H]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 19 Sellstr6m, A., Sj6berg, L.-B. and Hamberger, A., Neuronal and glial systems for ;~-aminobutyric acid metabolism, J. Neuroehem., 25 (1975) 393 398. 20 Simon, J. R., Martin, D. L. and Kroll, M., Sodium dependent effiux and exchange of GABA in synaptosomes, J. Neuroehem., 23 (1974) 981-991. 21 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119 181. 22 Wood, J. G., McLaughlin, G. and Vaughn, J. E., lmmunocytochemical localization of G A D in electron microscopic preparations of rodent CNS. In E. Roberts, T. N. Chase and D. B. Tower (Eds.), GABA in Nervous System Function, Vol. 5, Kroe Foundation Series, Raven Press, New York, 1974, pp. 133 148.
609
Further evidence for GABAergic afferents to the lateral habenula
ZEHAVA GOTTESFELD and DAVID M. JACOBOWITZ Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.)
(Accepted March 30th, 1978)
The stria medullaris (SM) is a massive fiber system which interconnects a variety of brain regions with the habenular nuclei (for review see refs. 3, 7 and 17). The identity of neurotransmitters associated with habenular afferents which pass via the SM is not known. In a recent work it has been demonstrated that the activity of glutamate decarboxylase (GAD), a marker for GA BAergic neurons, was decreased in the lateral habenula (LHb) as a result of SM lesion4,L This suggested that GABAergic fibers in the SM underwent anterograde degeneration with a subsequent loss of G A D at the projection site, the LHb. The most direct approach to identifying GABA-containing neurons and their pathways is to see them. The recent development of an immunocytochemical technique for G A D 1,e2 calls for a highly purified enzyme, and has been used to a limited extent only. A more widely used approach to visualizeS, 9 and biochemically trace GABAergic cells has been the high-affinity GABA uptake (ref. 21 for review). Whether the accumulation of GABA is by net uptake or by exchange diffusion has been questioned by several workers15, z0. Nevertheless, this process has been shown to be highly specific 10, and has been used as a tool to label neurotransmitter-specific neurons 2t. In the present study the GABA uptake process is used as an additional approach to tracing GABAergic pathways in the aM 4,5. If G A D is associated with GABAergic terminals, then its decreased activity in the L H b after SM lesion 4,5 should be correlated with a reduced high-affinity uptake of GABA. The results, indeed, demonstrate a decreased GABA accumulation in the LHb. Bilateral SM lesions were placed in male Sprague-Dawley rats (300-350 g) as described previously 4. Sham animals received similar treatments, except that the electrode was placed 0.2 mm above the SM and current was not delivered. The accuracy of the lesions was verified microscopically4,5. The experiment was designed to compare in parallel sham-treated and lesioned rats of identical survival periods. The rats were decapitated in alternate sequence and the brain was placed in a Vibratome (Oxford Laboratories, Foster City, Calif.) containing 0.32 M sucrose solution at 4 °C, and 300 #m sections were cut 1~. The habenular * Visiting Scientist to the National Institute of Mental Health.
610 TABLE 1 Kinetics and high-affTnity uptake of GABA and glutamate in the habenular nuclei Results are expressed as mean z~z S.E.M. For high-affinity uptake (8-10 experiments) the habenular nuclei were homogenized in 0.32 M sucrose and centrifuged at t000 × g for 10 min. Ten ixl aliquots (in triplicates) of the supernatant were incubated at 25 °C for 4 rain in the presence of GABA or glutamate (5 × 10-7 Mfinal concentration). The results were corrected after subtracting the blank values (see text). For the kinetics studies (3 separate experiments) similar aliquots of the 1000 g supernatant were incubated in the presence of 0.3-1 ltM of [aH]GABA or Jail]glutamate. Double reciprocal plots gave straight lines. Km (IzM)
GABA Lateral habenula (LHb) Medial habenula (MHb) Glutamate Lateral habenula (LHb) Medial habenula (MHb)
l/.mx
High-affTnity uptake
nmole/mg prot. ~rain
L Hb: MHb
dpm/llg prot./rain
LHb :MHb
5.0 ± 0.5
962 ± 110
1.51
1673 ± 128
3.6
3.3 ± 0.3
637 ± 72
2.5 ~- 0.3
909 ± 101
1.5 ~ 0.2
294 ± 50
470 ± 34
3.09
2318 ~ 255
3.8
603 i 47
TABLE II High-affinity GABA uptake and its inhibition by structural analogues Results are expressed as mean dz S.E.M. The number of experiments in parentheses. For further details see Table I and text. dpmlttg prot.lmin
% inhibition
Lateral habenula Control 2,4-Diaminobutyric acid (1 mM) fl-Alanine (0.5 mM)
1673 _k 128 (10) 467 :~- 56 (4) 1154 5- 107 (4)
72 31
Medial habenula Control 2.4-Diaminobutyric acid (1 mM) fl-Alanine (0.5 mM)
470 :~ 34 (10) 266 ± 30 (4) 204 _L 31 (4)
43 57
nuclei, lateral ( L H b ) a n d medial ( M H b ) , were dissected u n d e r the stereomicroscope, each nucleus was pooled f r o m 3 sections per rat and h o m o g e n i z e d in 0.32 M sucrose s o l u t i o n (final volume 75 #1). The h o m o g e n a t e s were centrifuged at 1000 × g for 10 m i n a n d 10 #1 s u p e r n a t a n t aliquots (in triplicates) c o n t a i n i n g 3-6 # g proteins were used. High-affinity uptake of G A B A or, for comparison, glutamate was measured in oxygenated Krebs phosphate buffer, p H 7.4 (140 m M NaC1, 5 m M KCI, 1.2 m M CaC12, 1.2 m M MgC12, 15 m M sodium phosphate a n d 5 m M glucose) in a final volume of
611 TABLE Ill Effect of bilateral stria medullaris lesions on GA BA and glutamate uptake by habenular nuelei
Results are expressed as mean ± S.E.M. of groups of 4-8 rats. Sham and lesioned rats werecompared in parallel for each survival period. For further details see text. Survival (days)
Uptake (dpm/l~gprot./min)
% Change
P*
Sham
Lesion
4 35 35
1994 ± 239 1199 =t=356 424 ± 46
1178 ± 264 452 ± 141 473 ± 76
41 --62
< 0.05 < 0.001
7 7
2318 ± 255 603 =~ 47
2864 ± 517 299 ± 54
24 --50
NS < 0.005
GA BA
Lateral habenula Medial habenula Glutamate
Lateral habenula Medial habenula
* Significant compared to sham using Student's t-test, two-tailed. 0.25 ml. The samples were preincubated in a shaking water bath for 5 min at 25 °C. Uptake was initiated by adding 50/~1 of 5 × 10 7 M (final concentration) of [3H]GA BA (spec. act. 43 Ci/mmole) or [3H]L-glutamate (spec. act. 40 Ci/mmole) with continued incubation for 4 rain. Uptake was stopped by excessive dilution with cold buffer. The radioactivity-containing particles ('crude synaptosomal fraction') were collected by filtration on Mill±pore filters (0.42/~m pore size), extracted with 2 ml cellosolve and counted in a liquid scintillation counter. Blanks (in triplicates) were treated identically but kept on ice, and their counts amounted to about one-fifth of the sample counts. The results were corrected by subtracting the blank values and expressed in dpm//~g prot./min. Protein was assayed in 5 #l aliquots of the 1000 g supernatant 1(~. Under the present experimental conditions the metabolic degradation of GABA is apparently insignificant, since l0 -s M aminooxyacetic acid, a GABA-transaminase inhibitor, did not affect the uptake. More than 90 ~o of the counts were in either GABA or glutamate (unpublished observations). GABA and glutamate accumulation were completely abolished by hypotonicity or in the presence of 0.2 % Triton X-100. In a Na+-free medium or in the presence of 10 7 M ouabain, uptake was greatly inhibited (by more than 88 ~i), as also shown by others2,1L The uptake of GABA was higher in the LHb compared to MHb (Table 1). This is consistent with previous observations of a markedly higher G A D activity in the LHb 4. Glutamate accumulation was also higher in the LHb. This differential uptake could be attributed, at least in part, to a higher Vm~x in the LHb (Table I). Both neurons and glial cells possess a high-affinity uptake mechanism for both GABA and glutamate6,S,lt,ls, 19. The uptake of GABA by neurons and gliae can be pharmacologically distinguished by 2,4-diaminobutyric acid and fl-alanine, respectively (see ref. 11 for review). Table II demonstrates that in the LHb a larger proportion of GABA uptake (72 %) was into nerve terminals. On the other hand, the accumulation of GA BA in neurons and gliae of the MHb did not markedly differ. As a result of SM lesions (Table Ill) the capacity of the LHb to take up GABA
612 was lost by 41 ~o a n d 62 ~ after survival o f 4 and 35 days, respectively. This coincides with a similar loss o f G A D activity, 4 4 % a n d 5 7 ~ after 4 a n d 14 days, respectively, following S M lesions 5. The possibility o f glial p r o l i f e r a t i o n in the L H b is unlikely, since the lesion was placed r e m o t e l y from the nucleus, and gliosis would have resulted in a n increase in b o t h G A D activity a n d G A B A uptake. In contrast, G A B A uptake in the M H b was n o t affected by the lesion in a c c o r d with lack o f change in G A D activity in this region 5. It is o f interest to note t h a t the u p t a k e o f g l u t a m a t e did not significantly change in the L H b after S M lesion (Table l l l ) , whereas a m a r k e d loss (50 ~ ) was f o u n d in the M H b . This could suggest a g l u t a m a t e r g i c i n p u t to the M H b t h r o u g h the SM, but a m o r e direct e x p e r i m e n t a l a p p r o a c h has yet to verify it. In conclusion, evidence based on G A B A high-affinity u p t a k e f u r t h e r s u p p o r t s the suggestion that G A B A e r g i c fibers within the stria medullaris project to the lateral habenula. The expert technical assistance o f Mr. R o b e r t P. M c D e v i t t a n d the excellent secretarial w o r k o f Ms. J u d y G. Blumenthal is gratefully a c k n o w l e d g e d .
1 Barber, R. and Saito, K., Light microscopic visualization of GAD and GABA-T in immunocytochemical preparations of rodent CNS. In E. Roberts, T. N. Chase and D. B. Tower (Eds.), GABA in Nervous System Function, Vol. 5, Kroc Foundation Series, Raven Press, New York, 1974, pp. 113-132. 2 Bennett, J. P., Jr., Logan, W. J. and Snyder, S. H., Amino acids as central nervous transmitters : the influence of ions, amino acid analogues, and ontogeny on transport systems for L-glutamic and L-aspartic acids and glycine into central nervous synaptosomes of the rat, J. Neurochem., 21 (1973) 1533-1550. 3 Cragg, B. G., The connections of the habenula in the rabbit, Exp. Neurol., 3 (1961) 388-409. 4 Gottesfeld, Z. and Jacobowitz, D. M., Neurochemical and anatomical studies of GABAergic neurons. In S. Garattini, J. F. Pujol and R. Samanin (Eds.), Interactions Between Putative Neurotransmitters in the Brain, Raven Press, New York, 1978, pp. I09-126. 5 Gottesfeld, Z., Massari, J. V., Muth, E. A., and Jacobowitz, D. M., Stria medullaris: a possible pathway containing GABAergic afferents to the lateral habenula, Brain Research, 130 (1977) 184-189. 6 Henn, F. A., Goldstein, M. N. and Hamberger, A., Uptake of the neurotransmitter candidate glutamate by glia, Nature (Lond.), 249 (1974) 663-664. 7 Herkenham, M. and Nauta, W. J. H., Afferent connections of the habenular nuclei in the rat. A horseradish peroxidase study, with a note on the fiber-of-passage problem, J. comp. Neurol., 173 (1977) 123-146. 8 H6kfelt, T. and Ljungdahl, A., Application of cytochemical techniques to the study of suspected transmitter substances in the nervous system. In E. Costa, L. L. Iversen and R. Paoletti (Eds.), Studies of Neurotransmitters at the Synaptic Level, Vol. 6, Advance. Biochem. Psychopharmacol.,
Raven Press, New York, 1972, pp. 1-36. 9 Iversen, L. L. and Bloom, F. E., Studies on the uptake of [ZH]GABA and [3H]glycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 10 Iversen, L. L. and Johnston, G. A. R., GABA uptake in rat central nervous system : comparison of uptake in slices and homogenates and the effects of some inhibitors, J. Neurochem., 18 (1971) 1939-1950. 11 Iversen, L. L. and Kelly, J. S., Uptake and metabolism of ~-aminobutyric acid by neurons and glial cells, Biochem. PharmacoL, 24 (1975) 933--938.
613 12 lversen, L. L. and Neal, M. J., The uptake of [aH]GABA by slices of rat cerebral cortex, J. Neurochem., 15 (1968) 1141 1149. 13 Jacobowitz, D. M., Removal of discrete fresh regions of the rat brain, Brain Research, 80 (1974) 111-115. 14 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Parts o f the Brain Stem, Williams and Wilkins, Baltimore, Md., 1963. 15 Levi, G. and Raiteri, M., Exchange of neurotransmitter amino acid at nerve endings can simulate high affinity uptake, Nature (Lond.), 250 (1974) 735-737. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 17 Marburg, O., The structure and fiber connections of the human habenula, J. comp. Neurol., 80 (1944) 211 233. 18 Schon, F. and Kelly, J. S., Autoradiographic localization of [Z3H]GABA and ['~H]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 19 Sellstr6m, A., Sj6berg, L.-B. and Hamberger, A., Neuronal and glial systems for ;~-aminobutyric acid metabolism, J. Neuroehem., 25 (1975) 393 398. 20 Simon, J. R., Martin, D. L. and Kroll, M., Sodium dependent effiux and exchange of GABA in synaptosomes, J. Neuroehem., 23 (1974) 981-991. 21 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119 181. 22 Wood, J. G., McLaughlin, G. and Vaughn, J. E., lmmunocytochemical localization of G A D in electron microscopic preparations of rodent CNS. In E. Roberts, T. N. Chase and D. B. Tower (Eds.), GABA in Nervous System Function, Vol. 5, Kroe Foundation Series, Raven Press, New York, 1974, pp. 133 148.