Brain Research, 573 (1992) 197-203 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
197
BRES 17463
Selective reduction of glutamate in the rat superior colliculus and dorsal lateral geniculate nucleus after contralateral enucleation Takashi Sakurai and Yasuhiro Okada Department of Physiology, School of Medicine, Kobe University, Kobe (Japan) (Accepted 8 October 1991)
Key words: Rat; Superior colliculus; Dorsal lateral geniculate nucleus; Amino acid; Retinotectal projection; Retinogeniculate projection; Glutamatergic
The effects of afferent lesions on the levels of glutamate, aspartate and y-aminobutyric acid (GABA) in the laminae of the superior coiliculus (SC) and dorsal lateral geniculate nucleus (dLGN) of the rat were studied, using microassay methods for these amino acids. The analysis was performed 12-14 days after left eye enucleation, or ablation of right visual cortical area, or both left eye enucleation and ablation of right visual cortex. Superficial gray layer (SGL) and deep layers in the SC were dissected out from the thin-sectioned, freeze-dried sample. In the dLGN, the outer and inner laminae were separately dissected. The glutamate contents in the upper half of SGL and outer lamina of dLGN contralateral to eye enucleation decreased significantly (15%). Combination of eye enucleation and visual cortical ablation further decreased the glutamate content in the upper half of the right SGL (29.3%). On the other hand, aspartate and GABA concentrations in the SC and dLGN exhibited no significant reduction after deafferentations. These results indicate that the retino-tectal and retino-geniculate pathway of the rat may be glutamatergic in nature.
INTRODUCTION The superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN) have a pivotal role in the visual system, processing visual auditory, sensory and m o t o r informations. SC and d L G N receive 3 main afferent projections, retinal and visual cortical fibers and fibers from the brainstem. The synaptic organization in the superficial layer in SC and d L G N have not been fully characterized in spite of several intensive studies 2°'22"25'36. Concerning the neurotransmitters in the d L G N , visual cortical afferents to d L G N are considered to be glutamatergic 6'16'23'37. In addition to glutamate, the existence of noradrenalin, 5-hydroxytryptamine (5-HT), acetylcholine and neuropeptides were detected immunohistochemically in the d L G N and their physiological role have been investigated as neurotransmitters 21'22. Nevertheless, a neurotransmitter in the retino-geniculate pathway has not been dearly determined. Although glutamate is probably a neurotransmitter in cortico-tectal projections 6'16'23'37, various endogenous substances such as catecholamine, 5-HT, acetylcholine, dipeptide and some other neuropeptides have been suggested as putative neurotransmitters in afferent fibers to SC 1'21'24'28'29. Cholinergic system in SC is now consid-
ered to originate in the connection between the parabigeminal nucleus and SC 11'29. Substance P- and enkephalin-like immunoreactivities were investigated in the SC after retinal deafferentation 24, but the functional meaning of these neuropeptides remains uncertain. Finally, there has been a lot of evidence in favour of a role for excitatory amino acids in retino-tectal pathway 35'37' 38. In the pigeon, eye enucleation causes a reduction of the high-affinity uptake of glutamate in the tectum 13 and optic nerve stimulation induced release of glutamate and aspartate from the optic tectum in vivo 4. In mammals, this was not confirmed in the observation of Fonnum et al. 6'16. However, we have recently showed a possibility of glutamatergic transmission in the rat retino-tectal pathway in the physiological examination 37. Thus, transmitters in the retino-tectal and retino-geniculate projections have not be fully determined. In this experiment, we performed quantitative analysis of glutamate, aspartate and 7-aminobutyric acid ( G A B A ) , in different layers of the SC and d L G N using microassay methods for the amino acids. Consequently, we observed the alteration occurred in the fine particular part of the SC and d L G N . The results show an evidence that the retino-tectal and the retino-geniculate pathway of the rat are glutamatergic.
Correspondence: Y. Okada, Department of Physiology, School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650¢ Japan.
198 MATERIALS AND METHODS
Experimental procedure Male albino (Wister) rats weighing 200-300 g were used and all operative procedures in this study were performed under anesthesia with intraperitoneal injection of sodium pentobarbitone (30 mg/ kg). For the lesion studies, three types of operations were performed, that is (1) unilateral enucleation, (2) unilateral visual cortical ablation and (3) a combination of the two lesions. Enucleation of the left eye was performed in I0 rats to produce optic nerve denervation to right SC and dLGN. Right visual cortical ablations were performed in 12 rats by aspiration to the depth of the subcortical white matter to produce degeneration of the corticotectal and the cortico-geniculate pathway. In 9 rats, both left eye enucleation and right visual cortical ablation were performed. Six rats were used as unoperated control animals. Twelve to 14 days after these surgical operations, rats were sacrificed by decapitation. After opening the skull, a tissue block including the midbrain and thalamus was rapidly removed and frozen in liquid nitrogen. The tissue block was cut into 20-/~m-thickcoronal sections in a cryostat (-26°C) and the sections were freeze-dried overnight. Cellular arrangement in the freeze-dried sections in the SC and dLGN were well visualized under stereomicroscopy and each layer of SC and dLGN was clearly identified, comparing with toluidine blue-stained sections, obtained every ten successive cutting. The superficial gray layer (SGL) of SC was dissected out at the edge between SGL and optic layer (OL). The width of SGL was about 250/~m from the surface. This dissected SGL was subdivided into two portions; upper and lower half of SGL. Remaining part of the SC including the OL, the intermediate gray layer, the intermediate white layer, the deep gray layer, and the deep white layer was perpendicularly cut 1.4 mm in width. D-LGN was well identified in the sectioned slices containing the thalamus and dissected out (the width of dLGN section was about 750 /~m from the surface). Dissected dLGN was equally separated into two parts, namely the outer lamina and the inner lamina. Each dissected piece of tissue was weighed with a fishpole quarts fiber balance (sensitivity 0.02/~g/mm) and each averaging about 0.9/tg dry weight. Assay of amino acids The glutamate and aspartate contents in each sample were determined using an enzymatic cycling method with NAD-NADH. The GABA concentration was measured enzymatically using NADP-NADPH cycling system. Details of these assay methods for glutamate and GABA have been described elsewhere3°'34. For the aspartate assay, the dried tissue samples were treated at 60°C for 30 min with 2/~m droplets of 0.05 N NaOH in the oil well to stop the enzyme activity and destroy NAD in the tissue. Four/A of aspartate assay reaction mixture containing 0.05 N imidazol-HCl buffer (pH 7.0), 0.15 mM a-ketoglutalate, 30/~M NADH, 5 ~g/ml malate dehydrogenase (MDH) and 30/~g/ml glutamat-oxalacetattransaminase (GOT) was added to the droplets and they were incubated at 38°C for 10 rain. After treatment with HC1, the mixture was transferred to 50/A of cycling reagent and incubated at 26°C for 30 min. The cycling reagent and following procedures were the same as that in glutamate assay mentioned above. Chemicals for standard solutions and the assay system for aspartate were obtained from Nakarai Co. and MDH and GOT from Boehringer Yamanouchi. Student's t-test (non-paired) was used for statistical analysis.
RESULTS
Glutamate content in the SC and d L G N In the rat, retinal efferent fibers cross at the optic chiasm (97-98%) and project to the contralateral SC and the d L G N TM. O n the other hand, cortico-tectal and cor-
tico-geniculate fibers terminate in the ipsilateral SC and dLGN. In all experiments, the left eye was enucleated and the concentrations of amino acids were compared in each side of SC and dLGN. The amino acids concentrations of non-denervated side were used as the control values, since there were no differences between concentrations of amino acids in the SC and the d L G N in each side of the control animals. The concentrations of glutamate in the SGL and the other portion of the SC are shown in Table I. The glutamate content of the SC of unoperated rats was in good agreement with those in various studies, assuming the dry weight is one fifth of the wet weight 16'23'38. Following left eye enucleation, glutamate content in upper half of fight SGL exhibited a significant reduction (15.3%). Ablation of the right visual cortical area resulted in approximately 20% loss of glutamate in both upper and lower half of the ipsilateral SGL. C o m b i n a t i o n of left eye enucleation and right visual cortical ablation led to a further reduction in the level of glutamate (29.3%) in the upper half of the SGL. Glutamate concentrations in the deep layers were a little less than those in the SGL, but exhibited no decrease following these operations. Mean glutamate concentrations in d L G N of unoperated animal showed no difference in the outer and inner lamina (Table II). Left eye enucleation reduced the glutamate content in the outer lamina of the fight d L G N (14.9%). Right visual cortical ablation reduced the glutamate content in both outer and inner lamina (approximately 41%) of right d L G N . C o m b i n a t i o n of the two lesions resulted in a significant reduction in the glutamate content of the outer lamina (38.4%) and inner lamina (42.9%).
Aspartate content in the SC and d L G N Mean concentration of aspartate in the SC of the unoperated animal was 21.4 mmoi/kg dry weight in the SGL and 16.8 mmol/kg dry weight in vertical sections containing deep layers (Table III). In spite of the various operative procedures, the aspartate content in the denervated side of the SC remained unchanged, compared with that of the non-operated side. The levels of aspartate in the d L G N were almost the same in the outer and inner laminae of unoperated animals (Table IV). Eye enucleation or ablation of visual cortical area did not produce decrease on the aspartate content. Combined lesions of both enucleation and cortical ablation reduced the aspartate concentration slightly (P < 0.05) in the outer lamina of fight SGL. G A B A content in the SC and d L G N G A B A concentration in the upper half of the SGL was 42.2 mmol/kg dry weight and mean concentration of
199 TABLE I
Glutamate concentrations in the superior colliculus after enucleation and visual cortical ablation Numbers of dissected samples of each layer were 9-18 obtained from 3 to 5 animals. P values for mean percentage change between each layer of left and right SC were obtained using a non-paired t-test. N.S. refers to difference with P > 0.05.
mmol/kg dry weight + S.E.M.
Mean change (%)
P
Left
Right
1. Unoperated control (n = 12-15) Upper half of SGL Lower half of SGL Deep layers
59.9 + 0.4 59.2 + 0.8 53.6 + 0.3
61.1 + 1.0 57.1 + 0.8 55.3 +__ 1.2
+2.0 -3.5 +3.2
2. Left eye enucleation (n = 12-17) Upper half of SGL Lower half of SGL Deep layers
60.7 + 0.9 60.8 + 1.5 54.8 + 0.7
51.4 + 1.1 57.1 + 1.0 55.0 + 0.5
-15.3 -4.8 +0.4
197
BRES 17463
Selective reduction of glutamate in the rat superior colliculus and dorsal lateral geniculate nucleus after contralateral enucleation Takashi Sakurai and Yasuhiro Okada Department of Physiology, School of Medicine, Kobe University, Kobe (Japan) (Accepted 8 October 1991)
Key words: Rat; Superior colliculus; Dorsal lateral geniculate nucleus; Amino acid; Retinotectal projection; Retinogeniculate projection; Glutamatergic
The effects of afferent lesions on the levels of glutamate, aspartate and y-aminobutyric acid (GABA) in the laminae of the superior coiliculus (SC) and dorsal lateral geniculate nucleus (dLGN) of the rat were studied, using microassay methods for these amino acids. The analysis was performed 12-14 days after left eye enucleation, or ablation of right visual cortical area, or both left eye enucleation and ablation of right visual cortex. Superficial gray layer (SGL) and deep layers in the SC were dissected out from the thin-sectioned, freeze-dried sample. In the dLGN, the outer and inner laminae were separately dissected. The glutamate contents in the upper half of SGL and outer lamina of dLGN contralateral to eye enucleation decreased significantly (15%). Combination of eye enucleation and visual cortical ablation further decreased the glutamate content in the upper half of the right SGL (29.3%). On the other hand, aspartate and GABA concentrations in the SC and dLGN exhibited no significant reduction after deafferentations. These results indicate that the retino-tectal and retino-geniculate pathway of the rat may be glutamatergic in nature.
INTRODUCTION The superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN) have a pivotal role in the visual system, processing visual auditory, sensory and m o t o r informations. SC and d L G N receive 3 main afferent projections, retinal and visual cortical fibers and fibers from the brainstem. The synaptic organization in the superficial layer in SC and d L G N have not been fully characterized in spite of several intensive studies 2°'22"25'36. Concerning the neurotransmitters in the d L G N , visual cortical afferents to d L G N are considered to be glutamatergic 6'16'23'37. In addition to glutamate, the existence of noradrenalin, 5-hydroxytryptamine (5-HT), acetylcholine and neuropeptides were detected immunohistochemically in the d L G N and their physiological role have been investigated as neurotransmitters 21'22. Nevertheless, a neurotransmitter in the retino-geniculate pathway has not been dearly determined. Although glutamate is probably a neurotransmitter in cortico-tectal projections 6'16'23'37, various endogenous substances such as catecholamine, 5-HT, acetylcholine, dipeptide and some other neuropeptides have been suggested as putative neurotransmitters in afferent fibers to SC 1'21'24'28'29. Cholinergic system in SC is now consid-
ered to originate in the connection between the parabigeminal nucleus and SC 11'29. Substance P- and enkephalin-like immunoreactivities were investigated in the SC after retinal deafferentation 24, but the functional meaning of these neuropeptides remains uncertain. Finally, there has been a lot of evidence in favour of a role for excitatory amino acids in retino-tectal pathway 35'37' 38. In the pigeon, eye enucleation causes a reduction of the high-affinity uptake of glutamate in the tectum 13 and optic nerve stimulation induced release of glutamate and aspartate from the optic tectum in vivo 4. In mammals, this was not confirmed in the observation of Fonnum et al. 6'16. However, we have recently showed a possibility of glutamatergic transmission in the rat retino-tectal pathway in the physiological examination 37. Thus, transmitters in the retino-tectal and retino-geniculate projections have not be fully determined. In this experiment, we performed quantitative analysis of glutamate, aspartate and 7-aminobutyric acid ( G A B A ) , in different layers of the SC and d L G N using microassay methods for the amino acids. Consequently, we observed the alteration occurred in the fine particular part of the SC and d L G N . The results show an evidence that the retino-tectal and the retino-geniculate pathway of the rat are glutamatergic.
Correspondence: Y. Okada, Department of Physiology, School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650¢ Japan.
198 MATERIALS AND METHODS
Experimental procedure Male albino (Wister) rats weighing 200-300 g were used and all operative procedures in this study were performed under anesthesia with intraperitoneal injection of sodium pentobarbitone (30 mg/ kg). For the lesion studies, three types of operations were performed, that is (1) unilateral enucleation, (2) unilateral visual cortical ablation and (3) a combination of the two lesions. Enucleation of the left eye was performed in I0 rats to produce optic nerve denervation to right SC and dLGN. Right visual cortical ablations were performed in 12 rats by aspiration to the depth of the subcortical white matter to produce degeneration of the corticotectal and the cortico-geniculate pathway. In 9 rats, both left eye enucleation and right visual cortical ablation were performed. Six rats were used as unoperated control animals. Twelve to 14 days after these surgical operations, rats were sacrificed by decapitation. After opening the skull, a tissue block including the midbrain and thalamus was rapidly removed and frozen in liquid nitrogen. The tissue block was cut into 20-/~m-thickcoronal sections in a cryostat (-26°C) and the sections were freeze-dried overnight. Cellular arrangement in the freeze-dried sections in the SC and dLGN were well visualized under stereomicroscopy and each layer of SC and dLGN was clearly identified, comparing with toluidine blue-stained sections, obtained every ten successive cutting. The superficial gray layer (SGL) of SC was dissected out at the edge between SGL and optic layer (OL). The width of SGL was about 250/~m from the surface. This dissected SGL was subdivided into two portions; upper and lower half of SGL. Remaining part of the SC including the OL, the intermediate gray layer, the intermediate white layer, the deep gray layer, and the deep white layer was perpendicularly cut 1.4 mm in width. D-LGN was well identified in the sectioned slices containing the thalamus and dissected out (the width of dLGN section was about 750 /~m from the surface). Dissected dLGN was equally separated into two parts, namely the outer lamina and the inner lamina. Each dissected piece of tissue was weighed with a fishpole quarts fiber balance (sensitivity 0.02/~g/mm) and each averaging about 0.9/tg dry weight. Assay of amino acids The glutamate and aspartate contents in each sample were determined using an enzymatic cycling method with NAD-NADH. The GABA concentration was measured enzymatically using NADP-NADPH cycling system. Details of these assay methods for glutamate and GABA have been described elsewhere3°'34. For the aspartate assay, the dried tissue samples were treated at 60°C for 30 min with 2/~m droplets of 0.05 N NaOH in the oil well to stop the enzyme activity and destroy NAD in the tissue. Four/A of aspartate assay reaction mixture containing 0.05 N imidazol-HCl buffer (pH 7.0), 0.15 mM a-ketoglutalate, 30/~M NADH, 5 ~g/ml malate dehydrogenase (MDH) and 30/~g/ml glutamat-oxalacetattransaminase (GOT) was added to the droplets and they were incubated at 38°C for 10 rain. After treatment with HC1, the mixture was transferred to 50/A of cycling reagent and incubated at 26°C for 30 min. The cycling reagent and following procedures were the same as that in glutamate assay mentioned above. Chemicals for standard solutions and the assay system for aspartate were obtained from Nakarai Co. and MDH and GOT from Boehringer Yamanouchi. Student's t-test (non-paired) was used for statistical analysis.
RESULTS
Glutamate content in the SC and d L G N In the rat, retinal efferent fibers cross at the optic chiasm (97-98%) and project to the contralateral SC and the d L G N TM. O n the other hand, cortico-tectal and cor-
tico-geniculate fibers terminate in the ipsilateral SC and dLGN. In all experiments, the left eye was enucleated and the concentrations of amino acids were compared in each side of SC and dLGN. The amino acids concentrations of non-denervated side were used as the control values, since there were no differences between concentrations of amino acids in the SC and the d L G N in each side of the control animals. The concentrations of glutamate in the SGL and the other portion of the SC are shown in Table I. The glutamate content of the SC of unoperated rats was in good agreement with those in various studies, assuming the dry weight is one fifth of the wet weight 16'23'38. Following left eye enucleation, glutamate content in upper half of fight SGL exhibited a significant reduction (15.3%). Ablation of the right visual cortical area resulted in approximately 20% loss of glutamate in both upper and lower half of the ipsilateral SGL. C o m b i n a t i o n of left eye enucleation and right visual cortical ablation led to a further reduction in the level of glutamate (29.3%) in the upper half of the SGL. Glutamate concentrations in the deep layers were a little less than those in the SGL, but exhibited no decrease following these operations. Mean glutamate concentrations in d L G N of unoperated animal showed no difference in the outer and inner lamina (Table II). Left eye enucleation reduced the glutamate content in the outer lamina of the fight d L G N (14.9%). Right visual cortical ablation reduced the glutamate content in both outer and inner lamina (approximately 41%) of right d L G N . C o m b i n a t i o n of the two lesions resulted in a significant reduction in the glutamate content of the outer lamina (38.4%) and inner lamina (42.9%).
Aspartate content in the SC and d L G N Mean concentration of aspartate in the SC of the unoperated animal was 21.4 mmoi/kg dry weight in the SGL and 16.8 mmol/kg dry weight in vertical sections containing deep layers (Table III). In spite of the various operative procedures, the aspartate content in the denervated side of the SC remained unchanged, compared with that of the non-operated side. The levels of aspartate in the d L G N were almost the same in the outer and inner laminae of unoperated animals (Table IV). Eye enucleation or ablation of visual cortical area did not produce decrease on the aspartate content. Combined lesions of both enucleation and cortical ablation reduced the aspartate concentration slightly (P < 0.05) in the outer lamina of fight SGL. G A B A content in the SC and d L G N G A B A concentration in the upper half of the SGL was 42.2 mmol/kg dry weight and mean concentration of
199 TABLE I
Glutamate concentrations in the superior colliculus after enucleation and visual cortical ablation Numbers of dissected samples of each layer were 9-18 obtained from 3 to 5 animals. P values for mean percentage change between each layer of left and right SC were obtained using a non-paired t-test. N.S. refers to difference with P > 0.05.
mmol/kg dry weight + S.E.M.
Mean change (%)
P
Left
Right
1. Unoperated control (n = 12-15) Upper half of SGL Lower half of SGL Deep layers
59.9 + 0.4 59.2 + 0.8 53.6 + 0.3
61.1 + 1.0 57.1 + 0.8 55.3 +__ 1.2
+2.0 -3.5 +3.2
2. Left eye enucleation (n = 12-17) Upper half of SGL Lower half of SGL Deep layers
60.7 + 0.9 60.8 + 1.5 54.8 + 0.7
51.4 + 1.1 57.1 + 1.0 55.0 + 0.5
-15.3 -4.8 +0.4
