Brain Research, 575 (1992) 231-237 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
231
BRES 17543
Effects of neonatal enucleation on catecholamine and serotonin turnover and amino acid levels in lateral geniculate nucleus and visual cortex of the adult rat M.L. Vizuete, V. Steffen, A. Machado and J. Cano Departamento de Bioquimica, Bromatologia y Toxicologia, Universidad de Sevilla, Sevilla (Spain)
(Accepted 5 November 1991) Key words: Lateral geniculate; Visual cortex; Turnover, Amino acid; Enucleation; Rat
Changes in turnover of dopamine (DA), noradrenaline (NA) and serotonin (5-hydroxytryptamine(5-HT)) and their metabolites, together with amino acid content, have been studied in dorsal lateral geniculate nucleus (LGNd) and visual cortex (VC) of neonatal enucleated rats. Enucleation increases the 5-HT turnover in LGNd and catecholamine turnover in VC. In contrast, enucleation decreases glutamate (and/or aspartate) content in LGNd and y-aminobutyricacid (GABA) in VC. These changes suggest an increase of the inhibitory action of the biogenic amines in LGNd after neonatal enucleation. The decrease of GABA in VC may reflect the importance of GABA in intracortical circuitry.
INTRODUCTION The measurement of morphological, functional and biochemical changes after uni- or bilateral enucleation in neonatal animals has significantly increased our knowledge of the developmental mechanism in the visual system and the plasticity of the brain in general 4°. Both anatomical and electrophysiological experiments have demonstrated substantial reorganization of sensory projections to the dorsal lateral geniculate nucleus (LGNd) 7 and visual cortex (VC) 31'39 following neonatal enucleation. The importance of noradrenaline (NA) and serotonin (5-hydroxytryptamine (5-HT)) in these structures has been assessed. The noradrenergic system in LGNd and VC was found to be highly plastic 34'38. It has also been reported that 5-HT metabolism increased in LGNd after enucleation 3s. These data obtained in biochemical studies are in good agreement with those obtained by qualitative assessment of sections through LGNd in neonatal and adult enucleates which have demonstrated an increase in the density of 5-HT innervation 26. Previous studies in other portions of the nervous system have also shown that 5-HT fibres exhibit remarkable plasticity after CNS lesions in either infancy or adulthood 1'8. Likewise, amino acids have been suggested as putative neurotransmitters in the visual system 12'15'3°. Excitatory amino acids 3 and taurine 9'22 are described as involved in
the transmission of optic inputs to the LGN. Recently, it has been reported that N-acetylaspartylglutamate (NAAG) is a better candidate as a neurotransmitter of rat optic nerve terminals 35. Glutamate (and/or aspartate) is now regarded as a candidate for the neurotransmitter of corticothalamic fibres 16. ~-Aminobutyric acid (GABA) has been localized in non-pyramidal neurons in the VC and in the interneurons in the LGNd. It has been shown that the GABAergic neurons in the visual cortex mediate inhibition and are involved in the processing of visual impulses a3. Studies in other amino acids have suggested that fl-alanine may have a neuroactive function 29 although the role of this compound is unknown. Our laboratory has investigated monoamine metabolism in LGNd and VC during postnatal development after neonatal enucleation. These studies showed an increase in N A and 5-HT metabolism in VC and LGNd, respectively 38. From these results we have previously raised the possibility that this reorganization may be due, at least in part, to an increased innervation of the LGNd and VC by inputs that normally reduce the responsivity of LGNd and VC neurons 38. The aim of this report was to investigate by different biochemical methods to what extent amino acid and monoaminergic parameters in the LGNd and VC of adult rats were affected 3 months after eye enucleation. We studied the turnover rate of NA, dopamine (DA)
Correspondence: J. Cano, Dept. Bioquimica, Bromatologia y Toxicologia, Fac. Farmacia, do Prof. Garcia Gonzalez s/n. 41012, Sevilla, Spain. Fax: (34) (5) 4233765.
232 and 5-HT, and their main metabolites, since changes in turnover rate may give a better understanding of the functional
activity
of
monoaminergic
neurons
than
Dopa and 5-HTP accumulation after inhibition of L-amino acid aromatic decarboxylase DOPA and 5-HTP levels were measured 30 rain after intraperitoneal injection of NSD-1015 (100 mg/kg).
changes in concentration of the amines, which might remain constant or even decline despite an increased rate of synthesis. M o r e o v e r , little is known about the effects of neonatal visual deprivation on m o n o a m i n e turnover in L G N d and VC. We used non-isotopic methods to estimate the turnover rate of D A , N A and 5 - H T as follows: (1) m e a s u r e m e n t of the rate of biosynthesis of D A and 5 - H T based on the accumulation of 3,4-dihydrox-
DA, NA, NMN and 5-HT accumulation after MAO inhibition DA, NA, NMN and 5-HT levels were determined at various times (0-20 min) after intraperitoneal injection of pargyline (75 mg/kg). DA, NA, NMN and 5-HT accumulation rates were obtained by calculating the slope (b) of the curve by linear regression. In this case, standard deviation of each accumulation rate equalled standard deviation of b (SDb). Slopes of the regression lines of control and enucleated rats were compared by using the Student's t-test.
yphenylalanine ( L - D O P A ) and 5-hydroxytryptophan (5H T P ) after central decarboxylase inhibition by 3-hydroxybenzylhydrazine (NSD-1015)4; (2) m e a s u r e m e n t of the rate of biosynthesis of D A , N A , N M N (normetanephfine) and 5 - H T as the rate of accumulation of D A , N A , N M N and 5 - H T after inhibition of m o n o a m i n e oxidase by pargyline2a; (3) determination of N A and 5 - H T metabolism as turnover of their acid metabolites: 3-methoxy-4-hydroxyphenylglycol ( M H P G ) , and 5-hydroxy-3indolacetic acid ( 5 - H I A A ) after M A O pargyline 36.
inhibition by
D u e to the role ascribed to the amino acids in the visual system we also carried out additional experiments
Turnover of acid metabolities: MHPG and 5-HIAA Concentrations of MHPG and 5-HIAA were determined at various times (0-20 min) after intraperitoneal injection of pargyline (75 mg/kg). MHPG and 5-HIAA declination rates were calculated by linear regression of logarithms of MHPG and 5-HIAA over the time periods specified. The slopes (b) of the regression lines were calculated by the method of least squares. The slopes obtained were used to estimate total MHPG and 5-HIAA turnover rate (T) as the product of fractional rate constant (k) k = b/0.434 and the estimated concentration of each metabolite at time 0 (steady-state) 2. Standard deviation of k (SDk) equalled standard deviation for b (SDb) divided by 0.434. Slopes of the regression lines and the corresponding turnover rates of controls and enucleated rats were compared by using the Student's t-test. Measurement of amino acids
to describe the possible changes of glutamate, aspartate, G A B A , glycine, fl-alanine and taurine levels after enucleation.
MATERIALS AND METHODS A group of 50 male Wistar rats of 3 months were used. We used control and enucleated animals; in the second case, both eyes were removed under ether anaesthesia from animals in the first postnatal day. We used 40 rats for measurement of turnover (20 control and 20 enucleated rats) and 10 for amino acid quantification (5 of each group). Animals were housed under controlled environmental conditions. Food and tap water were allowed ad libitum. Animals were killed by cervical dislocation and decapitation between 10.00 and 11.00 a.m. and the brains were quickly removed and dissected at 0°C using a dissecting microscope. The visual cortex was removed including the areas 17 and 18. Thereafter, the cerebral cortex was peeled back and with a dorsal view of the rat's diencephalon the lateral geniculate nucleus was removed. The total time for isolation of the tissue was < 3 min. They were frozen in liquid nitrogen and stored at -80°C until used. Pargyline HCI and NSD-1015 were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Both drugs were dissolved in 0.9% NaCI and injected intraperitoneally. Injection volumes were 5 ml/kg. Physiological amino acid standards were obtained from Pierce. Other chemicals were of the purest grade available from standard suppliers.
Measurement of turnover Analysis was performed by means of HPLC equipped with a Kontron 420 pump in conjunction with electrochemical detector (Bioanalytical Systems, Inc., LC-4B). Measurement of the biogenic amines and their metabolites was performed according to methods described elsewhere 38.
We used a precolumn derivatization technique that uses phenylisothiocyanate (PITC) as the tagging reagent, a Pico-Tag (Waters column) and UV detection. The solvents were prepared according to Maldonado et al.17: (eluent A: 70 mM acetate trihydrate pH 6.65 + 2.5% acetonitrile; eluent B: 45% acetonitrile + 40% H20 + 15% methanol). The detector was set at 254 nm. The method used is also that developed by Waters as Pico-Tag System (Millipore Corporation, MA, U.S.A.).
Statistical analyses A Student's t-test was used. When the P value was
231
BRES 17543
Effects of neonatal enucleation on catecholamine and serotonin turnover and amino acid levels in lateral geniculate nucleus and visual cortex of the adult rat M.L. Vizuete, V. Steffen, A. Machado and J. Cano Departamento de Bioquimica, Bromatologia y Toxicologia, Universidad de Sevilla, Sevilla (Spain)
(Accepted 5 November 1991) Key words: Lateral geniculate; Visual cortex; Turnover, Amino acid; Enucleation; Rat
Changes in turnover of dopamine (DA), noradrenaline (NA) and serotonin (5-hydroxytryptamine(5-HT)) and their metabolites, together with amino acid content, have been studied in dorsal lateral geniculate nucleus (LGNd) and visual cortex (VC) of neonatal enucleated rats. Enucleation increases the 5-HT turnover in LGNd and catecholamine turnover in VC. In contrast, enucleation decreases glutamate (and/or aspartate) content in LGNd and y-aminobutyricacid (GABA) in VC. These changes suggest an increase of the inhibitory action of the biogenic amines in LGNd after neonatal enucleation. The decrease of GABA in VC may reflect the importance of GABA in intracortical circuitry.
INTRODUCTION The measurement of morphological, functional and biochemical changes after uni- or bilateral enucleation in neonatal animals has significantly increased our knowledge of the developmental mechanism in the visual system and the plasticity of the brain in general 4°. Both anatomical and electrophysiological experiments have demonstrated substantial reorganization of sensory projections to the dorsal lateral geniculate nucleus (LGNd) 7 and visual cortex (VC) 31'39 following neonatal enucleation. The importance of noradrenaline (NA) and serotonin (5-hydroxytryptamine (5-HT)) in these structures has been assessed. The noradrenergic system in LGNd and VC was found to be highly plastic 34'38. It has also been reported that 5-HT metabolism increased in LGNd after enucleation 3s. These data obtained in biochemical studies are in good agreement with those obtained by qualitative assessment of sections through LGNd in neonatal and adult enucleates which have demonstrated an increase in the density of 5-HT innervation 26. Previous studies in other portions of the nervous system have also shown that 5-HT fibres exhibit remarkable plasticity after CNS lesions in either infancy or adulthood 1'8. Likewise, amino acids have been suggested as putative neurotransmitters in the visual system 12'15'3°. Excitatory amino acids 3 and taurine 9'22 are described as involved in
the transmission of optic inputs to the LGN. Recently, it has been reported that N-acetylaspartylglutamate (NAAG) is a better candidate as a neurotransmitter of rat optic nerve terminals 35. Glutamate (and/or aspartate) is now regarded as a candidate for the neurotransmitter of corticothalamic fibres 16. ~-Aminobutyric acid (GABA) has been localized in non-pyramidal neurons in the VC and in the interneurons in the LGNd. It has been shown that the GABAergic neurons in the visual cortex mediate inhibition and are involved in the processing of visual impulses a3. Studies in other amino acids have suggested that fl-alanine may have a neuroactive function 29 although the role of this compound is unknown. Our laboratory has investigated monoamine metabolism in LGNd and VC during postnatal development after neonatal enucleation. These studies showed an increase in N A and 5-HT metabolism in VC and LGNd, respectively 38. From these results we have previously raised the possibility that this reorganization may be due, at least in part, to an increased innervation of the LGNd and VC by inputs that normally reduce the responsivity of LGNd and VC neurons 38. The aim of this report was to investigate by different biochemical methods to what extent amino acid and monoaminergic parameters in the LGNd and VC of adult rats were affected 3 months after eye enucleation. We studied the turnover rate of NA, dopamine (DA)
Correspondence: J. Cano, Dept. Bioquimica, Bromatologia y Toxicologia, Fac. Farmacia, do Prof. Garcia Gonzalez s/n. 41012, Sevilla, Spain. Fax: (34) (5) 4233765.
232 and 5-HT, and their main metabolites, since changes in turnover rate may give a better understanding of the functional
activity
of
monoaminergic
neurons
than
Dopa and 5-HTP accumulation after inhibition of L-amino acid aromatic decarboxylase DOPA and 5-HTP levels were measured 30 rain after intraperitoneal injection of NSD-1015 (100 mg/kg).
changes in concentration of the amines, which might remain constant or even decline despite an increased rate of synthesis. M o r e o v e r , little is known about the effects of neonatal visual deprivation on m o n o a m i n e turnover in L G N d and VC. We used non-isotopic methods to estimate the turnover rate of D A , N A and 5 - H T as follows: (1) m e a s u r e m e n t of the rate of biosynthesis of D A and 5 - H T based on the accumulation of 3,4-dihydrox-
DA, NA, NMN and 5-HT accumulation after MAO inhibition DA, NA, NMN and 5-HT levels were determined at various times (0-20 min) after intraperitoneal injection of pargyline (75 mg/kg). DA, NA, NMN and 5-HT accumulation rates were obtained by calculating the slope (b) of the curve by linear regression. In this case, standard deviation of each accumulation rate equalled standard deviation of b (SDb). Slopes of the regression lines of control and enucleated rats were compared by using the Student's t-test.
yphenylalanine ( L - D O P A ) and 5-hydroxytryptophan (5H T P ) after central decarboxylase inhibition by 3-hydroxybenzylhydrazine (NSD-1015)4; (2) m e a s u r e m e n t of the rate of biosynthesis of D A , N A , N M N (normetanephfine) and 5 - H T as the rate of accumulation of D A , N A , N M N and 5 - H T after inhibition of m o n o a m i n e oxidase by pargyline2a; (3) determination of N A and 5 - H T metabolism as turnover of their acid metabolites: 3-methoxy-4-hydroxyphenylglycol ( M H P G ) , and 5-hydroxy-3indolacetic acid ( 5 - H I A A ) after M A O pargyline 36.
inhibition by
D u e to the role ascribed to the amino acids in the visual system we also carried out additional experiments
Turnover of acid metabolities: MHPG and 5-HIAA Concentrations of MHPG and 5-HIAA were determined at various times (0-20 min) after intraperitoneal injection of pargyline (75 mg/kg). MHPG and 5-HIAA declination rates were calculated by linear regression of logarithms of MHPG and 5-HIAA over the time periods specified. The slopes (b) of the regression lines were calculated by the method of least squares. The slopes obtained were used to estimate total MHPG and 5-HIAA turnover rate (T) as the product of fractional rate constant (k) k = b/0.434 and the estimated concentration of each metabolite at time 0 (steady-state) 2. Standard deviation of k (SDk) equalled standard deviation for b (SDb) divided by 0.434. Slopes of the regression lines and the corresponding turnover rates of controls and enucleated rats were compared by using the Student's t-test. Measurement of amino acids
to describe the possible changes of glutamate, aspartate, G A B A , glycine, fl-alanine and taurine levels after enucleation.
MATERIALS AND METHODS A group of 50 male Wistar rats of 3 months were used. We used control and enucleated animals; in the second case, both eyes were removed under ether anaesthesia from animals in the first postnatal day. We used 40 rats for measurement of turnover (20 control and 20 enucleated rats) and 10 for amino acid quantification (5 of each group). Animals were housed under controlled environmental conditions. Food and tap water were allowed ad libitum. Animals were killed by cervical dislocation and decapitation between 10.00 and 11.00 a.m. and the brains were quickly removed and dissected at 0°C using a dissecting microscope. The visual cortex was removed including the areas 17 and 18. Thereafter, the cerebral cortex was peeled back and with a dorsal view of the rat's diencephalon the lateral geniculate nucleus was removed. The total time for isolation of the tissue was < 3 min. They were frozen in liquid nitrogen and stored at -80°C until used. Pargyline HCI and NSD-1015 were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Both drugs were dissolved in 0.9% NaCI and injected intraperitoneally. Injection volumes were 5 ml/kg. Physiological amino acid standards were obtained from Pierce. Other chemicals were of the purest grade available from standard suppliers.
Measurement of turnover Analysis was performed by means of HPLC equipped with a Kontron 420 pump in conjunction with electrochemical detector (Bioanalytical Systems, Inc., LC-4B). Measurement of the biogenic amines and their metabolites was performed according to methods described elsewhere 38.
We used a precolumn derivatization technique that uses phenylisothiocyanate (PITC) as the tagging reagent, a Pico-Tag (Waters column) and UV detection. The solvents were prepared according to Maldonado et al.17: (eluent A: 70 mM acetate trihydrate pH 6.65 + 2.5% acetonitrile; eluent B: 45% acetonitrile + 40% H20 + 15% methanol). The detector was set at 254 nm. The method used is also that developed by Waters as Pico-Tag System (Millipore Corporation, MA, U.S.A.).
Statistical analyses A Student's t-test was used. When the P value was
