Development of neurotransmitter uptake in regions of the chick brain

SUMMARY The uptake of postulated neurotransmitters or their precursors into regions of the developing chick brain and retina has been examined. The transport of low con centrations (around 10-s M) of GABA, glutamic acid, choline, dopamine and sero tonin into homogenates was sodium and energy dependent and inhibited by a variety of pharmacological agents that are thought to act presynaptically. After morpholog ical fractionation, the high affinity transport mechanism was concentrated in the nerve ending fraction. Compounds were poorly accumulated by the cerebral regions of the 6 day in cubated chick embryo. After this time, the uptake capacity of each brain region studied exhibited a characteristic developmental profile. Mechanisms for


INTRODUCTION
The presence of synapses specific for a given neurotransmitter may be detected by assaying the extent to which that neurotransmitter can be taken up by homogenates or synaptosornes prepared from cerebral tissue21.Synapsotomal elements are responsible for much of such uptake by brain homogenates, but neuronal and glial cell body fragments could also play a role in this process.Such non-synaptic events are minimized by the use of purified synaptosomal preparations in uptake studies.
The advantage of using homogenates to study the energy dependent uptake of postulated neurotransmitter is the lack of preparative loss of tissue.Thus, the uptake capacities of different samples of brain can be precisely compared.For these reasons, we have examined this process in both homogenates and in synaptosomal fractions.These extracts are then incubated for 5 min with very low concentrations of radioactive compounds.The short incubation time minimizes any metabolic conversion of these compounds 1 7 ,23,37, and their low concentration ensures that any uptake is largely caused by high affinity mechanisms.There is evidence that a neurotransmitter is taken up by those synapses specific for it in a rather selective manner2 1 • The degree of uptake of a given compound in brain regions allows an estimate to be made of the relative preponderance of synapses specific for that neurotransmitter in those regions.We have examined the developmental profile of neurotransmitter uptake in various regions of the maturing chick brain.The cerebellum, optic lobes and cerebral hemispheres are readily distinguishable even in the early embryo.In addition, the uptake capacity of the retina was examined, since this is a neural tissue derived from the central nervous system and can easily be dissected out.The eye develops very early in embryogenesis.
The onset of uptake may reflect synaptic differentiation, but glial maturation may also possibly play a role.Very little high affinity uptake was found in the early embryo but, during growth, major increases occurred which were characteristic for each region.We have also examined the uptake capacity of various morphological fractions.Even in the young embryo, the nerve ending fraction had the greatest ability to transport possible neurotransmitters.

(a) Synaptosome preparation
Chick and chick embryos of a White Leghorn strain were used.After decapitation, tissues were rapidly dissected out and accurately weighed.Retinae were carefully removed largely free of the pigment layer.Tissues were then homogenized in 19 vols 0.32 M sucrose.In order to prepare synaptosomes, this homogenate was centrifuged at 1000 x g for 10 min; the supernatant was then recentrifuged at 14,000 x g for 20 min.The pellet from this latter centrifugation was rehomogenized in 0.32 M sucrose and recentrifuged (14,000 x g for 20 min).The precipitate was taken up in 2 ml 0.32 M sucrose and layered on a discontinuous sucrose gradient consisting of 5 ml 0.8 M sucrose and 5 ml 1.2 M sucrose12.After centrifugation in an SW 36 rotor ( 140,000 x g, 30 min), the material at the interface between the 0.8 Mand 1.2 M sucrose was drawn off with a disposable pipet and diluted with 5 vols 0.32 M sucrose.This suspension was then centrifuged in SW 36 rotor (88,000 x g, 30 min), and the pelleted material taken up in 2 ml 0.32 M sucrose.While this method was originally developed for the rat, a fraction considerably enriched in synaptosomes can also be made from avian brain 30 .

(b) Incubation
The standard incubation medium consisted of Krebs This medium was gassed with 95 % O:a--5 % C02, and 0.9 ml of this was mixed with 0.1 ml of either a 5% (v/v) tissue homogenate sucrose or of a synaptosomal suspension in 0.32 M sucrose.The GABA uptake mechanism was intensely active.Thus, in order to maintain linear kinetics, only a 0.5 % homogenate was used in these studies.In this case, 0.1 ml of 5 % liver homogenate was added as a carrier at the end of the incubation.We found no mechanism for GABA uptake by liver homogenates.Incubation was at 37 °C for 5 min with continuous shaking.To allow for non-energy dependent neurotransmitter-binding, identical mixtures were held at 0 °C, and these served as controls.Inhibitors of the uptake process, when used, were added to the medium prior to the addition of tissue preparations.In the case of dopamine and serotonin, incubations were performed in dim light in order to retard photodecomposition.
All samples were then centrifuged at 0 °C and 28,000 x g for 10 min.Supernatants were drawn off for determination of radioactivity remaining unbound to particulate matter.Pellets were resuspended in 4 ml isotonic (0.14 M) NaCl and recentrifuged (28,000 x g, 10 min).The washed pellets were then dissolved in 0.5 ml tissue solubilizer (NCS, Amersham Searle, Arlington Heights, Ill.) at 45 °C.Radioactivity in these samples was determined.It was thus possible to calculate what percentage of the total radioactive compound in each incubation tube was actively

Sodium dependence of high affinity uptake by optic lobe homogenates from 3-day-old chicks
The concentration of labeled compounds was between 1.0 x 10-s M and 2.3 x 10-s M. Tubes contained 0.1 ml of a 5% tissue homogenate except for the GABA uptake study where a 0.5% homogenate was used.Incubation was for 5 min at 37 °C in oxygenated Krebs-Ringer buffer.For detailed procedure see text.Standard errors of the mean are given.2.0 ± 0.3 12.9 ± 1.9 4.5 ± 0.5 80.9 ± 4. 6  12.4 ± 3.7 taken up by the particulate fraction.When the results were calculated on a protein basis, protein was assayed by the method of Lowry et al. 27.
(c) Binding of GABA Non-energy dependent binding of GABA to synaptic membranes was determined in synaptosomal preparations 43 .These were incubated with 10-s M [3H)GABA (10 Ci/mmole) at 0 °C for 5 min in 0.1 M Tris• HCl, pH 7.5.In order to minimize any active uptake, the synaptosomal preparations were frozen and thawed prior to use and the incubation medium contained no sodium ion or glucose.At the end of the incubation, radioactivity in the precipitate and supernatant was determined as described previously, the only difference being that the pellet was washed in 5 ml distilled water, not isotonic saline.

(a) Characteristics of the uptake system
The ability of optic lobe homogenates from 3-day-old chicks to take up the compounds studied was severely depressed when incubation was in sodium-free Krebs-Ringer buffer, where sucrose was submitted for Na+ (Table I).All high affinity transport systems were sodium-dependent.
The uptake of suspected neurotransmitters and of choline into homogenates of chick optic lobes was examined in the presence of a variety of pharmacological agents which have been reported to block their synaptic uptake2,3,16,36,42.The resulting inhibitions observed were consistent with a synaptic location of most of the uptake activity (Table II).
Transport of labeled neurotransmitter candidates and choline was examined in the presence of a large excess of the non-radioactive compounds (Table III).The

Inhibition of high affinity uptake by various agents
The concentration of labeled compounds was between 1.0 and 2.3 x 10-8 M. Tubes contained 0.1 ml of a 5 % homogenate of optic lobes from newly hatched chicks.In the case of GABA uptake, a 0.5 % homogenate was used.Incubation was for 5 min at 37 °C in oxygenated Krebs-Ringer buffer.Inhibitors and their final concentrations were: 10-3 M chlorpromazine (used with glutamate and GABA), 10-4 ML-amphetamine sulfate (used with dopamine), 10-3 M hemicholinium (used with choline), I0-4 M desipramine (used with serotonin).For detailed procedure see text.Standard errors of the mean are given.

Inhibition of uptake by non-radioactive homologous compounds
The concentration of labeled compounds was between 1.0 and 2.3 x 10-8 M. Tubes contained 0.1 ml of a 5 % homogenate of optic lobes from 2-day-old chicks.In the case of GABA, a 0.5 % homogenate was used.Incubation was for 5 min at 37 °C in oxygenated Krebs-Ringer buffer.Inhibition caused by unlabeled compounds present at 10-4 M was determined.For detailed procedure, see text.uptake of each radioactive chemical was severely inhibited by the unlabeled homologues while other compounds inhibited uptake to a much lesser extent.This suggested that these high affinity transport mechanisms were relatively specific for each substance studied.However there was considerable overlap and thus less selectivity between the dopamine and serotonin uptake systems.

( b) Localization of the transport mechanism
The ability of several morphological fractions to take up labeled compounds is shown in Table IV.The synaptosomal fraction was generally the most active toward compounds examined.This fraction contained the bulk of the total activity of the tissue.In the case of embryonic uptake, only GABA had an uptake that was active enough to allow cell fractions to be accurately tested (Table V).Even in the 11-day-old embryo, the nerve ending fraction was the most active.The nuclear fraction often had considerable activity; since this fraction was not further purified, it contained many unbroken cells and tissue fragments.The nerve ending fraction may thus represent the predominant site of uptake by brain homogenates.

High affinity uptake capacity of morphological fractions from chick optic lobes 8 days after hatch
Figures in parentheses give the specific activity of uptake relative to the original tissue homogenate.Standard errors of the mean are given.

Glutamate
GABA Serotonin 59 ± (1.0) 957 ± 7 (1.0) 149± 1 (1.0) 166 ± 2 (2.8) 337 ± 4 (0.35) 93 ± 4 (0.62) 4.3 ± 0.1 (0.07) 15.7 ± 0.3 (0.02) 24± 1 (0.16) 142 ± 20 (2.4)     ed at 0 °C in the presence of glucose and Na+ under our usual incubation conditions, was largely due to residual uptake activity rather than postsynaptic binding.The uptake of GABA was much greater than that of other compounds, and the kinetics of this process was examined in greater detail.Uptake was linear for at least 5 min at 37 °C and was proportional to tissue concentration up to the concentrations of optic lobe homogenates that we routinely used (see Methods).A series of incubations were carried out over a wide range of GABA concentrations, and Lineweaver-Burk curves26 were plotted (Fig. I).A high affinity uptake of GABA was found, with an apparent Km of 6.25 x lQ-6 M and a V max of 6.45 x 10-7 moles/min/g tissue.In addition, using higher GABA concentrations, a low affinity uptake mechanism was found.This had an apparent Km of 3.33 x 10-2 M and V max of 4.17 x 10-6 moles/ min/g tissue.

Figures in parentheses give
In view of the high Km of the lower affinity transport system, the major uptake pathway for physiological concentrations of GABA is the high affinity mechanism.Taking the different V max values into consideration, we have calculated that for 0.1 mM GABA, over 95 % of any uptake would be due to the high affinity system.Levi 2 5 reported embryonic chick brain to possess two mechanisms of differing affinities for GABA uptake.Only the high affinity system was present in 15-day-old chicks, and this had an apparent Km of 0.72 x 10-a M. Since the lowest GABA concentration used by Levi was 2 x 10-5 M, the very high affinity mechanism reported here may have not been observable under this conditions.

( c) Development of transport capacity
The uptake of a series of compounds during ontogenesis by various tissue homogenates was determined (Figs.2-6).The 6 day incubated embryos had a very small ability to accumulate isotopes.Subsequently, each region had a distinctive developmental profile.The uptake of GABA reached maximal values at hatch, while serotonin, dopamine and choline uptake mechanisms continued to increase significantly after hatch.Choline uptake was not detectable before the 18th day of incubation while GABA, glutamate and serotonin uptake were pronounced at 11 days.
Uptake mechanisms in the optic lobes and cerebral hemispheres generally developed in parallel except that the uptake of dopamine was much greater in the hemispheres than in any other brain region.After 11 days of incubation, the cerebellum showed no major increase in its capacity to transport any chemical examined.There was no clear uptake of choline, and the uptake of serotonin gradually diminished to background values in the 8-dayold chick.Cerebellar glutamate uptake also declined after the 11th day of incubation.No major accumulation of serotonin, dopamine or choline was seen in the retina of any stage.Retinal capacity to accumulate GABA increased steadily, ultimately reaching a value of about 25 % of the corresponding figure for the cerebral hemispheres.

DISCUSSION
A problem concerning neurotransmitter uptake studies is in the location of the active process since a Na+ dependent high affinity uptake mechanism may exist in glia as well as neurons15.The uptake of labeled isotopes that we have observed is at least in part neuronal since it takes place in cerebral areas of the 8-day-old embryo where glial cells are almost completely absent.In addition, the concentration of the uptake process within the synaptosomes fraction suggests its preponderantly synaptic location.One cannot completely eliminate the possibility of glial elements being present in the synaptosomal fraction which may contain membrane components that are not readily identifiable.The neuronal site of uptake is further supported by the inhibition of the reaction by neuropharmacological agents thought to act at nerve endings.The possibility of uptake by whole neuronal or glial cell bodies is minimal since homogenization breaks up intact cells.Iversen and Schon 1 7 reported 71 % of GABA uptake into substantia nigra to be into nerve terminals and only 2 % of uptake was glial.This uptake is depressed by the interruption of the nigro-striatal pathways and is thus thought to be synaptic 4 0.
A further question concerns the specificity of uptake.Such specificity has been reported (see Introduction) and is supported by our data on the differential developmental course of uptake of various radioactive compounds.Furthermore, there is a close parallel between the uptake of the compounds reported here and their concentration in avian brainl,33.Thus, serotonin, which is taken up to the greatest extent by the forebrain, is found in maximal concentrations in that region.The optic lobes, which take up choline to the largest degree, have the greatest endogenous acetylcholine levels.Choline uptake has been reported to be an early index of cholinergic innervation38 and is largely neuronal in chick embryos2 9 .This mechanism rather than endogenously synthesized choline may be the major source of substrate for synaptic acetylcholine synthesis13.Dopamine uptake and also its concentration are both over 4-fold greater in the forebrain than in any other area examined.This may be related to the fact that the avian cerebral hemispheres are largely homologous with the mammalian basal ganglia, which contain many dopamine neurons39.The relation between the uptake of a putative neurotransmitter and its concentration suggests that neurons synthesizing a specific neurotransmitter also possess a high affinity uptake mechanism for that transmitter.There is, however, data suggesting that norepinephrine and dopamine are taken up by both norepinephrine and dopamine synapses.The much greater concentration of norepinephrine than dopamine in optic lobes 4 suggests a preponderance ofnorepinephrine synapses in this region.Catecholaminergic uptake mechanisms matured relatively late, and so considerable posthatch elaboration of catecholamine neurons may occur.Vernadakis4 1 has showed that norepinephrine levels in several chick brain regions rise steeply after hatch.These neurons may then be more subject to environmental influences than systems which are largely developed at hatch.In the cat, the main increase in synaptic number of areas primarily or secondarily related to vision occurs while the visual system is in uses.
Considerable postnatal development of cerebral norepinephrine uptake has been reported in the rat7.In this case there was also a close relation between endogenous norepinephrine concentration and uptake capacity.However, the uptake of norepinephrine into chick cerebral hemisphere slices does not increase after the 15th day of incubation is.
The mechanism for the uptake of GABA is well developed in the new-hatched chick and declines somewhat in older birds.This is a similar result to that of Kelly et al.19 who found GABA uptake into mouse brain slices to decline during maturation.The uptake of GABA was detectable early in embryogenesis.Since GABA is thought to be an inhibitory neurotransmitter and since brain removal causes increased motility of the chick embryo 14 , it may be that the development of inhibitory pathway precedes that of excitatory ones.The development of GABA uptake in the optic lobes roughly parallels the rise of the levels of GABA and glutamic acid decarboxylase 3 5.
The retina of the new-hatched chick only takes up GABA to a marked extent.The location of this process has previously been shown to be largely neuronal (retinal ganglion and amacrine cells) rather than glia12s.There are conflicting data as to whether this uptake is neuronal or glial in mammals9,31.Our homogenization conditions would tend to favor nerve endings rather than cell body uptake, whereas the other studies were carried out with intact retinae.Although we found no differences in the GABA accumulation of light or dark adapted retinae, Lam and Steinman24 have reported that GABA uptake is increased by darkness in the goldfish retina.In addition, the GABA concentration of dark adapted frog retina is depressedll.The release of GABA by chick retina is stimulated by light39.However, several other retinal neurotransmitter candidates and their related enzymes may be also influenced by light.These include dopamine20, taurine32 and acetylcholine10.
The quantitatively predominant cerebellar uptake mechanism was that for GABA.All other compounds were very poorly taken up.This is consistent with the largely inhibitory functions of inputs to this region.However, GABA uptake capacity was well below that of the cerebral hemispheres or optic lobes.Therefore, the major neurotransmitter of the cerebellum may be an unknown compound whose uptake we have not examined.The transient ability of the immatme cerebellum to accumulate serotonin may reflect the appearance of a cell species that is only temporarily present.It may be significant that cerebellar serotonin concentrations are maximal in the 15day-old embryo and decline sharply thereafter41.Programmed neuronal death takes place to a considerable degree during the maturation of the chick brain6.
Profiles of neurotransmit'er uptake development have been expressed per unit wet weight.In view of the gradual decrease in water content during brain maturation3.irates of increase would be steeper if expressed on a dry weight basis.The major onset of transport mechanisms is at the time of cell differentiation rather than proliferation.The ability to transport postulated neurotransmitters may be a valid index of the selective maturation of distinct neuronal species.

Fig. l .
Figures in parentheses give the specific activity of uptake relative to the original tissue homogenate.Standard errors of the mean are given.