Effects of Glutamate and γ-Aminobutyric Acid on Spontaneously Active Intraocular Spinal Cord Graft Neurons

Pieces of fetal rat lumbar spinal cord were transplanted into the anterior eye chamber of adult rat hosts. At least seven months later, extracellular single-unit recordings of spontaneously active graft neurons were made prior to and during the superfusion of either glutamate or γ-aminobutyric acid (GABA). Superfusion of glutamate produced an increase (five cells), decrease (three cells), or had no effect (two cells) on the firing rate of neurons tested. Superfusion of GABA decreased the firing rate of all twelve neurons tested, while superfusion of the GABA receptor antagonist bicuculline increased the firing rates of all eight neurons tested. The latency and magnitude of the responses to glutamate and GABA were not related to depth of the recording electrode below the graft surface. Together, these data suggest that the intraocular spinal cord graft is suitable for the in vivo study of GABA and glutamate neuropharmacology.


INTRODUCTION
Fetal rat spinal cord tissue will develop in the anterior eye chamber of adult rat hosts/15,29,39/. The grafts are quickly vascularized by the host iris, increase in VOL. 2, NO. 2,1991 102 J.G. Broton, R.P. Yezierski First, the tissue is not dissociated prior to transplantation, resulting in the preservation of some neuronal organization. We have shown that neurons in intraocular spinal cord grafts are in many ways similar in appearance and organization to neurons in the intact spinal cord /6/. This contrasts with cultured spinal cord neurons, which develop neurites /32/ that do not resemble dendrites and axons in situ. Second, intraocular spinal cord grafts are nourished by the host's blood supply via a dense capillary network/6/ originating from the ground plexus of the host iris /15/. This is in contrast to the artificial medium which is fed to cultured spinal cord neurons or slice preparations. Third, intraocular grafts provide a means to study transplanted tissue for long time periods after manipulations of blood constituents or anterior eye chamber fluid. A previous study has described changes in the growth of spinal cord grafts after intravenous administration of thyrotropin releasing hormone to the adult hosts /19/. Also, colchicine has been injected into the anterior eye chamber several weeks after tissue transplantation in order to visualize neuropeptides in graft cell bodies /16/. These studies indicate that intraocular grafts provide a flexible model system to study neurotransmitter actions of developing tissue transplanted to the eye from different regions of the central nervous system, including the spinal cord. Previous electrophysiological studies have shown that spinal cord graft neurons respond to norepinephrine /14/and serotonin/18/. Two other putative neurotransmitter substances present in the spinal cord are glutamate and GABA. The present study was undertaken to evaluate the action of these substances on neuronal activity in intraocular spinal cord grafts.
Glutamate is an excitatory amino acid which is present both in the dorsal and ventral gray matter of the spinal cord/5,12/. Glutamate

Intraocular Grafting
The intraocular gratis used in this study were transplanted at the same time as the grafts described in a previous report/6/. The  Neurons tested for the effects of drug superfusion had regular spontaneous firing rates when calculated over a 30-90 sec period (see Results below). A drug was considered effective if the average firing rate of a neuron (calculated from a 30 sec pre-drug control period) increased or decreased by more than 20% during a 30, 60, or 90 see period after drug superfusion. The onset of the drug effect was determined by the bin-by-bin analysis of peristimulus time histograms. The magnitude of drug effects was calculated by comparing the neuronal activity during the 30 sec control period to that of the 30 sec period after the onset of each drug effect. Any changes in firing rate of the recorded neuron after drug superfusion are presumed to be the result of drug action on the entire population of graft neurons rather than a direct effect on the neuron under study.

RESULTS
The results described below are based on single-unit recordings of spinal cord graft neurons from twelve intraocular grafts. Three grafts were used to study the patterns of spontaneous activity of these neurons; no drugs were superfused onto these grafts. Four different grafts were used to determine the minimum concentrations of glutamate, GABA, and bicuculline needed to produce effects on graft neurons. Finally, five grafts were used to study in detail the effects of drug superfusions on spontaneous activity. An average of four neurons was recorded from each of the twelve grafts used in this study.

Spontaneous Activity
All neurons in this study were identified by the presence of spontaneous activity. This activity was easily distinguishable from that of neurons that were ir.ritated or killed by the advancing electrode, in that the former displayed action potentials which slowly increased in amplitude as the microelectrode was lowered into the graft, then remained at a relatively constant amplitude during the time they were studied. The s.pontaneous activity of 14 Figures 3 and 4. Two other spontaneously active neurons, Type C, fired with bursts of 3-5 spikes every 2-5 sec. This type of neuron was not tested for effects of drug superfusion.

Drug Concentrations
The concentrations of glutamate, GAB.A, and bicuculline used in this study werc determined in initial experiments where different drug concentrations were superfused while recording single-unit or multi-unit activity. Data from one experiment where different GABA concentrations were superfused are illustrated in Figure 2. GABA effects were evaluated after superfusion of 50/aM glutamate ( Fig. 2A). Subsequent superfusion of 3 mM GABA (Fig. 2B) resulted in an 11% decrease in activity when the 30 sec pre-GABA baseline period was compared to the 90 sec period after GABA superfusion. Superfusion of 15 mM GABA resulted in a 13% decrease in activity ( Fig. 2C), while superfusion of 30 mM GABA (Fig. 2D) decreased activity by 46% compared to pre-GABA baseline. The concentration of bicuculline used in this study was similarly determined in initial experiments evaluating drug effects on single-and multi-unit activity. In each case, superfusion of 1 mM bicuculline did not change the spontaneous firing, while subsequent superfusion of 2 mM bicuculline resulted in increased neuronal activity.

Effects of Glutamate and GABA Superfusions
The effects of superfusion of L-glutamate (50/aM), GABA (3 mM or 30 mM), or the GABA receptor antagonist bicuculline (2 mM) were evaluated on 22 neurons. Multiple and/or repeated superfusions were made on 11 neurons. The number of individual neurons that were tested for drug superfusion effects and the number of neurons that increased or decreased their basal firing rates by at least 20%, or were not affected, are shown in Table 1.
The effects of glutamate were evaluated on ten neurons. Glutamate superfusion increased the firing rates of five neurons. Figure 2A shows the effect of glutamate superfusion on a Type A neuron. The baseline firing rate of this neuron was 9.0 Hz. The mean firing rate for the 60 sec period after superfusion of 106 J.G. Broton, R.P. Yezierski, and/. Seiger  The firing rate returned to near baseline level by 100 see after drug superfusion. When the data were analyzed for all neurons displaying an increase in activity after glutamate superfusion, it was found that the latency to these increases varied from 1-35 sec (mean 14.2 sec) and the magnitude of the increase varied from 165-379% of the mean baseline activity. Increases in activity after glutamate superfusion were observed on both Type A (n--3) and Type B (n=2) neurons.
Glutamate administration resulted in a decrease in activity for three neurons, even though in two cases there were increases in the activity of neurons 107 recorded below the discriminator window. An example of this effect on a Type B neuron is illustrated in Figure 3. The neuron then resumed firing, initially in short bursts, then at a near-baseline level, at which time GABA was again administered. The second superfusion of GABA resulted in a longer cessation of activity lasting 110 sec, followed by a longer period of bursting activity before the neuron resumed firing at near-baseline level.
The effects of GABA superfusion on spontaneous activity were tested on four Type A and eight Type B neurons. The latency of effects observed on this group of neurons varied from 1-27 see (mean 9.2 sec). The magnitude of the inhibitory effects of GABA, calculated as above, ranged from 20-98%. Silent periods similar to those seen in Figures 4 and 5, and lasting at least 10 sec, were seen in 9/12 tested neurons after GABA superfusion. The GABA receptor antagonist bicuculline increased the firing rate of all eight neurons tested. Two different types of effects were observed following bicuculline administration. In four cases, the excitatory effect of bicuculline lasted less than 60 see and was followed by a decrease in unit activity. Figure 4 illustrates an example of this type of bicuculline effect.
In four other cases, the effect of bicuculline was long-lasting and included the appearance of bursts of unit firing superimposed on a neuron's regular pattern of activity. An example of this type of effect is shown in Figure 5. This effect occurred following the administration of GABA (30 mM) which produced a long-lasting (408 see) inhibition of the cell's spontaneous discharge. Superfusion of bicuculline (2 mM) brought about a return of the cell's discharge to a level 255% above the pre-GABA control level. After this initial burst of activity, which lasted nearly 200 see, the activity pattern of the cell consisted of periodic bursts of spikes superimposed on a background activity level similar to that observed during the pre-drug control period. After a second application of GABA, again there was a complete cessation of the cell's discharge lasting 184 see.
Recovery from this second GABA administration consisted of an initial return of the bursting activity followed by a later return to the cell's pre-drug discharge pattern.

Electrode Depth and Superfusion Effect
An attempt was made to determine if the variations in latency and magnitude of effects described above were due to differences in the depth of the recording electrode below the graft surface. For the 10 neurons where glutamate was superfused, the electrode depth ranged from 159-634/am (mean 399/am). No significant correlation was found when electrode depth was compared to the type of glutamate effect (i.e., increase or decrease of spontaneous firing rate), the latency to the glutamate effect, or the magnitude of effect. The electrode depths for the 12 neurons studied with GABA ranged from 94-624/am (mean 367/am). Again no significant correlation was found between the electrode depth and either the latency or magnitude of the GABA effect.

DISCUSSION
The technique of drug administration used in this study was chosen as an initial means of assessing the feasibility of studying glutamate and GABA effects on intraocular graft neurons. We were aware of potential problems of interpretation using this technique. Changes in the temperature of the chamber fluid surrounding the graft might change the spontaneous 108 J.G. Broton, R.P. Yezierski, and . Seiger firing rate of a neuron independent of the effect of superfused drugs. Furthermore, displacement of fluid by the drug might move the graft, thereby decreasing the amplitude of the recorded unit below the discriminator window. Initial experiments indicated that these were not serious problems. First, the monitored temperature of the solution did not noticeably change after drug superfusion. Further, in some instances drug superfusion did not have an effect (Table 1). Therefore it is unlikely that changes in fluid temperature produced spurious results. As for the possibility of pressure changes reducing the amplitude of recorded neurons, the observed amplitudes of action potentials did not change after drug superfusion. Similarly, after GABA-induced silent periods, action potentials eventually resumed which were the same amplitude as those observed pre-drug. Therefore the drug superfusion technique was determined to be appropriate for this initial study of glutamate and GABA effects on intraocular graft neurons.
Many neurons in intraocular spinal cord grafts were found which displayed repeated, often very regular rates of discharge. It is thought that this activity is generated within the graft itself, rather than as the result of innervation from the iris /15,29/. In this regard, spontaneously active neurons have also been observed in cultured spinal cord neurons in vitro /21,31,32/. In the present study we used this activity to identify neurons prior to superfusion of glutamate, GABA, and the GABA antagonist bicuculline. Superfusion of the graft with glutamate had an excitatory effect on five of ten neurons tested. The increase in firing rate is in accordance with studies of spinal cord neurons, where glutamate was iontophoresed/8,10,37/ or applied to a spinal cord slice preparation/22,33/. With three of ten tested neurons, superfusion of glutamate resulted in decreases in spontaneous firing rate. It is unlikely that this effect is due to a direct inhibitory action of glutamate, although such an effect has been demonstrated previously for cerebellar interneurons /38/ studied in vitro. Rather, the decrease in firing rate may be due to glutamate activation of GABA-ergic interneurons which influence N  Neurons in the present study were grouped into three classes according to their pattern of spontaneous activity. This has not been done in previous studies in which responses of intraocular spinal cord graft neurons were studied /14,15,18,29/. In the present study only Type B neurons, which had somewhat irregular rates of spontaneous activity, either were unaffected or decreased their firing rate after glutamate superfusion. It is not known whether different neurons types described in the present study represent neurons with different morphological characteristics. We have previously shown that graft neurons vary considerably in size and dendritic organization /6/. Further studies of neuronal responses to similar superfusions combined with intracellular marking techniques could help determine if there is a relationship between neuronal morphology and the effects described in the present study.
The variability in the latency and magnitude of effects of GABA superfusions and the independence of these parameters on the depth of the recording electrode, suggest that neurons in spinal cord grafts are affected in different ways, and to different degrees, by GABA. The superfusion technique used in this study precluded any conclusions being made regarding the preor post-synaptic action of GABA. Future studies where GABA and GABA receptor antagonists are administered iontophoretically while recording neuronal activity are needed to resolve this issue.
This initial study of glutamate and GABA sensitivity was carried out on grafts of immature rat spinal cord transplanted to the anterior eye chamber of adult rat hosts. An important question regarding the results concerns changes in drug sensitivity when neurons are in the presence of tissue that can provide afferent input and/or an efferent target. A major target of spinal cord projection neurons is the mesencephalon /40/. Presently we are studying the effects of cografting fetal mesencephalon with spinal cord neurons to determine the effects of this target tissue on the physiology and pharmacology of transplanted spinal cord neurons.
In conclusion, the results of the present study have shown that spinal cord graft neurons respond to glutamate and GABA. It has previously been shown that neurons in similar grafts display morphological and organizational characteristics similar to those found in situ /6/, and that graft neurons contain several neuropeptides present in the in situ spinal cord /16/. Further, intraocular graft neurons respond to norepinephrine /14/ and serotonin /18/. Together, these data suggest that the intraocular spinal cord graft is a useful model for the study of the physiology and pharmacology of simple neural networks in the developing and mature spinal cord. This model may be useful for the study of phenomena such as denervation supersensitivity, the influence and interaction of multiple transmitter systems on single neurons, and pharmacological changes that occur during the development of ascending and descending spinal pathways. 110 J.G. Broton, R.P. Yezierski, and/,. Seiger for technical assistance, and Theresa Whittingham for secretarial assistance. This work was supported by NIH Grant NS19509 to RPY and by The Miami Project to Cure Paralysis.