THE ANXIOMIMETIC PROPERTIES OF PENTYLENETETRAZOL IN THE RAT

Investigation of the biological basis of anxiety is hampered by the lack of an appropriate animal model for research purposes. There are no known drugs that cause anxiety in laboratory animals. Pentylenetetrazol (PTZ) produces intense anxiety in human volunteers (Rodin, 1958; Rodin and Calhoun, 1970). Therefore, it was the major objective of this dissertation to test the hypothesis that the discriminative stimulus produced by PTZ in the rat was related to its anxiogenic action in man. It was also an objective to suggest the neurochemical basis for the discriminative stimulus property of PTZ through appropriate drug interactions. In an operant procedure of lever pressing on a FR 10 schedule of food reinforcement, male hooded rats were trained to respond with a lever on one side of a food cup 15 min following a 20 mg/kg PTZ injection and to respond with a lever on the alternate side 15 min following a 1 ml/kg saline injection. All of the rats learned this discrimination reliably in a mean of 30 training sessions. The discriminative stimulus produced by PTZ was doseand time-dependent. The anxiogenic stimulants cocaine, R05-3663 and strychnine generalized to the PTZ discriminative stimulus whereas yohimbine partially generalized. The discriminative stimulus produced by cocaine, R05-3663 and yohimbine was antagonized by the anxiolytic drug, diazepam. The discriminative stimulus produced by cocaine was not antagonized by haloperidol. The non-anxiogenic psychomotor stimulants ~-amphetamine, methylphenidate or caffeine did not generalize to ~he PTZ discriminative stimulus. Bemegride was the only convulsant drug tested that generalized to the PTZ discriminative stimulus. Picrotoxin and 3-mercaptopropionic partially, but not significantly, generalized to the PTZ discriminative stimulus but bicuculline, garnma-hydroxybutyrate or nicotine did not generalize. The PTZ discriminative stimulus was dose-dependently antagonized by benzodiazepine-type, barbiturate-type, propanediol carbamate-type anxiolytics as well as valproic acid. Tolerance did not develop to antagonism of the PTZ of discriminative stimulus by diazepam or chlordiazepoxide. The discriminative stiIT'ulus produced by PTZ was not antagonized by an anticonvulsant or other central nervous system depressants that are not anxiolytics. There was a significant correlation between the potency of drugs effective in antagonizing the PTZ discriminative stimulus and their effective doses in a conflict test of anxiety as well as their clinically effective doses. The GABA antagonist ROS-3663 generalized to the PTZ discriminative stimulus whereas picrotoxin and 3-mercaptopropionic acid partially generalized. The PTZ discriminative stimulus was antagonized by the GABA mimetic, valproic acid. The glycine antagonist, strychnine, generalized to the PTZ discriminative stimulus. Neither acetylcholine nor serotonin agonists or antagonists generalized to or antagonized the PTZ discriminative stimulus. The catecholamine agonist, cocaine, generalized to the PTZ discri~inative stimulus whereas yohimbine and apomorphine partially generalized. However, neither a-amphetamine nor methylphenidate generalized to the PTZ discriminative stimulus and catecholamine receptor antagonists did not antagonize the PTZ discriminative stimulus. Generalization to the PTZ discriminative stimulus by anxiogenic drugs and lack of generalization by non-anxiogenic psychomotor stimulants or convulsants as well as antagonism of the PTZ discriminative stimulus by anxiolytics but not non-anxiolytic anticonvulsant or depressants supports the hypothesis that the discriminative stimulus produced by PTZ in the rat is related to its anxiogenic action in man. Generalization to the PTZ discriminative stimulus by GABA antagonists and antagonism by GABA agonists but lack of generalization to or antagonism by drugs affecting other neurotransmitter systems suggests that the PTZ discriminative stimulus might be ·mediated through decreased GABA neuronal ac-

The discriminative stimulus produced by cocaine was not antagonized by haloperidol.
The non-anxiogenic psychomotor stimulants ~-amphetamine, methylphenidate or caffeine did not generalize to ~he PTZ discriminative stimulus. Bemegride was the only convulsant drug tested that generalized to the PTZ discriminative stimulus. Picrotoxin and 3-mercaptopropionic partially, but not significantly, generalized to the PTZ discriminative stimulus but bicuculline, garnma-hydroxybutyrate or nicotine did not generalize. The PTZ discriminative stimulus was dose-dependently antagonized by benzodiazepine-type, barbiturate-type, propanediol carbamate-type anxiolytics as well as valproic acid. Tolerance did not develop to antagonism of the PTZ of discriminative stimulus by diazepam or chlordiazepoxide. The discriminative stiIT'ulus produced by PTZ was not antagonized by an anticonvulsant or other central nervous system depressants that are not anxiolytics. There was a significant correlation between the potency of drugs effective in antagonizing the PTZ discriminative stimulus and their effective doses in a conflict test of anxiety as well as their clinically effective doses. The GABA antagonist ROS-3663 generalized to the PTZ discriminative stimulus whereas picrotoxin and 3-mercaptopropionic acid partially generalized. The PTZ discriminative stimulus was antagonized by the GABA mimetic, valproic acid. The glycine antagonist, strychnine, generalized to the PTZ discriminative stimulus.
Neither acetylcholine nor serotonin agonists or antagonists generalized to or antagonized the PTZ discriminative stimulus.
The catecholamine agonist, cocaine, generalized to the PTZ discri~inative stimulus whereas yohimbine and apomorphine partially generalized. However, neither a-amphetamine nor methylphenidate generalized to the PTZ discriminative stimulus and catecholamine receptor antagonists did not antagonize the PTZ discriminative stimulus. Generalization to the PTZ discriminative stimulus by anxiogenic drugs and lack of generalization by non-anxiogenic psychomotor stimulants or convulsants as well as antagonism of the PTZ discriminative stimulus by anxiolytics but not non-anxiolytic anticonvulsant or depressants supports the hypothesis that the discriminative stimulus produced by PTZ in the rat is related to its anxiogenic action in man. Generalization to the PTZ discriminative stimulus by GABA antagonists and antagonism by GABA agonists but lack of generalization to or antagonism by drugs affecting other neurotransmitter systems suggests that the PTZ discriminative stimulus might be ·mediated through decreased GABA neuronal activity.  TABLE   1 Response emitted with the incorrect lever prior to lever selection during acquisition of the Pentylenetetrazol (2Q mg/kg) -Saline Discrimination. · 2 Effect of varying pentylenetetrazol treatment time on lever selection in rats trained to discriminate pentylenetetrazol from saline.
3 Generalization tests with rats trained to discriminate between pentylenetetrazol (20 mg/kg) and saline.

Generalization Tests:
Effect of test drugs on response rates in rats trained to discriminate between pentylenetetrazol (20 mg/kg) and saline.
5 Antagonism tests with rats trained to discriminate between pentylenetetrazol (20 mg/kg) and saline .
6 Antagonism Tests: Effect of test drugs on response rate in rats trained to discriminate between pentylenetetrazol and saline. 7 Antagonism Tests with diazepam and chlordiazepoxide after their chronic administration .
8 Tests for diazepam and haloperidol antagonism of the discriminative stimuli produced by cocaine yohimbine and ROS-3663 in rats trained to discriminate between pentylenetetrazol (20 mg/kg) and saline .
9 Response rates during tests for diazepam and haloperidol antagonism of the discriminative stimuli produced by cocaine and ROS-3663 in rats trained to discriminate between pentylenetetrazol and saline  (Rickels et al., 1978). Anxiety can be defined as that experience or feeling of an unpleasantness or impending doom (Matz and Nash, 1979).
Although we suffer from and can define anxiety we do not understand its biological basis. This is partly because investigations of the biological basis of anxiety have been hampered by the lack of an appropriate animal model for research purposes. Presently, conflict paradigms such as those described by Geller and Seifter (1962) and Howard and Pollard (1977) are believed to provide the best animal models of anxiety. These procedures have been used to investigate the biological basis of anxiety as well as the neurochemical mechanisms underlying the anxiolytic action of anti-anxiety drugs (for reviews see Sepinwall and Cook,19 7 8;Lippa ·et al.,19 79) .
However, with regard to elucidating the biological basis of anxiety these studies have not met with great success. In addition, conflict procedures are not without methodological problems that make interpretation of results difficult (Howard and Pollard, 1977) . Therefore, a more appropriate animal model to investigate the biological basis of anxiety is needed.
Pentylenetetrazol (PTZ) has been reported to produce intense anxiety in human volunteers (Rodin, 1958;Rodin and Calhou~, 1970). In a personal communication, Rodin wrote " ... within a matter of seconds [(after he had had himself injected with the drug by another physician)] I experienced catastrophic anxiety ... It was a sense of utter distress and impending catastrophe. There is no doubt that this was one of the most anxiety producing events of my life." Subjective effects of drugs cannot be measured directly in laboratory aniITals, however, it is believed that these effects are reflected in the discriminative stimulus properties of drugs in a number of cases (for review see Lal, 1977;Schuster and Balster, 1977;Colpaert and Rosecrans, 1978).
Therefore it was the major objective of this dissertation to test the hypothesis that the discriminative stimulus produced by PTZ in the rat was related to its anxiogenic action in humans. It was also an objective to suggest a neurochemical basis of the discriminative stimulus property of PTZ in the rat. Irwin and Benucazizi (1966) found that PTZ (1-30 mg/kg, p.o.) significantly facilitated one~trial learning and memory retention of CF 1 mice in a passive avoidance task. This effect occurred whether PTZ was injected before or immediately after the training trial and was greater than that observed with strychnine or picrotoxin.
Using a simple Y maze, Hunt and Krivanek (1966) and Krivanek and Hunt (1967) reported that post-trial injections of PTZ (_20 mg/kg) improved learning of a black-white brightness discrimination. Animals treated with PTZ reached the criterion of learning earlier and made fewer errors than did saline treated animals. Grossman (1969) bilaterally injected PTZ (5-10 ug) directly into the hippocamus of rats before as well as after traini~g to perform a brightness discrimination. PTZ facilitated the learning of this discrimination and post-trial injections of PTZ were found to produce a significantly greater effect than pre-trial injections. It was suggested that PTZ facilitated learning by activating neural pathways concerned with memory consolidation processes. Hunt and Bauer (1969) reported that subconvulsive doses (7.5-15 mg/kg) of PTZ, when injected immediately or 15 min following training of a black-white brightness discrimination, facilitated learning. Animals retested 24 h after training performed significantly better than saline treated animals. In a position discrimination task, Hunt and Bauer (1969) reported that PTZ (10 mg/kg) maximally facilitated learning when injected 10 min after training. Bauer (1969) injected subconvulsive doses of PTZ daily for 20 days and reported this dosage regimen to facilitate learning of a shuttle box avoidance or a brightness discrimination task when testing occurred 24 h after the last PTZ injection. Interestingly, Bovet et al. (1966) had reported that whereas strychnine or picrotoxin enhanced shuttle avoidance learning, PTZ (5 or 10 mg/kg) did not. Buckholtz (1974) also reported that PTZ (20 mg/kg) did not facilitate shuttle avoidance learning. Using a wheelturn avoidance procedure, Krivanek (1971) reported subconvulsive doses of PTZ facilitated learning.
For the most part, studies employing subconvulsive doses of PTZ demonstrate that this drug facilitates learning. It has been suggested that PTZ produces this effect by facilitating memory consolidation.

Impairment of Learning
An impairment or loss of memory for events preceding a trauma is referred to as retrograde amnesia. Whereas subconvulsive doses of PTZ are reported to facilitate learning, convulsive doses of this drug produce retrograde amnesia or impair learning. Palfai and Chillag (1971) reported that a convulsive dose (50 mg/kg) of PTZ, when injected up to 20 but not 60 min after training produced retroqrade ar.mesia in mice for a step-through passive avoidance task. The degree to which PTZ produced retrograde amnesia was directly related to the intensity of the foot shock indicating that the nature of the original traumatic experience contributed towards the temporal characteristics of the retrograde amnesia. Because electroconvulsive shock had previously been reported to produce retrograde amnesia Palfai and Chillag (1971) suggested that a "massive neural explosion" causes retrograde amnesia. Palfai and Kurtz (1973) reported that this convulsive dose of PTZ injected 15, 30 or 60 min but not 4 hr before a single step-through passive avoidance training impaired retention performance 24 h later. Interestingly, when this dose of PTZ was injected inunediately after the training trial it did not immediately produce retrograde amnesia, but rather the amnesia became apparent 4.5 h following the PTZ injection.
These data indicate that a convulsive dose of PTZ can affect memory for a long (24 h) time after injection. Palfai and Kurtz (1973) suggested that PTZ may produce dissociated memory consolidation and that PTZ did not immediately produce amnesia because the drug state in which memory consolidation took place was still present when the rats were tested. However, when the animals were tested 4.5 h after the PTZ injection a different brain state may have existed that resulted in impaired retention. Kurtz and Palfai (1973) tested for a dissociated or state-dependency hypothesis for the memory impairment produced by PTZ. Using the step-through passive avoidance task , results were suggestive for the state-dependency hypothesis but PTZ produced a general depression of activity that may have confounded this interpretation. Therefore, Kurtz and Palfai (1973) used a discriminated escape paradigm to test for dissociative properties of PTZ. When reversal training and testing occurred under similar physiological states, i.e., both following PTZ injection (PTZ-PTZ) or both in the non-drug state (salinesaline), test performance was unimpaired. However, when PTZ was injected before reversal training but not before testing (PTZ-Saline) or vice versa (Saline-PTZ) test performance was impaired. These data suggest that retrograde amnesia produced by a convulsive dose of PTZ was related to its dis-sociative or state-dependent effects. Furthermore, these data suggest the PTZ seizures may interfere with memory retrieval rather.than memory consolidation.
To determine if convulsions were necessary for PTZ to produce state-dependent learning, Palfai and Kurtz (1976) tested for the ability of a subconvulsive dose (30 mg/kg) of PTZ to produce this phenomenon. Unlike the convulsive dose of PTZ that produced symmetrical dissociation, the subconvulsive dose produced asymmetrical dissociation, that is, whereas training in the non-drug state did transfer to the drug state during testing, training in the drug state did not transfer to testing in the non-drug state. Electroencephalographic (EEG) changes were still evident at the time when training and testing took place after injection of the convulsive dose but not the subconvulsive dose. Therefore, the EEG correlates could explain the asymmetrical dissociation produced by the non-convulsive dose of PTZ.
Whereas it was suggested (Weissman, 1968) that only convulsive doses of PTZ produce retrograde amnesia, van Buskirk and McGaugh (1974) reported that in Ha/ICR mice subconvulsive as well as convulsive doses of PTZ, injected after training of a passive avoidance task, impaired retention of the task. However, in C57 BL/6J mice convulsive doses of PTZ did not impair retention. Not only do these data point out species differences with respect to the effect of PTZ in producing retrograde amnesia, these data also indicate that convulsions are not a prerequisite for PTZ to induce retro-grade amnesia. The data of Essman (1968) and Vinnitsky and Abulzade (1971) was cited by van Buskirk and McGaugh (1979) as supporting the suggestion that seizures are not required for the retrograde amnesia effect of PTZ. Essman (1968) reported that a post-trial convulsive dose (60 mq/kg) of PTZ produce retrograde amnesia even when the convulsions were blocked by lidocaine pretreatment. A similar finding was reported by Vinnitsky and Abulzade (1971) using other anesthetic agents. However, Palfai and Albula (1976a) criticized the data of Essman (1968), pointing out that lidocaine only partially blocked the seizures. Using a passive avoidance conditioning paradigm, Palfai and Albula (1976a) reported that convulsions were necessary for the development of PTZ-induced retrograde amnesia. These authors reported that subconvulsive doses (10 or 30 mg/kg) of PTZ did not produce retrograde amnesia and that animals pretreated with pentobarbital to block PTZ convulsions did not show retrograde amnesia.
Convulsive doses of PTZ have also been reported to produce retrograde amnesia for classically conditioned fear and taste aversions. Palfai and Cornell (1968) (1972) reported that convulsions induced by PTZ produced amnesia for a conditioned taste aversion to a n?vel taste produced by injections of apomorphine. Millner and Palfai (1975) injected PTZ (40 mg/kg) 2 min before, immediately or 3 min after injection of LiCl and reported conditioned aversion to the taste of saccharin. Kessler and Gellhorn (1943) had also reported that a convulsive dose of PTZ disrupted conditioned behavior. Putney and McCoy (1976) found that a subconvulsive dose (10 mg/kg) of PTZ enhanced the ability of subthreshold electroconvulsive (ECS) shock to produce retrograde amnesia.
Whereas ECS administration alone given 60 min after passive avoidance learning did not produce amnesia, injection of PTZ plus ECS produced amnesia.
Possible Chemical Basis of PTZ-Induced Retrograde Amnesia Essman (1968) reported that a convulsive dose (60 mg/kg) of PTZ injected immediately following a single trial to establish a passive avoidance response produced retrograde a~ne sia for the task but did not significantly change the brain ribonucleic acid (RNA) concentration. Therefore whereas the amnesia effect of some agents has been attributed to their ability to decrease brain RNA (for review see Seiden and Dykstra, 1977), retrograde amnesia produced by PTZ could not be correlated with a change in brain RNA. Palfai et al. (1974) reported a reduction in whole brain norepinephrine at 5 min but not one or 24 h after injection of a convulsive dose (50 mg/kg) of PTZ in mice trained in a passive avoidance paradigm. Because the time course of anterograde amnesia reported by Palfai and Kurtz (1973) was similar to the time course of the brain norepinephrine decrease and recovery it was suggested that normal norepinephrine levels in brain during the early phase of memory storage may be necessary for retention. However, convulsions produced by PTZ have been reported to alter the brain levels of several other neurotransmitters (see next section: Neurochemical Action of PTZ) therefore these substances must also be looked at with respect to their possible involvement in mediating the amnestic effect of PTZ before any conclusion can be made concerning norepinephrine. Iuvone et al. (1977) reported that immediate post-training administration of subconvulsive (50 mg/kg) or convulsive (60 mg/kg) doses of PTZ resulted in significant retention deficits on step-through inhibitory avoidance behavior. These doses also inhibited the incorporation of 3 tt-lysine into brain protein during the first 10 min after training and PTZ injection. These data suggest a relationship between inhibition of protein synthesis and PTZ-induced retrograde amnesia. Bookin and Pfeifer (1977) injected lysine vasopressin (_LVP). one hour prior to the acquisition trial and retention trial in a passive avoidance task and reported that LVP facilitated passive avoidance retention and antagonized amnesia for retention of the task produced by PTZ (50 mg/kg).
Because PTZ has been proposed to exert its amnestic effect by interfering with retrieval process  it was proposed that LVP may exert its anti-PTZ effect by fa-cilita~ing retrieval processes or blocking the PTZ-induced interference with retrieval.
PTZ-Induced Amnesia as a Tool to Screen Anti-Petit Mal Drugs Clincke and Wauquier (1979)  Pentylenetetrazol (PTZ) has been the most extensively studied analeptic, however its mechanism of action is poorly understood. Good reviews of the early literature (Hildebrandt, 1937;Hahn, 1960) and the chemistry of the tetrazole derivatives (Benson, 1947) are available. The present review will concern the effect of PTZ on CNS acetylcholine, GABA and monoamine metabolism as well as the effect of manipulation of brain monoamine levels on PTZ seizure threshold.

Effect of PTZ on Acetylcholine
Acetylcholine Levels Giarman and Pepeu (1962) sacrificed rats 12 minutes after a convulsant (75 mg/kg) dose of PTZ and reported a significant (23%) reduction in whole brain acetylcholine. Beani et al. (1969) sacrificed guinea pigs 30 sec., 3 min. and 6 min. after convulsions produced by PTZ (80 mg/kg) and measured total brain acetylcholine. The animals sacrificed 6 minutes post PTZ were almost dead at sacrifice with clear signs of post-seizure neurodepression. Determination of acetylcholine content in brains of guinea pigs sacrificed at 30 seconds or 3 minutes post-PTZ showed that the total amount of acetylcholine was significantly below control values. The reduction in brain acetylcholine produced by PTZ concerned only bound ~cetylcholine not free acetylcholine.
In contrast to the reports of Giarman and Pepeu (1962) and Beani· et al. (_1969),    Beleslin et al. (1965) found that intravenous infusion of PTZ (20-80 mg/kg) produced a dose-dependent increase in the output of acetylcholine into the cerebral ventricles. Acetylcholine output also increased (2-7 fold) from the cerebral cortex. Intraperitoneal injection of PTZ (150 aRd 300 mg/kg) produced a 2.9 and 7.5 fold increase in the release of acetylcholine from the cerebral cortex of anesthetized rats (Hemsworth and Neal, 1968). The increased release of acetylcholine was nearly maximal during the first 30 minute period of collection after injection and did not appreciably decrease over a period of 3 hours. The increase in release of acetylcholine was associated with increased electrical activity of the cerebral cortex.
Gardner and Webster (1973) found that intraperitoneal or intravenous administration of PTZ produced a dose-dependent ++ increase in Ca dependent acetylcholine efflux from the cerebral cortex of anesthetized rats which was proportional to EEG activation and convulsive activity. A single intraperitoneal injection (250 mg/kg) produced a 12-fold increase in acetylcholine efflux together with convulsive EEG activation. Trimethadione reduced the PTZ-induced convulsive activity, EEG activation and acetylcholine release. Phenobarbital, however, reduced the PTZ-induced EEG and convulsive activity but left the acetylcholine release relatively unaffected. Therefore the effect of PTZ on acetylcholine efflux may not be related to its convulsant effect.
Assuming that increased release of acetylcholine from brain leads to a reduction in its acetylcholine content the studies demonstrating PTZ-induced acetylcholine release are in agreement with the reports of Giarman and Pepeu (1962) and Beani et al. (1969) demonstrating a PTZ-induced reduction in brain acetylcholine.
Acetylcholinesterase Mahon and Brink (1970) reported that PTZ produced a concentration-dependent (Ki= 4.7 x 10-3 M) competitive inhibition of acetylcholinesterase in crude homogenates of rat brain. These authors point out, however, that kinetic constants calculated from their data indicate that the quantity of PTZ necessary to cause 50 percent inhibition of acetylcholinesterase in vitro is much higher than that amount likely to be present in the brain after administration of a convulsant dose of the drug. Furthermore inhibition of acetylcholinesterase by PTZ would elevate acetylcholine which is contradictory to existing data (Giarman and Pepeu, 1962;Beani et al., 1969).  administered PTZ (160 mg/kg) subcutaneously to frogs. Twenty minutes after PTZ administration, when convulsive symptoms were present, the animals were killed and spinal cord acetylcholinesterase activity was measured. No significant difference was found between controls and animals treated with PTZ.  suggested that the difference between the effect of PTZ on acetylcholinesterase in mammalian brain homogenates and amphibian spinal cord in vivo could reflect a higher resistance of the amphibian enzyme to PTZ.

Choline Uptake
When rats were sacrificed immediately after the onset of the . flexor stage of the convulsion, Simon et al. (1976) reported that the intravenous administration of PTZ (75 mg/ kg) produced a 45% increase in sodium-dependent high affinity choline uptake inrat hippocampal synaptosomes. This increase was not found, however, when cholinergic afferents to the hippocampus were interrupted by septal lesion prior to PTZ administration. Injection of a lower, non-convulsant (10 mg/kg) dose of PTZ did not result in a change in choline uptake.
Addition of PTZ (10-4 M) to hippocampal synaptosomal preparation from saline treated animals did not alter high affinity choline uptake . Because in vitro addition had no effect, Simon et al. (1976) suggested that the effect of PTZ is not a direct one synaptosomes but may be due to an increase in impulse flow.
PTZ increased only the Vmax and not the Km of high affinity choline uptake . PTZ is not al- suggesting that the alteration in uptake is not a secondary effect consequent to a change in choline acetyltransferase . Jenden et al. (_1976) and Klemm and Kuhar (1979) also reported that convulsant doses of PTZ activated high affinity choline uptake in hippocampal synaptosomes. Choline uptake rapidly returned toward normal in post-mortem tissue indicating the importance of measuring this process immediately after sacrificing (Klemm and Kuhar, 1979).
In summary, results of experiments investigating the effect of PTZ on acetylcholine function suggest that PTZ activates cholinergic neurons. Such an activation results in acetylcholine release, choline uptake and decreased brain acetylcholine levels.
EFFECT OF PTZ ON GABA GABA Levels Kamrin and Kamrin (1961) sacrificed mice during the tonic extension component of a PTZ-induced seizure and reported no difference in the concentration of brain GABA from control treated mice. There was also no difference between the cerebral concentration of glutamic acid, glycine, taurine or aspartic acid in PTZ or control treated mice (Kamrin and Kamrin, 1961). Tews et al. (1963) failed to detect any change in brain GABA concentration of dogs following tonic-clonic seizures produced by intravenous administration of PTZ (40 mg/kg). Similarly, Sytinskii and Priyatkina (1966) found no alteration of GABA concentration in rat brain following convulsions produced by PTZ (100 mg/kg). Wood and Peesker (1975) determined mouse brain GABA concentration at the onset of seizures induced by PTZ and found no difference between PTZ treated and saline-treated mice. Nahorski et al. (1970) examined rat brain GABA levels at various times after intraperitoneal administration of a convulsant dose (70 mg/kg) of PTZ and found no change in the levels of this amino acid until tonic extension when a significant rise was observed. A similar increase in GABA levels was also observed when the rats were sacrificed after the convulsion. These autho~s point out that the increase in GABA levels may be due to anoxic conditions which existed at the time of sacrifice.
In contrast to the above reports indicating either no change or an increase in cerebral GABA during convulsions induced by PTZ, Maynert and Kaji (1962) reported that mice given PTZ (85 mg/kg) and sacrificed during the first tonic seizure showed a small but significant decrease in brain GABA concentration. Wood et al. (1966) reported a slight but not statistically significant reduction brain GABA levels when rats were sacrificed 5 minutes after a convulsant dose (60 mg/kg) of PTZ.
GABA Release -4 -6 Johnston and Mitchell (1971) found that PTZ (10 -10 M) inhibited the resting but not the electrically evoked release of [ 3 HJ-GABA from rat brain cerebral cortical slices. By examining the release of [ 3 HJ-GABA from feline cortical slabs, Reiffenstein (1979)  GAD/GABA-T Wood et al. (1966) reported that PTZ (60 mg/kg) did not significantly reduce GAD activity in rat brain when rats were sacrificed 5 minutes after PTZ-induced seizures. Similarly, Syntinskii and Priyatkina (1966) found no alteration in GAD activity produced by PTZ either in vivo or in vitro. Tapia et al. (1969) also reported a lack of effect of PTZ on GAD activity. Sytinskii and Priyatkina (1966) reported that GABA-T activity in rat brain was not changed either by in vivo administration of a convulsant dose of PTZ or by the in vitro addition of PTZ. However, Wood et al. (1966) reported that PTZ (60 mg/kg) produced a significant increase in GABA-T activity.
The increase in GABA-T activity was not correlated, however, with decreased GABA levels .
Effectiveness of GABA and GABA-Mimetic Drugs Against PTZ Hawkins and Sarett (1957) found that orally adrninis-tered GABA protected mice from PTZ-induced convulsions. Wood et al. (1966) reported that GABA but not glycine in a dose of 120 rnrn9les/kg) administered intraperitoneally produced a slight protection against PTZ-induced convulsions in the rat.
However, several investigators (Purpura et al., 1958 a,b;Gulati and Stanton, 1960; were unable to demonstrate an anticonvulsant action of GABA following systemic administration. Kobrin and Seifter (1966) reported that intravenous administration of GABA (25-1000 mg/kg) to 1 day old chicks in which the blood-brain barrier was not complete produced a dose-dependent protection against convulsions induced by PTZ (35 mg/kg) also administered intravenously. Several other w-amino acids including glycine and B-alanine were ineffective. When GABA was administered to older chicks in which the blood-brain barrier was developed it was ineffective in protecting against PTZ-induced convulsions. Schlesinger et al. (1969) found that intracranial injection of GABA protected mice against PTZ-induced seizures.

vulsions.
Similarly, Schecter et al. (1977) reported that a six-fold elevation of brain GABA produced by gamma-acetylenic GABA did not protect mice against seizures induced by PTZ. Frey et al. (1979) examined the role of GABA mechanisms in PTZ convulsions by investigating the effect of inhibition of high affinity GABA uptake on PTZ-induced convulsions. The ability of inhibitors of high affinity GABA uptake blockers to increase the PTZ seizure threshold correlated well with the ability of these drugs to inhibit GABA uptake in vitro.
(-)-Nipecotic acid had the most pronounced effect. This effect of the GABA uptake blockers was not correlated with alteration in brain GABA levels or changes in GAD or GABA-T activities.
The GABA receptor agonist muscimol also elevated the PTZ convulsant threshold and this was accompanied by decreased brain GABA concentration and GAD activity (Frey et al., 1979).

Effectiveness of PTZ Against GABA
Presynaptic inhibition is thought to be mediated by GABA (Barker and Nicoll, 1972;Curtis and Johnston, 1974;Davidoff, 1972;Davidson and Southwick, 1971) and several investigations have been directed towards examining the effect of PTZ on GABA mediated presynaptic inhibition. Boyd et al. (1966) reported that PTZ blocked GABA mediated presynaptic inhibition in the cuneate nucleus. Similarly, Banna and Hazbun (1969) and Hill et al. (1974) found that PTZ reduced GABA-mediated presynaptic inhibition. Nicoll and Padjen (1976) found that PTZ in concentration of >l0-2 M antagonized the action of GABA at primary afferents of the isolated frog spinal cord.
While it appears clear that PTZ blocks GABA-mediated presynaptic inhibition, evidence on the effect of PTZ on GABA me- In contrast to th.e above studies showing lack of an effeet of PTZ on GABA-mediated post-synaptic inhibition, Mac-Donald and Barker (1977) reported that iontophoretically applied pTZ (_0.3Ml competitively inhibited GABA-mediated postsynaptic responses in cultured mammalian spinal cord neurons. Similarly, Scholfield (1979) found that PTZ antagonized the post~·synapt.tc action of GABA on 9uinea pig olfactory cortical neurons.
In summary, while PTZ does not alter brain GABA concentration, GAD or GABA-T activity it has been demonstrated that GABA and some GABA-mimetic drugs antagonize the convulsant action of PTZ. Furthermore, PTZ has been reported to block both pre-and post-synaptic inhibition mediated by GABA.
EFFECT OF PTZ ON MONOAMINE METABOLISM Bonnycastle et al. (1957) reported that a convulsant dose (75 mg/kg) of PTZ failed to alter rat brain serotonin levels.
However, Bertaccini (1959) found that rat brain serotonin concentration was elevated 20-30% when the animals were sacrificed 15-60 minutes after convulsions. Garattini et al. (1960) also reported that PTZ increased the brain serotonin content and that this effect was independent of convulsions. Kato et al. (1967) looked at the effect of acute and 11 day treatment of PTZ (50 mg/kg) in rats. When the animals were sacrificed 5 minutes after an acute injection there was an increase in 5-HT levels. At 2 hours 5-HT levels had returned to normal but were elevated 3-fold at 24 hours. Sacrificing the rats 5 minutes or 24 hours after 11 day PTZ treatment resulted in marked elevation of 5-HT levels at all times. Diaz (1970)  McMillen and Isaac (1978) later reported that the increase in 5-HIAA levels reported to occur after administration of 20 mg/kg PTZ (McMillen and Isaac, 1974) was probably secondary to a reduction in body temperature produced by the drug since ~aintaining body temperature prevented the increase in 5-HIAA produced by this dose of PTZ. Injection of a convulsant dose (40 mg/kg) of PTZ increased 5-HIAA levels and this effect was not prevented by controlling body temperature.
This indicates that convulsant doses of PTZ can increase 5-HT metabolism but after non-convulsant doses the increase in 5-HT metabolism is secondary to the hypothermic effect of PTZ.

Determination of PTZ concentration in plasma and CSF
showed that PTZ was not present to exert an effect at 24 hours, therefore the increased levels of 5-HIAA and HVA at this time was not due to the presence of PTZ (McMillen and Isaac, 1978).
PTZ (20 mg/kg) did not alter plasma tryptophan levels and a 40 mg/kg dose decreased total plasma tryptophan. !This change is opposite to the change observed in CSF 5-HIAA (McMillen and Isaac, 1978). These data suggest that the 24 hour increase in CSF 5-HIAA levels does not result from an increase in plasma tryptophan levels and may be a direct effect of PTZ on 5-HT neurons.
Pretreatment of cats with trimethadione (200 mg/kg) blocked the convulsions but not the EEG excitation produced by PTZ (40 mg/kg). This dose of PGZ elevated 5-HIAA levels whether or not the animals were pretreated with trimethadione suggesting that PTZ can increase 5-HT metabolism without causing convulsions (McMillen and Isaac, 1978).
The results of Diaz (1970) and Isaac (1974, 1978), with respect to the effect of PTZ on 5-HIAA levels, are opposite. This discrepancy can best be explained by differences in the period of measurement of 5-HT metabolism. In addition to its effect on 5-HT and dopamine metabolism, Kato et al. (1967) reported that acute administration of PTZ (50 mg/kg) resulted in elevated levels of brain histamine 24 hours after injection. Epinephrine levels were decreased at 5 minutes and 2 hours after PTZ, not changed after 1 hour and elevated after 24 hours. Norepinephrine levels were increased at 5 minutes, 1 hour and 2 hours but had returned to normal at 29 hours. Eleven day treatment with PTZ (50 mg/kg) produced marked elevation in brain histamine but not epinephrine or norepinephrine (Kato et al., 1967).
In summary, the above studies show that PTZ affects monoamine metabolism at both non-convulsant and convulsant doses.
Furthermore, these effects can last long after the immediate action of PTZ has stopped.
Effect of Monoamine Manipulation on PTZ Seizure Threshold Bonnycastle et al. (1957) reported that elevation of rat brain 5-HT levels by administration of either iproniazid or 5-HTP failed to protect rats against the convulsant or lethal effect of PTZ (75 mg/kg). However, Frey and Kilian (1973) found that 5-HTP increased the PTZ seizure threshold of both mice and rats. Rudzik and Johnson (1970) reported that administration of tranylcypromine, 5-HTP or 3,4-dihydroxyphenyl-alanine (DOPA) did not alter the PTZ seizure threshold. However, when 5-HTP was combined with tranylcypromine a significant elevation in the PTZ convulsive threshold occurred. In contrast the combination of tranylcypromine and DOPA did not increase the threshold for PTZ seizures.
The survival time of mice infused intravenously with PTZ is shortened by reserpine (Chen et al., 1954;Lessin and Parkes, 1959;Pfeifer and Galambos, 1967) or tetrabenazine (Lessin and Parkes, 1959) pretreatment. Administration of the MAO inhibitor, iproniazid, prevented the reduction in survival time produced by reserpine (Chen and Bohner, 1961;Lessin and Parkes, 1959;Pfeiffer and Galambos, 1967) or tetrabenazine (Lessin and Parkes, 1959). 5-HTP lengthened the survival time of mice pretreated with iproniazid but had no effect alone. In contrast, DOPA did not alter the survival time of mice pretreated with iproniazid (Lessin and Parkes, 1959). LSD but not 2-bromo-LSD antagonized the effect of reserpine on PTZ survival time. LSD did not alter the survival time of control mice (Lessin and Parkes, 1959) . These data suggest a relationship between sensitivity to PTZ and brain 5-HT levels. Chen and Bohner (1961) reported that in addition to 5-HTP, 5-HT, DOPA and dopamine reversed the lowering effect of reserpine on PTZ seizure threshold in iproniazid treated mice. Jones and Roberts (1968) found that intracerebroventricular injection of noradrenaline antagonized reserpine-induced facilitation of PTZ seizures but had no effect alone. Whereas small amounts of intraventricularly administered dopamine lowere~ the threshold for PTZ convulsions and were without effect on the reserpine facilitative effect, higher doses showed anti-convulsant action and antagonized the facilitative effect of reserpine. Schlesinger et al. (1969) reported that the combined intracranial injection of norepinephrine plus 5-HT protected mice against PTZ-induced seizures.
Pfeiffer and Galambos (1967) suggested that norepinephrine has a more important role in the change of susceptibility to PTZ seizures than 5-HT or dopamine. These investigators found that prenylamine decreased the PTZ convulsive threshold without altering 5-HT levels. Also guanethidine decreased only brain norepinephrine levels without altering dopamine or 5-HT and lowered the threshold for PTZ-induced convulsions. Alexander and Kopeloff (1970) found that pretreatment of rats with p-chlorophenylalanine (PCPA) to deplete brain serotonin lowered the threshold for PTZ-induced seizures. Frey and Kilian (197 3) pretreated mice with PCPA or cyproheptadine and found no change in the threshold for PTZ-induced clonic seizures. However, PCPA lowered the threshold in rats confirming the data of Alexan'.der and Kopeloff (1970). Rudzik and Johnson (1970) reported the PTZ convulsive threshold in mice was lowered only by drugs which decreased whole brain 5-HT and was not altered by catecholamine depleting drugs. Reserpine and P-CPA were found to lower the seizure threshold but U-14,624 and alpha-methyl-para-tyrosine produced no chage.
In contrast to these data, Frey and Kilian (1973) reported that alpha-methyl-para-tyrosine, disulfarim, FLA-63 and pr?pranolol but not haloperidol or phentolamine lowered the threshold for PTZ-induced clonic seizures. L-dopa elevated this threshold in mice but had no significant effect in rats. Because dopamine B-hydroxylase inhibitors were as effective as alpha-methyl-para-tyrosine and because of the lack of effectiveness of haloperidol these data suggest a more important role for norepinephrine than dopamine.
The threshold for PTZ-induced tonic extension was depressed by FLA-63, PCPA, cyproheptadine and propranolol but not alpha-methyl-p~ra-tyrosine or disulfarim (Frey and Kilian, 1973). Corcoran et al. (1973Corcoran et al. ( , 1974 found that selective destruction of catecholamine but not 5-HT neurons by intraventricular injection of 6-0HDA to rats pretreated with an MAO inhibitor increased the duration and intensity of convulsions induced by PTZ (70 mg/kg). The severity of seizures in rats with depletion of both norepinephrine and dopamine was not different from rats with preferential depletion of norepinephrine, suggesting that norepinephrine and not dopamine is important for the 6-0HDA induced exacerbation of PTZ convulsions.
A number of investigations have looked at the effect of amphetamines on PTZ seizure threshold. Kobinger (1958) found that methamphetamine raised the PTZ seizure threshold when administered 15 minutes before PTZ. Methamphetamine also antagonized the decreased PTZ seizure threshold produced by reserpine. Turner and Spencer (1968) however, reported that d-amphetamine had a pro-convulsant effect on PTZ convulsions in mice when administered 30-90 minutes before PTZ but had an anticonvulsant effect when administered 6 hours before PTZ.
BLockade of dopamine but not norepinephrine or 5-HT synthesis antagonized the pro-convulsant action of d-amphetamine (Spencer and Turner, 1969) indicating the importance of dopamine for this action. Rudzik and Johnson (1970) reported that neither d-amphetamine or metharnphetarnine significantly altered the PTZ convulsive threshold but administration of the chloroderivatives which have a unique effect on 5-HT disposition (Fuller et al., 1965) produced a significant increase of the PTZ seizure threshold. Gerald and Riffee (1973) found that acute administration of d-or 1-amphetamine increased susceptibility to PTZ-induced convulsions. d-Arnphetamine was about twice as potent as the 1-isomer. Whereas 1-arnphetamine increased tonic seizure susceptibility, d-amphetarnine decreased susceptibility to this seizure. Tolerance developed to these effects of amphetamine after seven consecutive daily injections (Gerald and Riffee (1973)).
Finally, Bhattacharya and Sanyal (1978) reported that prostaglandin E, (PGE 1 ) induced inhibition of PTZ-induced seizures in rats was antagonized by drugs which reduce brain 5-HT activity but not by drugs which decrease catecholamine activity. Pretreatment with reserpine, P-CPA, methysergide or 5,6-DHT significantly antagonized PGE 1 induced inhibition of convulsions produced by PTZ (60 mg/kg). Alpha-methyl-paratyrosine, diethyldithiocarbamate, phenoxybenzamine, propranolol or . haloperidol pretreatment did not significantly alter the effect of PGE 1 . These data suggest that this effect of PGE 1 is not a direct one but is mediated through serotoninergic mechanisms.
In summary, manipulation of CNS monoamines has provided information regarding the involvement of these neurotransmitters in the convulsant action of PTZ. The majority of evidence suggests that reduction of 5-HT or noradrenergic activity increases the susceptibility to PTZ seizures.

CONCLUSION
Studies to elucidate the action of PTZ on neurotransmitter metabolism have demonstrated that this drug affects the metabolism of acetylcholine, GABA and monoamines. A uniform neurochemical mechanism for the pharmacological actions of PTZ, however, is not apparent.
In addition to its effect on neurotransmitter function, PTZ has also been reported to have a direct effect on membrane excitability (Gross and Woodbury, 1972;Klee et al., 1973;David et al., 1974;Suguya and Onozuka, 1978) which may contribute to its action.

PTZ-Saline Discrimination Training
All of the rats were first magazine trained and shaped to lever press for food reinforcement. When the rats began to lever press at a rate of one response per minute they were shaped to learn a progressively increasing fixed ratio (FR) schedule of reinforcement until they consistently responded on an FR 10 schedule (10 lever presses for each 45 mg Noyes food pellet) .
In the beginning only responses with one of the levers was reinforced while responding with the other lever was not reinforced. When the rats began to respond with only one lever at a rate of 20 responses per minute the lever with which responses were reinforced was changed to the alternate lever. When the rats began to respond with this lever at a rate of 20 responses per minute, the lever with which responses were reinforced was again changed. After 4-5 such alternations without any injection, a PTZ-saline injection schedule was introduced.
In this phase of training, daily 15 minute sessions were preceded by injection of either PTZ (20 mg/kg) or saline (1 ml/kg). Each session began with one food pellet in the food cup so as to orient the rats to the manipulanda. The animals were trained to respond with one of the levers 15 minutes following a PTZ injection and the other lever 15 minutes following a saline injection ( Figure 1). Every tenth response (FR 10) with the appropriate lever was reinforced by delivery of a 45 mg Noyes food pellet. Responses with the incorrect lever (i.e., saline Diagrammatic representation of the PTZsa:iLi:ne discrimination . procedure. On an FR 10 schedule of food reinforcement, hungry rats were trained to respond with a lever on one side of a food cup 15 min following a 20 mg/kg PTZ injection and to respond with the lever on the alternate side 15 min following a 1 ml/kg saline injection. lever following PTZ injection or PTZ lever following saline injection) were recorded but were not reinforced by delivery of food.
To guard against a possible effect of lever or position preference the lever with which responses were reinforced following a PTZ injection was randomly assigned to be the lever on the right side of the food cup for half of the rats and the lever on the left side of the food cup for the remaining rats. For each rat, the position (i.e., right or left) of the PTZ lever remained constant on each subsequent session . To avoid the possibility that olfactory cues associated with the correct lever for rats previously tested in the Skinner boxes could serve as a cue for lever selection (Weissman, 1976), the sequence of PTZ-saline injections was varied separately for each group of rats trained successively on the same day.
Initially, the sequence of PTZ-saline injections alternated.
This training continued until five such alternations were achieved and responding was stabilized with the appropriate lever. Following this the rats entered the final phase of training where the PTZ-saline sessions were carried out seven days a week according to an irregularly alternating sequence of PTZ-saline injections. In this and all subsequent phases of the experiment, the session length was fixed at 10 minutes. The rats were trained to a criterion of emitting four or less responses with the incorrect lever prior to the first reinforcement (10 responses with the correct lever) on nine out of ten consecutive sessions.
Fbr each session data were recorded automatically to include the number of responses emitted with the incorrect lever prior to th_ e first reinforcement as well as the total number of responses emitted with. the correct and incorrect lever during the entire 10 minute session.

Discrimination Testing_
Wh.en the rats reached the c,riterion described above they were continuously and repeatedly used for generalization and anta9onism tes-ting-. These tests consisted of ten-minute sessions separ~ted by at least five practice sessions in which saline and PTZ wer-e correctly discriminated. For half of the rats, the sessions were ·preceded by a practice session in which saline was injected whereas for the remaining rats test sessions were preceded by a practice session in which PTZ was injected.
If the rats• pe~formance on these practice sess·ions seemed to deteriorate with respect to the number of responses on the incorrect lever prior to the first reinforcement, further training sessions were given before testing was reinstated. For generalization and antagonism testing, doses of each drug were administered in an irregular order.
A. Generalization Testi·ng ~ Following injection of the test drug (for pretreatment time see Table 3) each rat was placed in its assigned Skinner box and allowed to respond with the levers until ten nonreinforced responses were completed with one of the levers.
The lever with which ten responses were completed first was considered the selected lever and was subsequently fixed to be reinforced (FR 10) for the remainder of the session. Responses emitted with the other lever were recorded but not reinforced.
B. Antagonism Testing -Animals were injected with the appropriate dose of the test drug or saline. Following this pretreatment (for pretreatment time see Table 5), the rats were injected with pentylenetetrazol (20 mg/kg). Fifteen minutes after the pentylenetetrazol injection the animals were placed in their assigned Skinner boxes and tested for lever selection as described above.
C. Antagonism Testing with Diazepam and Chlordiapepoxide After Their Chronic Administration -The rats that were tested acutely with diazepam (2.5 or 10 mg/kg) for antagonism of the PTZ discriminative stimulus were treated with diazepam (10 mg/kg) for ten consecutive days. Similarly, the animals tested acutely with chlordiazepoxide (2.5 or 10 mg/kg) were treated with chlordiazepoxide (10 mg/kg) for ten consecutive days. Other rats that were tested acutely with solvent were treated with solvent for ten consecutive days. Results of previous studies (Cook and Sepinwall, 1975;Goldberg et al., 1967;Margules and Stein, 1968;Stein et al., 1975)     Generalization Testing (Tables 3 and 4) 1. Anxiogenic CNS Stimulants.
These doses of PTZ did not produce overt convulsions or myoclonus which were observed to occur when PTZ (40 mg/kg) was administered to naive rats. Following approximately 6 months of the PTZ-saline discrimination, however, some of the rats began to develop myoclonus after PTZ (20 mg/kg) injection.
When PTZ (2.5-20 mg/kg) was administered to these rats the percentage selecting the PTZ lever was not significantly All of the rats injected with aceperone (0.64 or 2.25 mg/kg) selected the saline lever and these rats did not emit any responses with the PTZ lever before saline lever selection. These rats were removed from the Skinner boxes after lever selection and therefore response rate is not given.
Twenty-five percent of eight rats selected the PTZ lever after a 100 mg/kg injection of DL-5-hydroxytryptophan (5-HTP). This was significantly different (Fisher Exact Probability, p<0.05) however, from 100% PTZ lever selection after PTZ (20 mg/kg). These rats emitted 0.3+0.25 responses with the nonselected lever before making a lever selection.
Two rats did not make a lever selection after this dose of 5-HTP. Response rate after 5-HTP was significantly less (Duncan's Multiple Range Test, P<0.05) than rates of responding during the previous PTZ and saline sessions.
After fluoxetine (5 mg/kg), 33 percent of six rats selected the PTZ lever and these rats did not emit any responses with the nonselected lever prior to lever selection.
Following injection of a higher (10 mg/kg) dose of fluoxetine, two rats selected the saline lever and two rats did not make a lever selection . The rats emitted 566~97 responses during the fluoxetine ( Thirty-three percent of six rats injected with naloxone (80 mg/kg) selected the PTZ lever and this was significantly different (Fisher Exact Probability, p<0.05) from 100% PTZ lever selection after PTZ (20 mg/kg), however. These rats did not emit any responses with the incorrect lever prior to lever selection. One rat did not make a lever selection after    1 Following pretreatment with the test drug the rats were placed in their assigned Skinner boxes and allowed to make a lever selection. The lever with which ten responses were completed first was considered the selected lever.
2 Number of rats tested.
3 % of rats selecting the pentylenetetrazol lever. "No Selection" indicates that 10 responses were not completed with either lever in the 10 min session due to behavioral toxicity of the drug. 4 Responses (Mean+S.E.) emitted with nonselected lever before lever selection. Where N <3 mean is given.
1 Test drugs were administered intraperitoneally. Following pretreatment with the test drugs the rats were placed in their assigned Skinner box and allowed to respond with the levers for 10 min. responses with the nonselected lever prior to lever selection.
Five o~t of six rats treated with etomidate (10 mg/kg) 5 minutes before PTZ (20 mg/kg) did not make a lever selection. The one rat that made a lever selection selected the PTZ lever and emitted one response with the saline lever before PTZ lever Range T~st, p<0.05) than response rates during the PTZ and saline ·sessions preceding these tests. One, two and four rats tested for lever selection at four, eight and 24 hours after GAG did not make a lever selection.
Gamma vinyl GABA (1000 mg/kg) did not angagonize the PTZ discriminative stimulus when the rats were tested for lever selection 72 hours after gamma-vinyl GABA (GVG) injection.
Three out of four rats tested at this time selected the PTZ lever and these rats did not emit any responses with the saline lever before PTZ lever selection. One rat did not make a lever selection. When tested at 2 and 8 hours after GVG the rats did not make a lever selection. One rat selected the PTZ lever when tested 24 hours after GVG, seven rats did not make a lever selection. Response rate for the three rats making a lever selection 72 hours after GVG was significantly less (Duncan's Multiple Range Test,p<0.05) than the rate of responding of these rats during the preceding PTZ and saline sessions.
All of six rats pretreated with 10 mg/kg amitriptyline selected the PTZ lever and these rats emitted l.8+0.87 responses with the saline lever before PTZ lever selection. Atropine (5-10 mg/kg) also failed to antagonize the PTZ discriminative stimulus. All of four rats treated with 5 mg/kg atropine 30 minutes before injection of pTZ (20 mg/kg) selected the PTZ lever and these rats did not emit any responses with the saline lever before PTZ lever selection.
Response rate of these rats during the test session was significantly less (Duncan's Multiple Range Test,p<0.05) than  1 Following pretreatment with the test drug, the rats were injected with pentylenetetrazol (20 mg/kg). 15 min following the pentylenetetrazol injection, animals were placed in the.ir assigned Skinner box and allowed to make a lever selection. The lever with which ten responses were completed first was considered the selected lever.
2 Number of rats tested.
3 % of rats selecting the pentylenetetrazol lever.
"No selection" indicates that ten responses were not completed on either lever in the 10 min session due to behavioral toxicity of the drugs.
4 Responses (Mean~S.E.) emitted with nonselected lever before lever selection. Where N < 3 mean is given.      response rate during the PTZ and saline sessions preceding the test session. One rat did not make a lever selection after pretreatment with 5 mg/kg atropine. Two rats pretreated with 10 mg/kg atropine selected the PTZ lever and these rats did not emit any responses with the saline lever before PTZ lever selection.
Response rate during the 10 mg/kg atropine test session appeared to be decreased compared to the response rate during the PTZ and saline sessions preceding the test but the small number of animals prohibited statistical analysis.
Lack of Tolerance Development to Diazepam and Chlordiazepoxide in Antagonism of the PTZ Discriminative Stimulus (Table 7) Chronic administration of solvent, diazepam or CDP to rats tested acutely with these drugs for antagonism of the PTZ discriminative stimulus did not alter their ability to antagonize the discriminative stimulus produced by PTZ. All of the rats pretreated acutely with solvent for antagonism of the PTZ discriminative stimulus selected the PTZ lever after chronic solvent administration. Whereas 67 and 17% of the rats pretreated acutely for antagonism of the PTZ discriminative stimulus with 2.5 and 10 mg/kg diazepam selected the PTZ lever, 67 and 33% of these rats selected the PTZ lever after chronic diazepam administration. Similarly, whereas 30 and 20% of the rats pretreated acutely for antagonism of the PTZ discriminative stimulus with 2.5 and 10 mg/kg CDP selected the PTZ lever, 40 and 20% of these rats selected the PTZ lever after chronic CDP treatment.
Tests for Diazepam and Haloperidol Antagonism of the Discriminative Stimuli Produced by Cocaine, Yohimbine and R05-3663 in Rats Trained to Discriminate Between Pentylenetetrazol (20 mg/kg) and saline (Tables 8 and 9).

Cocaine
As stated previously (  1 Rats were injected with the appropriate dose of either diazepam, chlordiazepoxide or solvent. 30 min later PTZ (20 mg/kg) was injected. 15 min following the PTZ injection, animals were placed in their assigned Skinner box and allowed to make a lever selection. The lever with which ten responses were completed first was considered the selected lever.
lever when haloperidol was injected before cocaine. P.rior to lever selection the rats emitted 0."8±0.33 responses with the nonselected lever. Response rate during the haloperidol plus cocaine test session was significantly less (Duncan's Multiple Range Test, p<0.05) than response rate during the PTZ and saline session preceding the test.

R05-3663
R05-3663 (0.64-5 mg/kg) dose-dependently generalized to the discriminative stimulus produced by PTZ (Table 3) . Diazepam (5 mg/kg) pretreatment antagonized the discriminative stimulus produced by R05-3663 so that higher doses of this drug were required to produce PTZ lever selection. Whereas 100% of the rats injected with 5 mg/kg R05-3663 selected the PTZ lever, none of the rats selected the PTZ lever when this dose of R05-3663 was preceded by a 5 mg/kg diazepam injection.
However, 40 and 80% of the rats selected the PTZ lever when injected with 10 and 20 mg/kg R05-3663, respectively 30 min after diazepam (5 mg/kg) injection. Prior to lever selec-

Yohimbine
Yohimbine (2.5 mg/kg) generalized to the PTZ discriminative stimulus in six of twelve rats (Table 3). Diazepam (5 mg/kg) pretreatment antagonized the yohimbine discriminative stimulus in three out of four of these rats that selected the PTZ leger after yohimbine alone.   1 Following injection of the test drug the rats were placed in their assigned Skinner box and allowed to make a lever selection. The lever with which ten responses were completed first was considered the selected lever.
2 Number of rats tested.
3% of rats selecting the PTZ lever.
4Responses (Mean±.S . E.) emitted with nonselected lever before lever selection. Following pretreatment and allowed to re-3 Total responses (Mean+S.E.) emitted during the 10 min session following pretreatment with the test drug and during the preceding pentylenetetrazol (20 mg/kg) and saline session. 4 values were obtained by dividing the total number of responses emitted following test pretreatment by the total number of resonses during the precedi.ng pentylenetetrazol ( 20 mg/kg) and saline sessions. lOA DISCUSSION Acquisition of PTZ-Saline Discrimination A number of drugs are known to function as discriminative stimuli which reliably control operant behavior (for reviews see Schuster and Balster, 1977;Lal, 1977;Colpaert and Rosecrans, 1978). Usually in these experiments laboratory animals are trained to emit one response when treated with a drug and an alternate response when treated with the drug vehicle, another dose of the same drug or a different drug. When acquisition of such response differentiation is reliably established, the drug is said to produce a discriminative stimulus which controls the differential response -emission in the trained subjects.
The present experi~ent demonstrates that rats can learn to reliably discriminate between the effect of PTZ (20 mg/kg) and saline. A mean of 30 sessions was required for the rats to reach the discrimination criterion. A similar number of trials to reach a similar criterion was previously reported for many other psychoactive drugs (Weissman, 1978). As was the case with other drugs (Lal, 1976;Shearman et al., 1978) the discriminative stimulus produced by PTZ was dosedependent.

Time Course of PTZ Discrimination
In addition to its anxiogenic action, PTZ has also been reported to produce hallucinations in man (Rodin, 1958;Winter and Wallach, 1969). However, whereas the anxiogenic effect of PTZ diminishes soon after injection of PTZ is discontinued the hallucinations persist for up to 24 h after PTZ injection. In order to test the hypothesis that the discriminative stimulus produced by PTZ in the rat was related to its anxiogenic action and not its hallucinatory effect, discrimination of PTZ was tested at various times after injection of the drug.
Pharmacokinetic studies of PTZ in rats show that 70-95% of this drug appears in the urine within 24 h (Rowles et al., 1971;Vohland and Koransky, 1972). The cerebrospinal fluid and plasma half-life of a 20 mg/kg PTZ injection in cats has been reported to be approximately one hour (McMillen and Isaac, 1978).
In the present study the discrimination of PTZ diminished as the reported tissue levels of this drug decreased. A significant percentage of the rats no longer selected the PTZ lever when the reported blood levels of PTZ were significantly decreased. These data support the hypothesis that the discriminative stimulus produced in the rat is related to its anxiogenic property and not its hallucinatory effect.

Generalization Tests with Other Anxiogenics
To test the hypothesis that the discriminative stimulus produced by PTZ in the rat was related to its anxiogenic action in man (Rodin, 1958;Rodin and Calhoun, 1970;Winter and Wallach, 1969), other anxiogenic stimulants were tested for generalization to the PTZ discriminative stimulus.
Cocaine has been well recognized to induce anxiety in man (Cohen, 1975;Siegel, 1977;Wesson and Smith, 1977). Dosedependent generalization by cocaine to the PTZ discriminative stimulus supports the hypothesis that the discriminative stimulus property of PTZ in the rat is related to its anxiogenic action in man. Additional support for this was provided by the finding that the anxiolytic drug, diazepam, antagonized the cocaine discriminative stimulus. Whereas the nonanxiolytic drug, haloperidol, did not antagonize the cocaine discriminative stimulus.
In addition to its anxiogenic action, however, cocaine is also a psychomotor stimulant.
In order to determine that psychomotor stimulation was not the basis of the cocaine generalization to PTZ, nonanxiogenic psychomotor stimulants were tested for generalization to PTZ. Neither d-amphetamine, methylphenidate nor caffeine generalized to the PTZ discriminative stimulus suggesting that the psychomotor stimulant property of cocaine was not the basis of its discrimination to PTZ.
Several investigators (Holmberg and Gershon, 1961;Ingram, 1962;Gershon and Lang, 1962;Garfield et al., 1967) have reported the anxiety-like effects produced by yohimbine in man . Amobarbital was reported to reduce the anxiety response to yoh~mbine while imipramine and epinephrine potentiated this effect (Holmberg and Gershon, 1961).
In the present study, yohimbine partially generalized to the discriminative stimulus property of PTZ. Furthermore, diazepam antagonized the yohimbine discriminative stimulus in those rats that perceived the yohimbine stimulus as similar to the PTZ discriminative stimulus. These data support the hypothesis that the discriminative stimulus property of PTZ in the rat is related to its anxiogenic action in man .
Recently, O'Brien and Spirt (1980) speculated that if the anxiolytic action of benzodiazepines were considered to be mediated through an antagonistic action then a molecule structurally similar to ROS-3663 could exist in brain to function as an endogenous anxiety producing li~and.
In the present study, ROS-3663 dose-dependently generalized to the PTZ discriminative stimulus. Furthermore, the discriminative stimulus strength of ROS-3663 was attenuated by diazepam.
These data provide behavioral evidence that ROS-3663 may structurally resemble a naturally occurring anxiety-inducing ligand · and support the hypothesis that the discriminative stimulus produced by PTZ in the rat is related to the anxiogenic action in man.
It has been reported that patients poisoned with strychnine are extremely apprehensive and fearful of impending death (Polson and Tattersall, 1969;Franz, 1975). Strychnine generalization to the discriminative stimulus produced by PTZ supports the hypothesis that the discriminative stimulus property of PTZ in the rat is related to its anxiolytic action in man.
In addition to the anxiogenic action of strychnine and ROS-3663, however, these drugs are also convulsants. In order to determine that the convulsant action of strychnine Also, gamma-hydroxybutyrate that produces a hypersynchronous EEG in the chick (Osuide, 1972), rat (Marcus et al., 1967;Godschalk et al., 1976Godschalk et al., , 1977, cat (Winters and Spooner 1965), rabbit and man (Schneider et al., 1963), that is selectively antagonized by anti-petit rnal drugs (Godschalk et al., 1976) and has been compared to petit mal epilepsy (Winters and Spooner, 1965;Snead et al., 1976;Godschalk et al., 1977), did not generalize to the PTZ discriminative stimulus. These data suggest that the convulsant action of strychnine and R05-3663 was probably not the basis of their generalization to the PTZ discriminative stimulus and that a subconvulsant or petit mal like brain state likely to be produced by these drugs was not the basis of the discriminative stimulus produced by PTZ. The finding that bemegride generalized to the PTZ discriminative stimulus suggests that this drug may be anxiogenic.

Blockade of the PTZ Discriminative Stimulus by Anxiolytics
As another approach to test the hypothesis that the discriminative stimulus produced by PTZ in the rat was related to its anxiogenic action in man, antagonism of the PTZ discriminative stimulus by clinically effective anxiolytic drugs was tested.

~10
Several benzodiazepine anxiolytics, barbiturate type anxiolytics as well as a propanediol carbamate anxiolytic effectively antagonized the PTZ discriminative stimulus in a dose-dependent manner and in nonsedative doses. In addition, valproic acid, which was recently found to have anxiolytic properties in a conflict test  also dosedependently antagonized the PTZ discriminative stimulus.
These data support the hypothesis that the discriminative stimulus produced by PTZ in the rat is related to its anxiogenic action in man.
The potency of the various drugs effective in antagonizing the PTZ discriminative stimulus was highly correlated with their effective doses in a widely accepted conflict test used to measure anxiolytic activity ( Figure 3) as well as their clinically effective doses ( Figure 4). These data also support the hypothesis that the discriminative stimulus property of PTZ in the rat was related to its anxiogenic action in man.
In addition to anxiolytic activity, however, all of the drugs that antagonized the PTZ discriminative stimulus are also central nervous system depressants. Several investigators (Warner, 1965;Goldberg et al., 1967;Margules and Stein, 1969;Stein et al., 1975;Cook and Sepinwall, 1975) have reported that tolerance develops only to the depressant action benzodiazepines. Therefore, lack of tolerance development to other actions of these drugs in experimental animals has often been employed as a tool to identify pharmacological actions for their predictive value for clinical efficacy Lippa et al., 1979). Because benzodiazepines have been the most widely studied anxiolytic drugs with respect to tolerance development to their anxiolytic actions, it was investigated whether tolerance would not develop to two prototype benzodiazepines, chlordiazepoxide and diazepam, in antagonism of the PTZ discriminative stimulus.
It was hypothesized that if the antagonistic property of these benzodiazepines in the PTZ discrimination paradigm was related to their anxiolytic action tolerance would not develop. Previously, lack of tolerance development to the prevention of PTZ-induced convulsions by diazepam was reported (Fuxe et al., 1975;Juhasz and Dairman, 1977;Lippa and Regan, 1977).
The ability of either diazepam or chlordiazepoxide to antagonize the PTZ discriminative stimulus was not affected by their chronic administration even though this treatment was more than sufficient (Cook and Sepinwall, 1975;Goldberg et al., 1967;Margules and Stein, 1968;Stein et al., 1975) to produce tolerance to the depressant action of these drugs.
The present data are consistent with previous findings (Fuxe et al., 1975;Jahasz and Dairman, 1977;Lippa and Regan, 1977) that show lack of tolerance development to prevention of PTZ induced convulsions by diazepam. Because tolerance develops to the depressant but not anxiolytic action of benzodiazepines these data support the hypothesis that antagonism of the PTZ discriminative stimulus by these drugs is related to their anxiolytic action.
A~ditional data which supports the suggestion that antagonism of the PTZ discriminative stimulus was related to anxiolytic action and not to the central nervous system depressant activity of these drugs was provided through antagonism tests with nonanxiolytic central nervous system depressants. Etomidate is a nonbarbiturate hypnotic which is used for the induction of anesthesia because of its short duration of action (Jannsen et al., 1975). The anxiolytic action of etomidate has not been reported to date. Although ethanol is widely considered an anxiolytic, the anti-anxiety effect of this drug has not been clearly demonstrated  • Whereas morphine is known to alleviate anxiety during opiate withdrawal (Redmond, 1979) it does not relieve anxiety in opiate free individuals. Chlorpromazine and haloperidol, useful in the treatment of psychoses, do not decrease anxiety (Rickels et al., 1979).
None of these central nervous system depressants were effective in antagonizing the discriminative stimulus produced by PTZ. These data indicate that drugs that depress the central nervous system but do not possess anxiolytic activity do not antagonize the PTZ discriminative stimulus.
Therefore these data support the hypothesis that the discriminative stimulus produced by PTZ in the rat is related to its anxiogenic action.
In addition to their anxiolytic and depressant actions, all of the drugs that were effective in antagonizing the PTZ discriminative stimulus are also effective against PTZ convulsions (Childress and Gluckman, 1964;Banziger, 1965;Swingard and Castellion, 1966;Ludwig and Potterfield, 1971;Dren et al., 1978;Lippa et al., 1979). Based upon these data, the finding that tolerance does not develop to diazepam in antagonizing PTZ convulsions (Lippa and Regan, 1977) as well as other considerations (Lippa et al., 1979), it has been suggested that the ability of drugs to prevent or antagonize or prevent PTZ convulsions may reflect their anxiolytic property (Hill and Tedeschi, 1971;Lippa and Regan, 1977;Lippa et al., 1979).
Therefore, to test the hypothesis that antagonism of the PTZ discriminative stimulus by these drugs was not related to their anticonvulsant action, antagonism of the PTZ discriminative stimulus with anticonvulsants that lack clinical anxiolytic actions was tested.
Trimethadione and ethosuxirnide are known to protect laboratory animals against PTZ produced convulsions (Everett and Richards, 1944;Toman andGoodman, 1948, Swinyard andCastellion, 1966;Woodbury and Fingl, 1975). These drugs are effective in the treatment of petit-mal epilepsy (Woodbury and Fingl, 1975) but not in the treatment of anxiety.
In the present experiment neither trimethadione nor ethosuximide antagonized the discriminative stimulus produced by PTZ.
Etomidate like trimethadione and ethosuximide, is known to protect mice and rats against the convulsant action of PTZ (Desmedt et al., 1976). However, etomidate also did not antagonize the PTZ discriminative stimulus.
There is a high correlation (R=0.95; p < 0.05) between the potency of drugs effective in antagonizing the PTZ discriminative stimulus and their effective doses against PTZ-induced convulsions ( Figure 5). However, whereas all drugs that antagonize the PTZ discriminative stimulus prevent PTZ convulsions not all drugs that are effective against PTZ convulsions antagonize the PTZ discriminative stimulus. These data suggest that the anticonvulsant action of the drugs effective in antagonizing the PTZ discriminative stimulus is not the property that is responsible for this effect. These data also suggest that the mechanism of action of the PTZ in producing convulsions is different from that underlying its mechanism for producing a discriminative stimulus. Furthermore these data suggest that antagonism of the PTZ discriminative stimulus is a better measure of anxiolytic activity than is antagonism of PTZ seizures. The exact neurochemical basis for anxiety is unknown.
However, it is possible that some indication of this may be obtained from the postulated mechanism of action of anxiolytic drugs with the assumption that the neurochemical basis for anxiety is opposite to that for the anti-anxiety action of anxiolytic drugs.
Benzodiazepines are the most effective and thus most widely prescribed drugs for the treatment of anxiety (Howard and Pollard, 1977). Also, this class of anxi9lytics has been the most extensively studied with respect to their mechanism of action (Costa and Greengard, 1975;Sepinwall and Cook, 1978;Koe, 1979).
In the present study, it was hypothesized that the neurochemical basis of the anxiogenic discriminative stimulus property of PTZ was opposite to that of the postulated neurochemical mechanism(s) responsible for the anxiolytic action of benzodiazepines.
To test this hypothesis, drugs known to have effects opposite to those of benzodiazepines on the neurotransmitter systems postulated to mediate the anxiolytic action of benzodiazepines and drugs known to have a similar effect as benzodiazepines on these neurotransmitter systems were tested for generalization to and antagonism of the anxiogenic discriminative stimulus property of PTZ.

Garruna-aminobutyric Acid (GABA)
A functionally significant interaction between benzo-diazep~nes and brain gamma-aminobutyric acid (GABA) became apparent from a variety of neurochemical (for reviews see Costa et al., 1978, Costa and and neurophysiological (for review see Haefly, 1978) experiments. However, the pharmacological significance of such an inteiaction is unclear. Whereas the anticonvulsant, sedative, and muscle relaxant actions of benzodiazepines seem to be related to GABAergic mechanisms (Cook and Sepinwall, 1975;Lippa-et al., 1979), a similar relationship for the anxiolytic action of benzodiazepines is not clear.
To measure anxiolytic actions of drugs in experimental animals, one test that is considered quite specific is the conflict paradigm where reduction of response suppression by "confliotful" stimuli is measured (Lippa' et al., 1979, Sepinwall and . Inthis test, benzodiazepines are known to show high potency and efficacy in nonsedative doses. Therefore, the conflict model has been used to evaluate various neurochemical hypothesis for anxiolytic activity. Cook and Sepinwall (1975) reported that the GABA-T inhibitor, aminooxyacetic acid (AOAA), was without anti-conflict activity alone and did not potentiate the anticonflict effect of diazepam. Tye et al. (19_79) also reported that AOAA lacked anticonflict effect. These data might suggest that alteration of brain GABA is not related to anti-anxiety action. However, i-20 recent studies of Iadrola and Gale (1979,a,b) show that the neuroanatomical sites of action of this GABA-T inhibitor is not appropriate for anxiolytic activity. First of all this drug elevates brain GABA outside of nerve terminals. Increasing the concentrations of GABA at these sites may actually inhibit neuronal GABA release because of its action on presynaptic autoreceptors.
In addition, the elevation of GABA by AOAA is more pronounced in extrapyramidal areas. The GABA-T inhibitor, valproic acid, on the other hand, increases brain GABA levels predominantly within nerve terminals and in brai~ sites in the limbic areas (Iadrola and Gale, 1979a,b).
Therefore, the lack of anxiolytic action by some GABA-T inhibitors may be due to the inappropriate site of their action.
Valproic acid's efficacy as an anxiolytic drug may be due to an action at more appropriate sites. Sullivan et al. (1978) and Cook and Sepinwall (1979) systemically administered the GABA receptor agonists muscimol and 4,5,6,7-tetrahydroisoxazolo (5, 4-c) pyridin-3-ol (THIP) to examine their effect on conflict behavior. Whereas muscimol was weakly active, THIP was inactive. Neither muscimol nor THIP potentiated the anticonflict effect of benzodiazepines in this test. However, several investigators (Baraldi et al., 1979;Maggi and Enna, 1979;Enna et al., 1979) have reported that muscimol does not penetrate into the brain after systemic administration. This evidence may explain the lack of anticonflict activity of muscimol and and its structural analogue, THIP.
Recently, Guidotti (personal communication) injected muscimol and THIP intraventricu~ larly and reported that both of these drugs produced anticonflict activity in rats~ Earlier data which suggested that the anticonflict activity of benzodiazepines might be -mediated oy GABA were provided by Stein et al. ll975) and Zakusov et· al. (1977). These investigators reported that the anti~conflict activity of benzodiazepines was reduced by the GABA antagonists picrotoxin, bicuculline or thiosemicarbizide.
Therefore, using the conflict model much data has been generated to suggest that th.e anxiolytic action of benzodiaze..pines may be mediated by enhancing GABA activity. It may be hypothesized then, that anxiety is due to a deficit in GABA neuronal function.
In order to test the hypothesis that decreased GABA neuronal activity was the neurochemical basis for the anxiogenic discriminative stimulus property of PTZ, generalization to the PTZ discriminative stimulus with GABA antagonistic drugs and antagonism of the PTZ discriminative stimulus with GABA minetic drugs was tested.
Picrotoxin and bicuculline were used as antagonists of tne postsynaptic inh1Bitory action of GABA (Curtis and Johnson, 1974). Whereas bicuculline acts at the receptor sites (Curtis and Johnson, 1974) , picrotoxin seems to act at chloride channels (Olsen· et ·a1., 1978a,b). Schecter and Tranier (1977) found that GABA-transaminase inhibitors prolong the onset of seizure and death following picrctoxin but not following bicucuclline. Other differences between picrotoxin and bicuculline have been reported with respect to GABA-receptor sensitivity (Krnjevic, 1974), blockade of GABA binding to brain receptors , stimulation of [ 3 H]-GABA release from brain tissue (Collins, 1973;Johnston and Mitchell, 1971), alteration of specific r 3 H]-diazepam binding ih cortical membranes (Tallman et al., 1978) and .
-induction of epileptic spikes following topical application and systemic injection (Edmonds and Bellin, 1976) .
Whereas, picrotoxin partially generalized to the PTZ discriminative stimulus, bicucuclline did not generalize.
Therefore, these data do not conclusively support the hypothesis that the anxiogenic discriminative stimulus property of PTZ is related to decreased GABA neuronal activity.
3-Mercaptopropionic acid is a convulsant drug that acts by inhibiting glutamate decarboxylase and producing a subsequent decrease in the concentration of brain GABA (Lamar, 1970). Like picrotoxin, 3-mercaptopropionic acid partially generalized to the PTZ discriminative stimulus. Therefore these data although suggestive do not fully support the hypothesis that decreased GABA function is the basis of the anxiogenic discriminative stimulus property of PTZ.
R0-3663 is a convulsant benzodiazepine that selectively antagonizes the effect of GABA at spinal and peripheral neuronal sites (Schlosser and Franco, 1979). In addition, this drug has been reix>rted to inhibit the GABA enhancement of 3 H-diazepam binding (O'Brien and Spirt, 1980) Roth and Nowycky, 1977). Pretreatment of the animals with gaITlll1a-hydroxybutyrate did not antagonize the discriminative stimulus produced by PTZ also suggesting that the PT~ discriminative stimulus is not related to decreased GABA neuronal activity.
Gamma-acetylentic GABA (GAG) and gamma-vinyl-GABA (GAG) are inhibitors of GABA-T which increase the brain GABA levels of mice and rats Jung et al., 1977b) and protect animals against seizures induced by audiogenic stimulation, strychnine, isoniazid, thiosemicarbizide and electric shock (Schecter et al., 1977a,b), but not picrotoxin, bicuculline nor PTZ (Schecter et al., 1977a,b) antagonized the discriminative stimulus produced by PTZ. Thus these data do not support the hypothesis that the PTZ discriminative stimulus is related to a deficit of brain GABAergic functioning.
However, it may be premature to consider the above data as going against the hypothesis that the anxiogenic discriminative stimulus property of PTZ is related to decreased GABA neuronal activity. Recently, Iadrola and Gale (1979a,b) have shown that the GABA-T inhibitors GAG and GVG increase brain GABA primarily in nonsynaptosomal sites such as glia.
In order to overcome these shortcomings the indirectly acting GABA mimetic drug, valproic acid, was tested for antagonism of the PTZ discriminative stimulus. Although valproic acid inhibits GABA-T (Godin et al., 1969, Simler et al . , 1973Fowler et al., 1975), enzyme-kinetic studies show that it increases brain GABA levels primarily by causins an accumulation of succinic semialdehyde through inhibition of succinic semialdehyde dehydrogenase ( Van der Lann et al., 1979). The succinic semialdehyde in turn blocks the conversion of GABA to succinic semialdehyde through product inhibition. These actions are preferentially localized within the nerve terminal ( Van der Lann et al., 1979;. In addition, whereas other GABA-T inhibitors ca.use GABA elevation primarily in the compartments outside of nerve terminals, GABA elevation produced by valproic acid is primarily located ih nerve terminals (Iadrola and Gale, 1979;Sarhan and Seilar, 1979). Valproic acid has also been reported to inhibit the reuptake of GABA into nerve terminals {ftarvey, 1976). Valproic acid dose-dependently antagonized the anxiogenic discriminative stimulus produced by PTZ. These data support the hypothesis that a deficit in GABA neuronal activity is the neurochemical basis of the anxiogenic discriminative stimulus property of PTZ. These data also suggest that valproic acid may be a clinically effective anxiolytic drug and that deficits i.n GABA functioning are important for anxiety. Furthermore these data suggest that antagonism of the PTZ discriminative stimulus by benzodiazepines, and thus their anxiolytic action might be related to thei.r property of enhancing GABA transmission.

Glycine
A significant correlation between the ability of a series of J5enzodiazepines to reduce 3 H-strychnine binding to glycine receptors and their clinical effects led to the proposal that these drugs might produce their muscle relaxant and anti-anxiety action by mimicking glycine at its receptor ~ites Snyder and Enna, 1975).
However, when Cook and Sepinwall (1975), using the data of Young et al. (1974), compared the 3 H-strychnine displacement potency of ten benzodiazepines with their anticonflict patency, they did not £ind a significant correlation. Because there was a significant correlation between anticonflict potency and potency in a human bioassay it was concluded that the affinity of benzodiazepines £or the glycine receptor was not correlated with anxiolytic activity. Furthermore, Stein et al. (1975) reported that although strychnine reduced the anticon£lict effect of oxazepam it also depressed unpunished responding suggesting that this effect was nonselective.
In order to test the hypothesis that decreased glycinergic activity was the neurochemical basis of the anxiogenic discriminative stimulus property of PTZ, the glycine antagonist (Curtis and Johnson, 1974) strychnine, was tested for generalization to the PTZ discriminative stimulus.
Although not completely strychnine generalized to the PTZ discriminative stimulus thus supporting the hypothesis that decreased glycinergic activity was the basis £or the anxiogenic discriminative stimulus property of PTZ.
McDonald and Barker (1977) reported that whereas PTZ antagonized the post-synaptic inhibitory responses to GABA in a mammalian tissue culture system it did not affect glycine responses. Therefore, unless it is de:rronstrated that PTZ antagonizes glycine the hypothesis that the neurochemical basis of the PTZ discriminative stimulus is due to decreased glycin~rgic activity might not be tenable. There was considerable evidence presented earlier that suggests that · the discriminative stimulus produced by PTZ was related to decreased GABA Neuronal activity. Because both GABA and glycine are inhibitory neurotransmitters it may be suggested that interference with either of these inhibitory systems produces a discriminative stimulus that provides the basis for the common discriminative stimulus properties of these drugs.

Phosphodiesterase Inhibition
Based upon a significant correlation between the in vitro potency of several drugs in inhibiting phosphodiesterase and their potency in the thirsty rat conflict test, Beer et al. (1972) proposed that the anxiolytic activity of drugs may be related to their ability to inhibit this enzyme. In support of this hypothesis, Weinryb et al. (1975), found that a series of substituted pyrazoF--(3, 4b) -pyr idines that were potent inhibitors of phosphodiesterase had significant anticonflict activity. However, Morrison (1969) and Cook and Sepinwall (1975) did not find significant anticonflict effeet of phosphodiesterase inhibitors in a lever press conflict test. Furthermore, Collins et al. (1976) reported that the potent phosphodiesterase inhibitor, SQ 65,396 was without anxiolytic effect compared to diazepam and placebo in psychoneurotic patients.
In order to test the hypothesis that the ability of benzodiazepines to antagonize the discriminative stimulus property pf PTZ was not related to their ability to inhibit phosphodiesterase, antagonism of the PTZ discriminative stimulus with the phosphodiesterase inhibitor, caffeine, was tested.
Caffeine did not antagonize the anxiogenic discriminative stimulus property of PTZ. This finding supports the hypothesis that the ability of benzodiazepines to antagonize the PTZ discriminative stimulus was not related to their ability to inhibit phosphodiesterase. These data also support previous work (Morrison 1969;Cook and Sepinwall, 1975;Collins et al., 1976) that suggests that the anxiolytic action of benzodiazepines is not related to their inhibition of phosphodiesterase.

Acetylcholine
There is no clear correlation between the effect of benzodiazepines on cholinergic function and their antianxiety action. Benzodiazepines were reported to increase rat brain acetylcholine levels (Consolo et al., 1975). The functional significance of this, however, is unclear as there was no correlation between the muscle relaxant or anti-PTZ of the benzodiazepines and their effect on acetylcholine even though PTZ has been reported to cause a decrease in rat brain acetylcholine (Giarrnan and Pepeu, 1962;Beani et al . , 1969;Longoni et al., 1974;Consolo et al., 1975) by causing a massive release of this neurotransmitter. In one behavioral study, Soubrie et al. (1976) reported that diazepam antagonized the hyperactivity elicited by anticholinergics in mice.
This effect was blocked by picrotoxin implicating the involvement of GABA mechanism for this action of diazepam.
In donflict tests, cholinergic agonists (Morrison, 1969) or antagonists (Hanson et al., 1970;Miczek, 1973) were without significant anticonflict effect. In order to test the hypothesis that the anxiogenic discriminative stimulus property of PTZ was related to its effect on cholinergic function, generalization to and antagonism of the PTZ discriminative stimulus with cholinergic agonistic and antagonists was tested.
None of the cholinergic drugs tested either generalized to or antagonized the PTZ discriminative stimulus. These data do not support the hypothesis that the neurochemical basis of the anxiogenic discriminative stimulus property of PTZ was related to its action on cholinergic function. Furthermore , these data suggest that the ability of benzodiazepines to antagonize the PTZ discriminative stimulus was not related to their affect on cholinergic function.

Serotonin
There is considerable biochemical (Stein et al . , 1975) and behavioral (for reviews see Sepinwall and Cook, 1978;Koe, 1979) evidence obtained from animal experiments that the benzodiazepines may exert their anti-anxiety action by decreasing serotonin activity . Stein et al. (1975) reported that whereas tolerance developed to the depressant action and effect of benzodiazepines on decreasing norepinephrine turnover, tolerance did not develop to their anticonflict effect or their effect of decreasing serotonin turnover. Using conflict paradigms it has been demonstrated that reduction serotonin activity by inhibition serotonin synthesis (Robichaud and Sledge, 1969;Geller and Blum, 1970;Schoenfeld, 1976), blockade of serotonin receptors (Graef£ and Schoenfeld, 1970;Graeff, 1974;Stein et al., 1973;Winter, 1972) or destruction of serotonin nerve terminals (Stein et al., 1975) produces significant anticonflict effects. However, there have been some reports (Winter, 1972;Blakely and Parker, 1973;Cook and Sepinwall, 1975a) that show a lack of anticonflict effect of serotonin antagonistic drugs. Whereas serotonin antagonists are usually reported to have anticonflict effects, serotonin agonists have been reported (Graeff and Schoenfeld, 1970;Winter, 1972;Geller et al., 1974) to significantly suppress conflict behavior.
In order to test the hypothesis that the anxioqenic discriminative stimulus property of PTZ was related to an agonistic effect of this drug on serotonin function, generalization to the PTZ discriminative stimulus with serotonin agonists and antagonism of the PTZ discriminative stimulus with serotonin antagonists was tested.
Neither of the serotonin agonists used in this study (5-HTP and fluoxetine) generalized to the PTZ discriminate stimulus. Furthermore, the serotonin receptor antagonist, methysergide, did not antagonize the discriminative stimulus proper~y of PTZ. These data do not support the hypothesis that the neurochemical basis of the anxiogenic discriminative stimulus property of PTZ was related to an agonistic effect on serotonin function. Furthermore, these data suggest that the ability of benzodiazepines to antagonize the PTZ discriminative stimulus is not related to their ability to decrease serotonin transmission.
It is interesting that serotonin antagonists are not clinically effective anxiolytics therefore reduction of serotonin function by these drugs might not be the neurochemical basis for their anxiolytic action as suggested by other animal studies.

Catecholamines
Benzodiazepines are known to decrease the turnover of both norepinephrine and dopamine (for review see Koe, 1979). There is no clear evidence, however, that this effect is related to the anxiolytic activity of these drugs. Fuxe et al. (1975) reported that diazepam antagonized increases in norepinephrine turnover produced by stress or injection of the anxiogenic drug yohirnbine. Gray (1976) reported similar effects for chlordiazepoxide as well as norepinephrine synthesis inhibitors or lesions of the dorsal noradrenergic bundle against stress. Lader (1974) reviewed evidence for the involvement of catecholamines in anxiety and more recently Redmond (1979) summarized considerable evidence for a role of norepinephrine in anxiety.
I~ activation of catecholaminergic system causes anxiety then drugs effective in decreasing catecholaminergic transmission should decrease anxiety. The beta-adrenergic receptor blockers, propranolol and oxprenolol have been reported to have anxiolytic activity (Jefferson, 1974;Krishman, 1976).
However, whereas standard anxiolytics are effective against both the somatic and psychic components of anxiety, these drugs appear to be effective only against the somatic anxiety.
In the conflict test, Sepinwall et al. (1973) reported propranolol to be without significant anticonflict effect.
To test the hypothesis that the anxiogenic discriminative stimulus property of PTZ was related to an action of increasing catecholaminergic transmission, catecholamine agonists were tested for generalization to the PTZ discriminative stimulus and catecholamine antagonists were tested for antagonism of the PTZ discriminative stimulus. Of the catecholamine agonists tested for generalization only cocaine significantly generalized to the PTZ discriminative stimulus.
The dopamine receptor agonist, apomorphine, as well as yohimbine partially generalized; however, a-amphetamine and methylphenidate did not.
Propranolol that was previously reported to generalize to the discriminative stimulus property of cocaine (Silverman and Ho, 1977), did not generalize to the PTZ discriminative stimulus. These data leave it unclear whether the anxiogenic discriminative stimulus property of PTZ was related to an action of activating catecholaminergic mechanisms. However, when several catecholamine antagonists including clonidine, aceperone, propranolol, haloperidol or chlorpromazine were tested for antagonism of the PTZ discriminative stimulus they were found to be without effect.
Therefore, these data do not conclusively support the hypothesis that the anxiogenic discriminative stimulus prospects of PTZ is related to an effect of increasing catecholaminergic activity.

Endorphins
The effect of benzodiazepines on endorphins is still being worked out. Therefore, the possible involvement of these newly discovered endogenous substances in mediating the anxiolytic activity of benzodiazepines on anxiety remains to be determined. Some of the behavioral effects of benzodiazepines, including their anticonflict effect, however, have been reported to be antagonized by the narcotic antagonist, naloxone (Billingsly and Kibena, 1978;Gylys et al., 1979).
--Thesedata might suggest that anxiolytic activity is mediated by activation of endorphinergic systems.
To test the hypothesis that the anxiogenic discriminative stimulus property of PTZ was related to an antagonistic effect on endorphin systems, generalization to the PTZ discriminative stimulus with naloxone was tested as was antagonism of the PTZ discriminative stimulus with morphine. The failure of naloxone to generalize to the PTZ discriminative stimulus and the failure of morphine to antagonize this stimulus does not support the hypothesis that the anxiogenic discriminative stimulus property of PTZ is related to an antagonistic action on endorphin systems.

Endogenous Benzodiazepine Receptor (Ligand(s)
The demonstration of saturable, high-affinity, stereospecific benzodiazepine binding sites in the central nervous system (Squires and Braestrup, 1977;Mohler and Okada, 1977) suggested the presence of endogenous ligands capable of regulating the neurophysiological processes subserved by these receptors. Several such substances have been suggested as endogenous ligands for benzodiazepine receptors (Colello et al., 1978;Karobath et al., 1978;Skolnick et al., 1978;Mohler et al., 1979;Nixon and Wolf, 1979;Skolnick et al., 1979), however, their potency is too weak to satisfy a neurotransmitter or neuromodulator role.
Although none of the candidates to date satisfy all the criteria for an endogenous ligand of the benzodiazepine receptor, purines and nicotinamide do have benzodiazepine-like properties in vivo. Despite the relatively low affinity, Skolnick et al. (1979) reported that inosine produced a doseand time-related antagonism of PTZ-induced convulsions and proposed that endogenously occurring purines such as inosine could be naturally occurring ligands for benzodiazepine receptors. Although the affinity of nicotinamide for the benzodiazepine receptor is approximately four to five fold lower than affinlties reported for purines (Mohler ~t al., 1979), this naturally occurring substance has been reported to partially restore behavior suppressed by punishment in rats. Marangos et al. (1979) reported that pentylenetetrazol inhibited 3 H-diazepam binding in vitro and suggested that PTZ may exert pharmacological actions by an interaction with the benzodiazepine receptor. Based upon the antagonism of the PTZ discriminative stimulus by benzodiazepines it is possible that the anxiogenic discriminative stimulus property of PTZ is related to its interaction with this receptor. It is possible that PTZ may exert its angiogenic action by inhibiting the binding of a naturally occurring anxiolytic ligand to benzodiazepine receptors or mimicking the action of a naturally occurring anxiety-inducing ligand.
ROS-3663 is structurally related to diazepam, however, unlike diazepam it does not bind to benzodiazepine receptors (Speth ·et · a1., 1979;O'Brien and Spirt, 1980). Furthermore, whereas diazepam has GABAmimetic activity ROS-3663 antagonizes the effect of GABA (Schlosser and Franco, 1979;O'Brien and Spirt, 1980). O'Brien and Spirt (1980) speculated that if the anxiolytic action of benzodiazepines were considered to be mediat~d through an antagonistic action then a molecule structurally similar to ROS-3663 could exist in brain to function as an endogenous anxiety-inducing ligand.
ROS-3663 dose-dependently generalized to the PTZ discriminative stimulus. Furthermore, the discriminative strength of ROS-3663 was attenuated by diazepam. These data provide behavioral evidence that ROS-3663 may structurally resemble a naturally occurring anxiety-inducing ligand.
If this is the case PTZ may act by a similar mechanism as this naturally occurring anxiety-inducing ligand.

SUMMARY AND CONCLUSIONS
In summary, it was deITK)nstrated that laboratory rats can learn to discriminate between a subconvulsive dose (20 mg/kg) of PTZ and saline. A mean of 30 training sessions were required for the animals to reach the discrimination criterion.
The discriminative stimulus produced by PTZ was dose-and time-dependent.
The anxiogenic stimulants cocaine, ROS-3663 and strychnine generalized to the PTZ discriminative stimulus whereas yohimbine partially generalized. The discriminative stimu-1 us produced by cocaine, ROS-3663 and yohimbine was antagonized by diazepam. The discriminative stimulus produced by cocaine was not antagonized by haloperidol. The non-anxiogenic psychomotor stimulants a-amphetamine, methylphenidate and caffeine did not generalize to the PTZ discriminative stimulus. These data support the hypothesis that the discriminative stimulus produced by PTZ in the rat is related to its anxiogenic action in man.
Of the several convulsant drugs tested, only bemegride significnatly generalized to the PTZ discriminative stimulus whereas picrotoxin and 3-mercaptopropionic acid partially generalized. Bicuculline, nicotine or gamrna-hydroxybutyrate did not generalize to the PTZ discriminative stimulus. These data suggest that the PTZ discriminative stimulus is probably not relate<l to a subconvulsant brain state likely to be produced by these drugs.
Tpe PTZ discriminative stimulus was dose-dependently antagonized by benzodiazepine-type, barbiturate-type and propanediol carbamate-type anxiolytics as well as valproic acid.
Because tolerance develops to the sedative but not anxiolytic benzodiazepines, lack of tolerance development to diazepam or chlordiazepoxide in antagonism of the PTZ discriminative stimulus suggests that this property w&s related to their anxiolytic action.
There was a significant correlation between the potency of the drugs effective in antagonizing the PTZ discriminative stimulus and their effective doses in a conflict test used to measure anxiolytic activity as well as their clinically effective doses.
The PTZ discriminative stimulus was not antagonized by the nonanxiolytic anticonvulsants ethosuximide, etomidate, trimethadone or diphenylhydantoin nor the nonanxiolytic central nervous system depressants morphine, ethanol, chlorprornazine, or haloperidol. These data suggest that the PTZ discriminative stimulus is not related to a subconvulsant brain state or nonspecific central nervous system stimulation.
The GABA antagonist, ROS-3663, dose-dependently generalized to the PTZ discriminative stimulus, whereas picrotoxin and 3-mercaptopropionic acid partially generalized. The GABAmimetic drug, valproic acid, antagonized the PTZ dis-crirninative stimulus. These data suggest that the PTZ discriminative stimulus might be mediated through decreased GABA neuronal activity. Because of the known GABA ·mimetic property of benzodiazepines it was suggested that antagonism of the PTZ discriminative stimulus by these drugs may be related to their effect on GABA transmission.
The glycine antagonist, strychnine, generalized to the PTZ discriminative stimulus suggesting the possible involvement of decreased glycinergic activity for the discriminative stimulus property of PTZ. However, in view of the considerable evidence for the involvement of GABA and the lack of effect of PTZ on glycine in vitro it was suggested that general interference with inhibitory transmission -may generate a discriminative stimulus like that produced by PTZ.
Drugs affecting acetylcholine or serotonin systems did not generalize to or antagonize the PTZ discriminative stimulus. These data suggested that the PTZ discriminative stimulus was not related to an effect on these neurotransmitter systems.
Cocaine generalized to the PTZ discriminative stimulus whereas yohimbine and apomorphine partially generalized.
The PTZ discriminative stimulus was not blocked by drugs with anti-catecholaminergic activity. Therefore these data do not support the involvement of catecholamine mechanisms for the discriminative stimulus property of PTZ.
In view of the recent demonstration of benzodiazepine receptors in brain, it was suggested that PTZ may produce its anxiogenic action by interacting with an unknown ligand for the benzodiazepine receptor. PTZ may either mimic a naturally o?curring anxiety-inducing ligand or inhibit the action of a naturally occurring anxiolytic ligand.
In conclusion the data presented in this dissertation support the hypothesis that the discriminative stimulus property of PTZ in the rat is related to its anxiogenic action in man.
It is suggested that the anxiogenic stimulus produced by PTZ is related to deficits in GABA neuronal activity. Tedeschi (1969) suggested that for animal tests to have predictive value as models for a particular human condition, they must fulfill the following criteria: 1. Tests should be selective enough to differentiate false positives, and to distinguish side effects from therapeutic activity.

2.
Tests should be sensitive enough to detect activity of reference agents within a reasonable dose range.
3. The relative potency of reference agents in the animal test should compare with their relative potency in man.

4.
Tolerance should not develop to the measure presumed to reflect therapeutic efficacy.
The PTZ-saline discrimination paradigm described in this dissertation fulfills all of these criteria. Therefore it is suggested that this procedure may represent a new animal model of anxiety that can be used to detect anxiolytic activity of new compounds.
Currently, prevention of PTZ-induced seizures is used as a screening test for anxiolytic drugs. However, all of the drugs that prevent these seizures are not anxiolytics. To date there have been no false positives in the PTZ-saline discrimination procedure. All of the drugs that antagonize the PTZ discriminative stimulus are clinically effective anxiolytics. Currently, there is no animal model to detect anxiogenic side effects of drugs; the PTZ-saline discrimination may provide such a model.
In addition to these potential benefits for drug development, the PTZ-saline discrimination may be used to investigate the neurochemical mechanisms and behavioral factors related to anxiety as well as the mechanism of action of anxiolytic drugs. It is the author's hope that the work reported in this dissertation will contribute towards these ends.