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Tactile discrimination performance deficits following unilateral microinjections of catecholaminergic blockers in the rat
Physiology & Behavior, Vol. 23, pp. 1057-10~3.Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Tactile Discrimination Performance Deficits Following Unilateral Microinjections of Catecholaminergic Blockers in the Rat a LEE HOYMAN
Psychology Department, Indiana University, Bloomington, I N 47401 R e c e i v e d 25 A p r i l 1978 HOYMAN, L. Tactile discrimination performance deficits Jbllowing unilateral microinjections of catecholaminergic blockers in the rat. PHYSIOL. BEHAV. 23(6) 1057-1063, 1979.--Unilateral microinjections of aqueous solutions of acetylcholine, scopolamine, serotonin, methysergide, dopamine, haloperidol, or norepinephrine into the lateral hypothalamic region had no measurable effects on neurological tests or on an appetitive discrimination in which the rats were trained to turn toward the side opposite to a touch to the flank. Unilateral injections of the adrenergic blockers phentolamine or propranolol produced pronounced neurological asymmetries and selectively disrupted only those discrimination trials in which the touch was presented ipsilateral and the required response was a turn contralateral to the injection. The unimpaired performance seen on the remaining trials when the stimulus was contralateral to the injection suggested that the adrenergic blockers disrupted response output processes rather than sensory processes. In a second experiment using different rats and a slightly different task, the dopaminergic blocker haloperidol when injected into the caudate produced a deficit closely similar to that produced by phentolamine in the lateral hypothalamus. Tactile discrimination Phentolamine Caudate Response deficit
Propranolol
IN R E C E N T studies of the effects of nigrostriatal 6-hydroxydopamine (6-OHDA) injections [11] gross neurological deficits similar to those following lateral hypothalamic (LH) lesions [12,13] have been observed. The present study attempted to expand upon these observations by examining the effects of unilateral intrahypothalamic injections of short-acting drugs on both a battery of neurological tests similar to those used in previous studies and on an appetitive lateralized tactile discrimination. Both an agonist and an antagonist of each of the four major known neurotransmitters, acetylcholine (Ach), serotonin (5HT) dopamine (DA), and norepinephrine (NE), were tested although only three of these neurochemicals are thought to have active synapses within the L H region. Cholinergic [8], serotonergic [20], dopaminergic [10,20], and noradrenergic [20] fibers have been identified anatomically within the L H and all but the DA systems are thought to have terminals in the LH. Behavioral studies of the effects of injections of 6-OHDA into the L H and related areas have shown profound deficits including loss of orienting responses to various sensory stimuli [ 11], loss of visual placing [ 15], decreased predatory aggression [9], deficits in instrumental responding [4], deficits in responding for food and water [14], and loss of active
Haloperidol
LH
Orienting Response
avoidance [17]. However, these effects, which seem to suggest sensorimotor deficits, cannot be attributed specifically to a change in DA-containing or NE-containing neurons since 6-OHDA affects both types of neurons approximately equally. Experiments which have alternately protected the N E system or the DA system from the toxic effects of the 6-OHDA have suggested that DA is the relevant transmitter with respect to these deficits. F o r instance, it has been reported that selective reduction of DA in young or mature rats interferes with the acquisition and performance of an active avoidance response, while selective depletion of N E either has no effect or facilitates avoidance [3,16]. However, the use of a chronic treatment like 6-OHDA opens up the possibility of recovery of function effects. Thus, an alternative explanation for these observed differences between the DA and NE systems might be that these systems show different physiological or behavioral recovery of function effects. The present study sought to avoid this problem by only using drugs with a short duration of action. EXPERIMENT 1 In Experiment 1 the effects of unilateral microinjections were examined on a tactile discrimination task which was
1I would like to thank my dissertation committee (Drs. G. P. Frommer, G. A. Heise, J. E. Kelsey and R. P. Maickel) for their assistance. This report is a modified version of the dissertation.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/121057-07502.00/0
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HOYMAN
designed to separate operationally stimulus input from response output processes on each trial [7]. Rats were trained to make a head-turn response toward the side opposite the flank touched on each of a randomized succession of trials in order to receive sucrose solution reinforcement. The animals were trained to a near perfect accuracy baseline and consequently it was only possible to measure impairments in performance. At least four different outcomes are possible for such a task. If, for instance, the unilateral injection disrupted sensory processes exclusively, then an impairment would be predicted only on trials in which the touch was presented on the presumably affected side, the side contralateral to the injection, and the required response was a head-turn toward the ipsilateral side. Similarly, a purely motor impairment should be evident only on trials requiring a response contralateral to the injection following an ipsilateral touch. A combined impairment of both sensory and motor processes should affect all trials because either the stimulus or the response occurs contralateral to the injection on every trial, Finally, no impairment on any trial would be predicted if the injection disrupted only intrahemispheric sensorimotor integration [19].
METHOD
Su/~jects Seven female albino rats weighing approximately 250 g at the time of surgery served as subjects. The rats were singly housed, were allowed 30 min access to water after each day's session, and had constant access to Purina rat chow.
Apparatus Discrimination training took place in a 17x9x 11 cm clear plastic chamber, the sides of which had 10×8 cm holes interrupted by vertical plastic rods spaced 3 cm apart. Three 1.3-cm-dia. holes, one centered on the front wall 6 cm from the floor and one on each side wall 6 cm from the floor and 2 cm from the front wall, had photocells and 28 volt miniature lamps positioned to detect nose-poke responses. Behind each side hole was a drinking tube filled with 8% sucrose solution which was accessible only when a circular aluminum door was rotated open by a 30 RPM clock motor. A drinkometer circuit between the drinking tubes and the grid floor recorded licks on the tubes. Tactile stimuli were presented manually by the experimenter by means of Von Frey hairs made of 0.15-mm-dia. piano wires approximately 15 cm long with 2-mm-dia. styrofoam balls cemented on the ends. During each experimental session the experimenter sat with Von Frey hairs poised in both hands which were resting on soft pads situated at the level of the open sides of the chamber. Colored lights cued the experimenter to touch the rat's flank through the bars of the chamber on either the right or left side. BRS 200 series solid state programming equipment controlled access to the drinking tubes, cued the experimenter, and recorded response events in such a manner that no auditory cues were confounded with the tactile stimuli.
Sltl~-'~'l:v Prior to training each rat was implanted with bilateral double-barreled cannulae in the LH. Each rat was anes-
thetized with 2.5 cc/Kg Equi-Thesin and 5 mg methyl atropine. The rat was then blinded by enucleation, and its head was fixed in a stereotaxic instrument which oriented the top of the skull horizontally [5]. The skull was then bared and the outer 25 ga cannulae were implanted 3.0 mm caudal to bregma, 2.0 mm lateral to the midline, and 7.6 mm below dura. The cannulae were fixed to the skull with dental cement. The inner cannulae were made of 31 ga stainless steel tubing which resulted in a very loose fit inside the 25 ga outer tubing. A 5 cm length of 0.023-in-dia. tubing was cemented to the top of each inner cannulae and the other end was attached to a 25/zl syringe. When not in use the outer cannulae were plugged with dummy inner cannulae and the entire assembly was covered with silicone glue.
Drugs The effects of an agonist and a relatively short-acting receptor blocker of the cholinergic, serotonergic, dopaminergic, and noradrenergic systems were examined. The agonist in each case was selected to be the amine itself in the forms: acetylcholine chloride, serotonin hydrogen oxalate, dopamine hydrochloride, and norepinephrine bitartrate. The antagonists of the respective neurotransmitters selected were: scopolamine, methysergide, haloperidol, and phentolamine and propranolol (~- and/3 adrenergic blockers). All drugs were prepared as solutions in distilled water with the exception of haloperidol which was prepared in a solution of lactic acid (pH=5.5). Four of the saline injections also had lactic acid added to control for the haloperidol vehicle. Drug solutions were typically administered in volumes of 2 p~l or 3 /~1. The requirement of having very loose fitting inner cannulae so that insertion could be made very quickly to avoid traumatizing the rat meant that drug dosages were necessarily imprecise. A large proportion of every injection flowed into the space between the inner and outer cannulae. Thus, the amount of drug which reached the brain during the short testing period was probably always considerably less than the amount injected. All dosages were between 5/~g and 90 p~g. Four 40 p~g, three 60 /~g, and one 80 /~g dosage of phentolamine were given; while four 40 p~g, two 60/~g, two 80 ~g, and five 90 /~g dosages of propranoloi were given. Solubility limits limited methysergide to five 10/zg and five 5 /~g administrations, and haloperidol to one 15 p.g, one 30/~g, four 40/~g, and six 60/~g administrations. The occurrence of seizures limited Ach to a mean dose of 17 ~g. The mean doses of the remaining drugs were: scopolamine, 33 t~g; NE, 67/xg; DA, 56/zg; and 5HT, 32/zg.
Procedure During initial training, each session continued until the rat had obtained 12 reinforcements on each side tube (correction procedure). A center hole observing response which consisted of the rat holding its nose in the hole for at least 0.5 sec was required to initiate trials. Each trial began with the experimenter presenting a single brief 0.5 g Von Frey hair touch to the rat's middle flank. After being touched the rat was required to break the photocell beam in the hole on the side opposite to that touched within 4 sec in order to be reinforced with 4 sec of licking. An error of omission was defined as a failure to respond in either side hole within 4 sec after the touch. Responding in the hole on the same side as
T A C T I L E DISCRIMINATION F O L L O W I N G MICROINJECTIONS the touch within the 4 sec trial period was an error of commission. All nontrial side hole responses were without effect and were counted as intertrial (ITI) responses. As each of ths rats approached the baseline accuracy criterion of no more than 3 errors per session for two consecutive days, a noncorrection procedure was put in effect for the remainder of the experiment. At least two consecutive baseline sessions with 3 or fewer errors always preceeded each drug session. The order of presentation of the different drugs and dosages was quasirandomized so that each drug was given approximately equal representation as the first drug given in a particular cannula, second drug, last drug, etc. To minimize handling trauma to the rat, the injections were made rapidly in 1-3 min.
sections were mounted and stained with cresyl violet. Photographic prints were made by using the stained sections as negatives in a photographic enlarger which had a No. 8k2 Wratten filter on the light source to remove the blue and violet wavelengths.
Data Analyses
Neurological Tests Each of the rats underwent a battery of neurological tests [ 11, 12, 13] to assess the effects of the injections. Orientation tests were performed in the home cage with a small soft long-handled paintbrush. The rat was lightly touched on either the snout, shoulder area, lower foreleg, middle flank, rear flank, or lower hindleg. Orientation responses were judged as strong if the rat localized the stimulus by turning toward or biting the probe, as weak if any movement at all in the direction of the probe occurred, and as absent if no movement or movement away from the probe occurred. Normal rats rarely failed to respond strongly to a touch presented to this manner. Further tests included observations of open field locomotion, noting body side preference in climbing the tail, and behavior hanging from a suspended rod.
Histology After testing had been completed the rats received an overdose of anesthetic and were perfused intracardially with isotonic saline followed by 10% formol-saline. The brains were extracted, and frozen slices 40 p. thick were made. The
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Statistical analyses were made by means of matched group two-tailed t-tests which simply compared the performance of each of the rats on the day before the drugging to its performance under the influence of the drug for each particular drug. All dosage levels of each drug were pooled in the data analyses to simplify presentation. Because of programming constraints the response latency measure was always the time it took the rat to make any response, correct or incorrect, following a touch contralateral or a touch ipsilateral to the injection.
RESULTS AND DISCUSSION Figure 1 provides a summary of the effects of the unilateral drug injections into the LH region. Control injections of saline (or saline and lactic acid) did not produce any significant changes in performance and similarly the drugs Ach, scopolamine, 5HT, methysergide, DA, haloperidoi, and NE all produced no significant changes in the number of correct responses at any of the dosages administered. Of course any tendency to improve performance as might be predicted following NE or DA administration would not show up on the present discrimination since it was designed to measure only decrements. The two adrenergic blockers, however, produced large selective disruptions of one type of trial. Phentolamine, the a adrenergic blocker, caused a significant decrement in correct responses on those trials when the touch was ipsilateral to the unilateral injection and the required response was con-
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Propranolol
Phentolamine
FIG. 1. Mean change in correct responses contralateral and correct responses ipsilteral to unilateral injections into the LH on a discrimination task which required a response on the side opposite to a touch. Mean change was between previous day's baseline performance and performance beginning about 5 min following injection.
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HOYMAN
tralateral (p<0.001) while causing no change in correct responses on those trials when the touch was contralateral and the response ipsilateral to the injection (p<0.4). Similarly, propranolol, the/3 adrenergic blocker, produced a large decrement on trials when the required response was contralateral to the injection (p<0.001) and no change on trials when the stimulus was contralateral (p<0.5). Of the total number of contralateral errors, 8(1% were errors of commission following phentolamine and 83% were errors of commission following propranolol. The pattern of results produced by the adrenergic blockers corresponds to the prediction made for a deficit in response output processes because the rats failed to respond by turning contralateral to the injection on trials when the touch was on the presumably unaffected ipsilateral side. The results are inconsistent with an interpretation based on a deficit in sensory processing or sensory attention because the rats were unimpaired when the touch was on the presumably affected side, contralateral to the injection. No dose-response relationship was apparent for any of the drugs given. The grand mean baseline response latency was about 0.66 sec. Table 1 shows the effects of the adrenergic blockers on response latency. Out of the nine drugs only the two adrenergic blockers produced any effects on the neurological battery. Orientation responses were scored as absent following 71% of those touches contralateral to the injection and as weak following the remainder. Touches ipsilateral to the injection produced 87% strong responses. The rats were also observed to make more ipsiversively directed movements on the open field, in the home cage, while hanging by the tail, or while hanging by the forepaws. Histological analysis revealed all of the cannulae tips to be located either in the LH itself or in the tissue immediately lateral to the LH. Figure 2 presents the cannulae tip placements in four typical rats. As can be seen, the cannulae themselves caused considerable damage. Very little mechanical damage, however, was observed at the sites of the injections immediately below the cannulae. This lack of damage was probably due in part to the tendency for the fluid to flow back up into the outer cannulae rather than being forced into the tissue. EXPERIMENT 2 Experiment 2 was designed to test the effects of an adrenergic blocker and a dopaminergic blocker on a control tactile discrimination in which both the touch stimulus and the required response occurred on the same side of the body on each trial. The effects of both phentolamine and
FIG. 2. Representative Nissl-stained sections showing cannulae tip placements in four rats.
TABLE 1 MEAN DRUG-INDUCEDCHANGE IN RESPONSE LATENCY Saline
Phentolamine
Propranolol
Latency to respond to ipsilateral touches
0.054 sec (p<0.2)
+0.87 q~<0.01}
+0.57 (p<0.008)
Latency to respond to contralateral touches
-0.071 sec (p<0.07)
+0.39 (p<0.03)
+0.22 qy<0.1)
TACTILE
DISCRIMINATION
FOLLOWING
1061
MICROINJECTIONS
y,h”,“l’n”,
LH
Sal ine
:y/iOp;
Phentolami ne
CAUDATE
FIG. 3. Mean change in correct responses contralateral and correct responses ipsilateral to unilateral central injections on a discrimination task which required a response on the same side as a touch.
haloperidol in the LH and in the caudate nucleus were examined on this task in order to test several possibilities left over from Experiment 1, including, most notably, the possibility interfere with that haloperidol in the LH might intrahemispheric sensorimotor integration, an effect which would not be predicted to show up in the task of Experiment 1. METHOD
Subjects
Four naive female albino rats similar to those used in Experiment 1 served as subjects. Apparatus
The apparatus was the same as that used in Experiment 1 except that the lights which cued the experimenter to touch one side or the other were reversed. Surgery
The surgical procedures were identical to those in Experiment 1 except that each rat received four cannulae. Besides bilateral cannulae in the LH each rat received additional bilateral cannulae in the caudate nucleus at 1.0 mm rostral to bregma, 2.8 mm lateral to the midline, and 4.3 mm below dura. Procedure
Shaping, training and testing were identical to that in Experiment 1 except that the rats were trained to turn toward the side touched by a probe. Injections of haloperidol, phentolamine, saline, and saline and lactic acid were made into
both the caudate and LH. Mean dosages were: haloperidol in the LH, 50 pg; phentolamine in the LH, 50 pg; haloperidol in the caudate, 51 pg; phentolamine in the caudate, 48 pg. RESULTS AND DISCUSSION
Figure 3 summarizes the effects of the injections into the two brain areas. As in Experiment 1, haloperidol had no effect when injected into the LH. Phentolamine injections into the LH produced a large decrement in correct responses on trials in which the touch and required response were both contralateral to the injection @<0.003), but produced no significant effects when the stimulus and required response occurred on the side ipsilateral to the injection (p
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HOYMAN
FIG. 4. Representative Nissl-stained sections showing cannulae tip placements in the caudate nucleus in two rats.
DISCUSSION In Experiment 1, it was found that only the a and/3 adrenergic blockers phentolamine and propranolol caused neurological asymmetries when unilaterally injected into the LH, while Ach, 5HT, DA, NE, scopolamine, methysergide, or haloperidol injections produced no observable changes in behavior. Injection of either of the adrenergic blockers into the LH caused a disruption of discrimination trials in which the touch stimulus was ipsilateral and the required response was a turn toward the side contralateral to the injection. Between the tasks in Experiment 1 and Experiment 2 the adrenergic blocker phentolamine always prevented the execution of contraversive head-turn responses regardless of whether the touch was ipsilateral or contralateral to the in-
jection. This pattern of deficits suggests that an appropriate characterization of the function of the adrenergic system in the LH might be as a response generating or response mediating system. Also a lateralized loss of the incentive value or desirability of the sucrose solution could produce the observed results. The present results and interpretation are consistent with various theories of a catecholaminergic response-mediating or arousal system in the brain [1,2]. The present data are also consistent with several theories of LH function including, for example, one [6] which suggested that LH stimulation activates species-specific approach response sequences, and one that suggested that the LH mediates incentive motivation ]181. An important question in any study of central drug injections is the extent to which the drugs diffuse and spread in the brain. In the present experiment extremely large amounts of drugs were injected opening up the possibility of widespread diffusion. Thus, the present study does not claim to have localized the locus of the behavioral effects in the LH merely because that was where the cannulae were placed. The main emphasis of the present study was behavioral, and the only important behavioral requirement for the drug injections was that they be reasonably unilateral. However, the observation that the onset of the easily observed postural asymmetries became evident at times while the experimenter was still in the process of injecting the rat, with the longest latencies being on the order of 5 rain, suggests that the site of action of the effective drugs may have indeed been relatively near the cannulae tips. One final issue which must be addressed is the question of the neurochemical specificity of particularly the rather high doses of phentolamine, propranolol, and haloperidol used in this experiment. Certainly the present methods do not exclude the possibility that one or more of the aforementioned drugs produced their effects through a nonspecific anesthesis. Indeed, in pilot work, procaine in the LH was shown to be effective in producing the response deficit. However, the fact that haloperidol was ineffective in the LH and the fact that phentolamine was equally ineffective in the caudate together with the fact that these results agree with histochemical mappings of the N E and DA systems argues strongly that these drugs produced their behavioral effects through a neurotransmitter-specific action.
REFERENCES I. Campbell, B. A., L. D. Lytle and H. C. Fibiger. Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science 166: 637-638, 1969. 2. Carlton, P. L. Cholinergic mechanisms in the control of behavior by the brain. Psychol. Rev. 70: 1%39, 1963. 3. Cooper, B. R., G. R. Breese, L. D. Grant and J. L. Howard. Effects of 6-hydroxydopamine treatments on active avoidance responding: evidence for involvement of brain dopamine. J. Pharmac. exp. Ther. 185: 358-370, 1973. 4. Fibiger, H. C., A. G. Phillips and A. P. Zis. Deficits in instrumental responding after 6-hydroxydopamine lesions of the nigrostriatal dopaminergic projection. Pharmac. Biochem. Behav. 2: 87-96, 1974. 5. Frommer, G. P. Modified non-traumatic headholder. Behav. Meth. Res. lnstrum. 8: 225-226, 1971. 6. Glickman, S. E. and B. B. Schiff. A biological theory of reinforcement. Psychol. Rev. 74: 81-109, 1967.
7. Hoyman, L., G. D. Weese and G. P. Frommer. Tactile discrimination performance deficits following neglect-producing unilateral lateral hypothalamic lesions in the rat. Physiol. Behay. 22: 13%147, 1979. 8. Jacobowitz, D. M. and M. Palkovits. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. J. comp. Neurol. 157: 13-28, 1974. 9. Jimerson, D. and D. J. Reis. Effects of intrahypothalamic injection of 6-OHDA on predatory aggression in rat. Brain Res. 61: 141-152, 1976. 10. Lindvall, O. and A. Bjorklund. The organization of the ascending catecholamine neuron systems in the rat brain. Acre physiol. scand. Suppl. 412: 1-48, 1974. II. Marshall, J. F., J. S. Richardson and P. Teitelbaum. Nigrostriatal bundle damage and the lateral hypothalamic syndrome. J. comp. physiol. Psychol. 87: 808-830, 1974. 12. Marshall, J. F. and P. Teitelbaum. Further analysis of sensory inattention following hypothalamic damage in rats. J. comp. physiol. Psychol. 86: 375-395, 1974.
TACTILE DISCRIMINATION
FOLLOWING
MICROINJECTIONS
13. Marshall, J. F., B. H. Turner and P. Teitelbaum. Sensory neglect produced by lateral hypothalamic damage in rats. Science 174: 523--525, 1971. 14. Martin, G. E. and R. D. Myers. Action of intrahypothalamic 6-hydroxydopamine on motivated responding for food and water in the rat. Pharmac. Biochern. Behav. 2: 393-399, 1974. 15. Sechzer, J. A., G. N. Ervin and G. P. Smith. Loss of visual placing in rats after lateral hypothalamic microinjections of 6-hydroxydopamine. Expl. Neurol. 41: 723-737, 1973. 16. Smith, G. P., B. E. Levin and G. N. Ervin. Loss of active avoidance responding after lateral hypothalamic injections of 6-hydroxydopamine. Brain Res. 88: 483-498, 1975.
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17. Smith, R. D., B. R. Cooper and G. R. Breese. Growth and behavioral changes in developing rats treated intracisternally with 6-hydroxydopamine: evidence for involvement of brain dopamine. J. Pharmac. exp. Ther. 185: 609-619, 1973 18. Trowill, J. A., J. Panksepp and R. Gandelman. An incentive model of rewarding brain stimulation. Psychol. Rev. 76: 264281, 1969. 19. Turner, B. H. Sensorimotor syndrome produced by lesions of the amygdala and lateral hypothalamus. J. cornp, physiol. Psychol. 82: 37-47, 1973. 20. Ungerstedt, U. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta physiol, scand. Suppl. 367: 1-48, 1971
Tactile Discrimination Performance Deficits Following Unilateral Microinjections of Catecholaminergic Blockers in the Rat a LEE HOYMAN
Psychology Department, Indiana University, Bloomington, I N 47401 R e c e i v e d 25 A p r i l 1978 HOYMAN, L. Tactile discrimination performance deficits Jbllowing unilateral microinjections of catecholaminergic blockers in the rat. PHYSIOL. BEHAV. 23(6) 1057-1063, 1979.--Unilateral microinjections of aqueous solutions of acetylcholine, scopolamine, serotonin, methysergide, dopamine, haloperidol, or norepinephrine into the lateral hypothalamic region had no measurable effects on neurological tests or on an appetitive discrimination in which the rats were trained to turn toward the side opposite to a touch to the flank. Unilateral injections of the adrenergic blockers phentolamine or propranolol produced pronounced neurological asymmetries and selectively disrupted only those discrimination trials in which the touch was presented ipsilateral and the required response was a turn contralateral to the injection. The unimpaired performance seen on the remaining trials when the stimulus was contralateral to the injection suggested that the adrenergic blockers disrupted response output processes rather than sensory processes. In a second experiment using different rats and a slightly different task, the dopaminergic blocker haloperidol when injected into the caudate produced a deficit closely similar to that produced by phentolamine in the lateral hypothalamus. Tactile discrimination Phentolamine Caudate Response deficit
Propranolol
IN R E C E N T studies of the effects of nigrostriatal 6-hydroxydopamine (6-OHDA) injections [11] gross neurological deficits similar to those following lateral hypothalamic (LH) lesions [12,13] have been observed. The present study attempted to expand upon these observations by examining the effects of unilateral intrahypothalamic injections of short-acting drugs on both a battery of neurological tests similar to those used in previous studies and on an appetitive lateralized tactile discrimination. Both an agonist and an antagonist of each of the four major known neurotransmitters, acetylcholine (Ach), serotonin (5HT) dopamine (DA), and norepinephrine (NE), were tested although only three of these neurochemicals are thought to have active synapses within the L H region. Cholinergic [8], serotonergic [20], dopaminergic [10,20], and noradrenergic [20] fibers have been identified anatomically within the L H and all but the DA systems are thought to have terminals in the LH. Behavioral studies of the effects of injections of 6-OHDA into the L H and related areas have shown profound deficits including loss of orienting responses to various sensory stimuli [ 11], loss of visual placing [ 15], decreased predatory aggression [9], deficits in instrumental responding [4], deficits in responding for food and water [14], and loss of active
Haloperidol
LH
Orienting Response
avoidance [17]. However, these effects, which seem to suggest sensorimotor deficits, cannot be attributed specifically to a change in DA-containing or NE-containing neurons since 6-OHDA affects both types of neurons approximately equally. Experiments which have alternately protected the N E system or the DA system from the toxic effects of the 6-OHDA have suggested that DA is the relevant transmitter with respect to these deficits. F o r instance, it has been reported that selective reduction of DA in young or mature rats interferes with the acquisition and performance of an active avoidance response, while selective depletion of N E either has no effect or facilitates avoidance [3,16]. However, the use of a chronic treatment like 6-OHDA opens up the possibility of recovery of function effects. Thus, an alternative explanation for these observed differences between the DA and NE systems might be that these systems show different physiological or behavioral recovery of function effects. The present study sought to avoid this problem by only using drugs with a short duration of action. EXPERIMENT 1 In Experiment 1 the effects of unilateral microinjections were examined on a tactile discrimination task which was
1I would like to thank my dissertation committee (Drs. G. P. Frommer, G. A. Heise, J. E. Kelsey and R. P. Maickel) for their assistance. This report is a modified version of the dissertation.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/121057-07502.00/0
1058
HOYMAN
designed to separate operationally stimulus input from response output processes on each trial [7]. Rats were trained to make a head-turn response toward the side opposite the flank touched on each of a randomized succession of trials in order to receive sucrose solution reinforcement. The animals were trained to a near perfect accuracy baseline and consequently it was only possible to measure impairments in performance. At least four different outcomes are possible for such a task. If, for instance, the unilateral injection disrupted sensory processes exclusively, then an impairment would be predicted only on trials in which the touch was presented on the presumably affected side, the side contralateral to the injection, and the required response was a head-turn toward the ipsilateral side. Similarly, a purely motor impairment should be evident only on trials requiring a response contralateral to the injection following an ipsilateral touch. A combined impairment of both sensory and motor processes should affect all trials because either the stimulus or the response occurs contralateral to the injection on every trial, Finally, no impairment on any trial would be predicted if the injection disrupted only intrahemispheric sensorimotor integration [19].
METHOD
Su/~jects Seven female albino rats weighing approximately 250 g at the time of surgery served as subjects. The rats were singly housed, were allowed 30 min access to water after each day's session, and had constant access to Purina rat chow.
Apparatus Discrimination training took place in a 17x9x 11 cm clear plastic chamber, the sides of which had 10×8 cm holes interrupted by vertical plastic rods spaced 3 cm apart. Three 1.3-cm-dia. holes, one centered on the front wall 6 cm from the floor and one on each side wall 6 cm from the floor and 2 cm from the front wall, had photocells and 28 volt miniature lamps positioned to detect nose-poke responses. Behind each side hole was a drinking tube filled with 8% sucrose solution which was accessible only when a circular aluminum door was rotated open by a 30 RPM clock motor. A drinkometer circuit between the drinking tubes and the grid floor recorded licks on the tubes. Tactile stimuli were presented manually by the experimenter by means of Von Frey hairs made of 0.15-mm-dia. piano wires approximately 15 cm long with 2-mm-dia. styrofoam balls cemented on the ends. During each experimental session the experimenter sat with Von Frey hairs poised in both hands which were resting on soft pads situated at the level of the open sides of the chamber. Colored lights cued the experimenter to touch the rat's flank through the bars of the chamber on either the right or left side. BRS 200 series solid state programming equipment controlled access to the drinking tubes, cued the experimenter, and recorded response events in such a manner that no auditory cues were confounded with the tactile stimuli.
Sltl~-'~'l:v Prior to training each rat was implanted with bilateral double-barreled cannulae in the LH. Each rat was anes-
thetized with 2.5 cc/Kg Equi-Thesin and 5 mg methyl atropine. The rat was then blinded by enucleation, and its head was fixed in a stereotaxic instrument which oriented the top of the skull horizontally [5]. The skull was then bared and the outer 25 ga cannulae were implanted 3.0 mm caudal to bregma, 2.0 mm lateral to the midline, and 7.6 mm below dura. The cannulae were fixed to the skull with dental cement. The inner cannulae were made of 31 ga stainless steel tubing which resulted in a very loose fit inside the 25 ga outer tubing. A 5 cm length of 0.023-in-dia. tubing was cemented to the top of each inner cannulae and the other end was attached to a 25/zl syringe. When not in use the outer cannulae were plugged with dummy inner cannulae and the entire assembly was covered with silicone glue.
Drugs The effects of an agonist and a relatively short-acting receptor blocker of the cholinergic, serotonergic, dopaminergic, and noradrenergic systems were examined. The agonist in each case was selected to be the amine itself in the forms: acetylcholine chloride, serotonin hydrogen oxalate, dopamine hydrochloride, and norepinephrine bitartrate. The antagonists of the respective neurotransmitters selected were: scopolamine, methysergide, haloperidol, and phentolamine and propranolol (~- and/3 adrenergic blockers). All drugs were prepared as solutions in distilled water with the exception of haloperidol which was prepared in a solution of lactic acid (pH=5.5). Four of the saline injections also had lactic acid added to control for the haloperidol vehicle. Drug solutions were typically administered in volumes of 2 p~l or 3 /~1. The requirement of having very loose fitting inner cannulae so that insertion could be made very quickly to avoid traumatizing the rat meant that drug dosages were necessarily imprecise. A large proportion of every injection flowed into the space between the inner and outer cannulae. Thus, the amount of drug which reached the brain during the short testing period was probably always considerably less than the amount injected. All dosages were between 5/~g and 90 p~g. Four 40 p~g, three 60 /~g, and one 80 /~g dosage of phentolamine were given; while four 40 p~g, two 60/~g, two 80 ~g, and five 90 /~g dosages of propranoloi were given. Solubility limits limited methysergide to five 10/zg and five 5 /~g administrations, and haloperidol to one 15 p.g, one 30/~g, four 40/~g, and six 60/~g administrations. The occurrence of seizures limited Ach to a mean dose of 17 ~g. The mean doses of the remaining drugs were: scopolamine, 33 t~g; NE, 67/xg; DA, 56/zg; and 5HT, 32/zg.
Procedure During initial training, each session continued until the rat had obtained 12 reinforcements on each side tube (correction procedure). A center hole observing response which consisted of the rat holding its nose in the hole for at least 0.5 sec was required to initiate trials. Each trial began with the experimenter presenting a single brief 0.5 g Von Frey hair touch to the rat's middle flank. After being touched the rat was required to break the photocell beam in the hole on the side opposite to that touched within 4 sec in order to be reinforced with 4 sec of licking. An error of omission was defined as a failure to respond in either side hole within 4 sec after the touch. Responding in the hole on the same side as
T A C T I L E DISCRIMINATION F O L L O W I N G MICROINJECTIONS the touch within the 4 sec trial period was an error of commission. All nontrial side hole responses were without effect and were counted as intertrial (ITI) responses. As each of ths rats approached the baseline accuracy criterion of no more than 3 errors per session for two consecutive days, a noncorrection procedure was put in effect for the remainder of the experiment. At least two consecutive baseline sessions with 3 or fewer errors always preceeded each drug session. The order of presentation of the different drugs and dosages was quasirandomized so that each drug was given approximately equal representation as the first drug given in a particular cannula, second drug, last drug, etc. To minimize handling trauma to the rat, the injections were made rapidly in 1-3 min.
sections were mounted and stained with cresyl violet. Photographic prints were made by using the stained sections as negatives in a photographic enlarger which had a No. 8k2 Wratten filter on the light source to remove the blue and violet wavelengths.
Data Analyses
Neurological Tests Each of the rats underwent a battery of neurological tests [ 11, 12, 13] to assess the effects of the injections. Orientation tests were performed in the home cage with a small soft long-handled paintbrush. The rat was lightly touched on either the snout, shoulder area, lower foreleg, middle flank, rear flank, or lower hindleg. Orientation responses were judged as strong if the rat localized the stimulus by turning toward or biting the probe, as weak if any movement at all in the direction of the probe occurred, and as absent if no movement or movement away from the probe occurred. Normal rats rarely failed to respond strongly to a touch presented to this manner. Further tests included observations of open field locomotion, noting body side preference in climbing the tail, and behavior hanging from a suspended rod.
Histology After testing had been completed the rats received an overdose of anesthetic and were perfused intracardially with isotonic saline followed by 10% formol-saline. The brains were extracted, and frozen slices 40 p. thick were made. The
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Statistical analyses were made by means of matched group two-tailed t-tests which simply compared the performance of each of the rats on the day before the drugging to its performance under the influence of the drug for each particular drug. All dosage levels of each drug were pooled in the data analyses to simplify presentation. Because of programming constraints the response latency measure was always the time it took the rat to make any response, correct or incorrect, following a touch contralateral or a touch ipsilateral to the injection.
RESULTS AND DISCUSSION Figure 1 provides a summary of the effects of the unilateral drug injections into the LH region. Control injections of saline (or saline and lactic acid) did not produce any significant changes in performance and similarly the drugs Ach, scopolamine, 5HT, methysergide, DA, haloperidoi, and NE all produced no significant changes in the number of correct responses at any of the dosages administered. Of course any tendency to improve performance as might be predicted following NE or DA administration would not show up on the present discrimination since it was designed to measure only decrements. The two adrenergic blockers, however, produced large selective disruptions of one type of trial. Phentolamine, the a adrenergic blocker, caused a significant decrement in correct responses on those trials when the touch was ipsilateral to the unilateral injection and the required response was con-
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FIG. 1. Mean change in correct responses contralateral and correct responses ipsilteral to unilateral injections into the LH on a discrimination task which required a response on the side opposite to a touch. Mean change was between previous day's baseline performance and performance beginning about 5 min following injection.
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tralateral (p<0.001) while causing no change in correct responses on those trials when the touch was contralateral and the response ipsilateral to the injection (p<0.4). Similarly, propranolol, the/3 adrenergic blocker, produced a large decrement on trials when the required response was contralateral to the injection (p<0.001) and no change on trials when the stimulus was contralateral (p<0.5). Of the total number of contralateral errors, 8(1% were errors of commission following phentolamine and 83% were errors of commission following propranolol. The pattern of results produced by the adrenergic blockers corresponds to the prediction made for a deficit in response output processes because the rats failed to respond by turning contralateral to the injection on trials when the touch was on the presumably unaffected ipsilateral side. The results are inconsistent with an interpretation based on a deficit in sensory processing or sensory attention because the rats were unimpaired when the touch was on the presumably affected side, contralateral to the injection. No dose-response relationship was apparent for any of the drugs given. The grand mean baseline response latency was about 0.66 sec. Table 1 shows the effects of the adrenergic blockers on response latency. Out of the nine drugs only the two adrenergic blockers produced any effects on the neurological battery. Orientation responses were scored as absent following 71% of those touches contralateral to the injection and as weak following the remainder. Touches ipsilateral to the injection produced 87% strong responses. The rats were also observed to make more ipsiversively directed movements on the open field, in the home cage, while hanging by the tail, or while hanging by the forepaws. Histological analysis revealed all of the cannulae tips to be located either in the LH itself or in the tissue immediately lateral to the LH. Figure 2 presents the cannulae tip placements in four typical rats. As can be seen, the cannulae themselves caused considerable damage. Very little mechanical damage, however, was observed at the sites of the injections immediately below the cannulae. This lack of damage was probably due in part to the tendency for the fluid to flow back up into the outer cannulae rather than being forced into the tissue. EXPERIMENT 2 Experiment 2 was designed to test the effects of an adrenergic blocker and a dopaminergic blocker on a control tactile discrimination in which both the touch stimulus and the required response occurred on the same side of the body on each trial. The effects of both phentolamine and
FIG. 2. Representative Nissl-stained sections showing cannulae tip placements in four rats.
TABLE 1 MEAN DRUG-INDUCEDCHANGE IN RESPONSE LATENCY Saline
Phentolamine
Propranolol
Latency to respond to ipsilateral touches
0.054 sec (p<0.2)
+0.87 q~<0.01}
+0.57 (p<0.008)
Latency to respond to contralateral touches
-0.071 sec (p<0.07)
+0.39 (p<0.03)
+0.22 qy<0.1)
TACTILE
DISCRIMINATION
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MICROINJECTIONS
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FIG. 3. Mean change in correct responses contralateral and correct responses ipsilateral to unilateral central injections on a discrimination task which required a response on the same side as a touch.
haloperidol in the LH and in the caudate nucleus were examined on this task in order to test several possibilities left over from Experiment 1, including, most notably, the possibility interfere with that haloperidol in the LH might intrahemispheric sensorimotor integration, an effect which would not be predicted to show up in the task of Experiment 1. METHOD
Subjects
Four naive female albino rats similar to those used in Experiment 1 served as subjects. Apparatus
The apparatus was the same as that used in Experiment 1 except that the lights which cued the experimenter to touch one side or the other were reversed. Surgery
The surgical procedures were identical to those in Experiment 1 except that each rat received four cannulae. Besides bilateral cannulae in the LH each rat received additional bilateral cannulae in the caudate nucleus at 1.0 mm rostral to bregma, 2.8 mm lateral to the midline, and 4.3 mm below dura. Procedure
Shaping, training and testing were identical to that in Experiment 1 except that the rats were trained to turn toward the side touched by a probe. Injections of haloperidol, phentolamine, saline, and saline and lactic acid were made into
both the caudate and LH. Mean dosages were: haloperidol in the LH, 50 pg; phentolamine in the LH, 50 pg; haloperidol in the caudate, 51 pg; phentolamine in the caudate, 48 pg. RESULTS AND DISCUSSION
Figure 3 summarizes the effects of the injections into the two brain areas. As in Experiment 1, haloperidol had no effect when injected into the LH. Phentolamine injections into the LH produced a large decrement in correct responses on trials in which the touch and required response were both contralateral to the injection @<0.003), but produced no significant effects when the stimulus and required response occurred on the side ipsilateral to the injection (p
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FIG. 4. Representative Nissl-stained sections showing cannulae tip placements in the caudate nucleus in two rats.
DISCUSSION In Experiment 1, it was found that only the a and/3 adrenergic blockers phentolamine and propranolol caused neurological asymmetries when unilaterally injected into the LH, while Ach, 5HT, DA, NE, scopolamine, methysergide, or haloperidol injections produced no observable changes in behavior. Injection of either of the adrenergic blockers into the LH caused a disruption of discrimination trials in which the touch stimulus was ipsilateral and the required response was a turn toward the side contralateral to the injection. Between the tasks in Experiment 1 and Experiment 2 the adrenergic blocker phentolamine always prevented the execution of contraversive head-turn responses regardless of whether the touch was ipsilateral or contralateral to the in-
jection. This pattern of deficits suggests that an appropriate characterization of the function of the adrenergic system in the LH might be as a response generating or response mediating system. Also a lateralized loss of the incentive value or desirability of the sucrose solution could produce the observed results. The present results and interpretation are consistent with various theories of a catecholaminergic response-mediating or arousal system in the brain [1,2]. The present data are also consistent with several theories of LH function including, for example, one [6] which suggested that LH stimulation activates species-specific approach response sequences, and one that suggested that the LH mediates incentive motivation ]181. An important question in any study of central drug injections is the extent to which the drugs diffuse and spread in the brain. In the present experiment extremely large amounts of drugs were injected opening up the possibility of widespread diffusion. Thus, the present study does not claim to have localized the locus of the behavioral effects in the LH merely because that was where the cannulae were placed. The main emphasis of the present study was behavioral, and the only important behavioral requirement for the drug injections was that they be reasonably unilateral. However, the observation that the onset of the easily observed postural asymmetries became evident at times while the experimenter was still in the process of injecting the rat, with the longest latencies being on the order of 5 rain, suggests that the site of action of the effective drugs may have indeed been relatively near the cannulae tips. One final issue which must be addressed is the question of the neurochemical specificity of particularly the rather high doses of phentolamine, propranolol, and haloperidol used in this experiment. Certainly the present methods do not exclude the possibility that one or more of the aforementioned drugs produced their effects through a nonspecific anesthesis. Indeed, in pilot work, procaine in the LH was shown to be effective in producing the response deficit. However, the fact that haloperidol was ineffective in the LH and the fact that phentolamine was equally ineffective in the caudate together with the fact that these results agree with histochemical mappings of the N E and DA systems argues strongly that these drugs produced their behavioral effects through a neurotransmitter-specific action.
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TACTILE DISCRIMINATION
FOLLOWING
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