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Functional aging of the nigro-striatal system
Peptides, Vol. 1, Suppl. 1, pp. 17%184. Printed in the U.S.A.
Functional Aging of the Nigro-Striatal System PATRICK K. RANDALL
Andrus Gerontology Center, University o f Southern California, Los Angeles, CA 90007
RANDALL, P. K. Functional aging of the nigro-striatal system. PEPTIDES 1: Suppl. 1, 177-184, 1980.--A number of movement disorders in the elderly are suggestive of age-associated extrapyramidal dysfunction. In addition, the onsets of two major movement disorders of better-known extrapyramidal pathophysiological locus, Huntington's chorea and Parkinson's disease, are highly age related. While slight to moderate declines in a number of presynaptic dopaminergic markers in the stfiatum have generally supported the view of declining dopaminergic function in the aged, a clear picture of the relationship between these biochemical parameters and function of this important extrapyramidal pathway has yet to emerge. Recent results from a number of laboratories representing combined behavioral, electrophysiological, and biochemical approaches have allowed a more analytical approach to the problem. First, age-related changes in this pathway appear to be coordinated between the pre- and post-synaptic components of the system. Dopamine receptor binding and dopamine-activated adenyl cyclase decrease at an earlier age and to a greater extent than do the presynaptic markers of the dopamine neurons themselves. Second, functional disorder of the nigro-striatal system may be more regulatory in nature than simple neuronal loss would suggest. Aging C57BL/6J mice, for example, did not show behavioral or biochemical supersensitivity following a chronic haloperidol regime which resulted in both phenomena in young- and middle-aged mice. Regulatory decreases in receptor number and response to dopamine agonists appear to be intact in the aging mouse. Lastly, the increased vulnerability of the elderly to seemingly incompatible motoric side-effects to the neuroleptic drugs indirectly suggest alterations in the control system parameters of the nigro-striatal pathway. Electrophysiologicalapproaches are now opening this avenue of investigation. Nigro-striatal system
Functional aging
IT is now generally agreed that a slight to moderate reduction of the biochemical markers of the dopaminergic nigrostriatal pathway occurs during aging, both in the human and in the laboratory rodent [1, 7, 18, 19, 27, 40, 41, 47]. While the specific degree of the DA loss is still a matter of some controversy, it is clear that its magnitude is less than is generally necessary in disease states or experimental preparations to produce functional impairments on physiological or behavioral levels. Post-mortem material from Parkinson's patients, for example, suggests a DA depletion of greater than 80% before functional impairment occurs [36]. Similarly, most of the severe behavioral consequences of lesions of the DA containing pathway are not apparent in animals with depletions of less than 80-90% [66]. It is apparent, then, that the nigro-striatal pathway has a high compensatory capability for the normalization of function in the face of all but the most severe damage. There is, however, evidence that given the appropriate context, eg. disease state or pharmacological challenge, age-related changes in the nigrostriatal pathway may be of functional significance. The well-known increase in the symptomatology of Parkinson's disease with age is suggestive of an interaction between a normal aging process and an underlying pathophysiological lesion of as yet unknown origin [19]. Additionally, the aged are much more likely to develop two, often severe, extrapyramidal side-effects to neuroleptic drugs [2, 12, 13, 28]. The increased incidence of drug-induced extrapyramidal dysfunction overlaps consid-
erably with the age distribution of onset of Parkinson's symptomatology [2]. The high degree of association between these side-effects to the major tranquilizers and age strongly suggests an increased vulnerability to pharmacological blockade of striatal DA receptors---even in a population with no obvious pathology of the nigro-striatal system. If placed in functional context, the aging deficits in nigro-striatal function must be considered relative to the adequacy of compensatory mechanisms rather than a simple loss of neuronal elements or biochemical machinery. Pharmacological challenge may reveal compensated aging deficits much as it does in Parkinson's disease or Huntington's chorea [34]. Environmental challenge in the form of severe task requirements may also reveal underlying deficits. Marshall and Berrios [39], for example, have recently reported that aging deficits in swimming behavior in rats are diminished by apomorphine. In this experiment, the swimming paradigm itself may provide sufficient environmental stress to unmask the underlying nigrostriatal deficit. Also arguing against the view of simple presynaptic loss of DA function is the apparent coordinated aging of the system as a whole, including non-dopaminergic elements. Recent evidence from a number of laboratories have suggested that impairments of function in the cells post-synaptic to the nigrostriatal pathway may be more severe and occur earlier than those in the nigro-striatal pathway itself. Losses as great as 50% have been reported in the binding of 3Hhaloperidol and 3H-spiroperidoi in the striatum of old mice
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and rats [27, 38, 57]. Dopamine-sensitive adenyl-cyclase has been reported to show substantial losses at relatively early ages [25, 38, 48, 56].
MOTORIC RESPONSE OF AGING RODENTS TO ACUTE DRUG TREATMENT
Our initial work in this area was simply to determine whether the aging rodent, as well as the human, shows increased vulnerability to DA blockade. A convenient test of motoric sensitivity to neuroleptic drugs in the rodent is the induction of catalepsy. This response, thought to result from blockade of striatal DA receptors [11], consists of a sustained immobility following a slight postural displacement. The cataleptic response may last from several seconds to approximately 20 minutes and is usually inducible within 20 minutes of intraperitoneal haloperidol. The time course of the drug is several hours. In this experiment we simply placed the animal, head downward, on a 45° ramp of wire mesh and gently pulled the tail. This procedure reliably produces an immobility roughly proportional to the dose of the neuroleptic. Contrary to our expectations, 24-month-old C57BL/6J mice were less, rather than more, sensitive to the extrapyramidal effects of the drug than were 4-month-old mice, ie. they showed shorter, rather than longer, immobility durations. A progressive change across the life-span was suggested by intermediate values in 12-month-old animals. The short duration of catalepsy in aging mice and rats on first exposure to haloperidol or spiroperidol is a reliable finding in our laboratory. It is unlikely that the aging animals metabolize the drug more rapidly than the young, making differential drug metabolism an improbable explanation. In addition, the motoric requirements of the response with this procedure are slight. In other experiments (see below) old mice show profoundly longer cataleptic durations, so that motoric inability to perform the response is also an unlikely interpretation. The diminished response to the neuroleptic in aging animals is potentially explainable by a deficit (or perhaps compensatory change) in another opposing system, which might mask any dopaminergic deficit. The catalepsy produced by neuroleptic drugs can be blocked by cholinergic antagonists, and cholinergic agonists produce a catalepsy quite similar to that produced by DA blockade [3,78]. The dopamine-acetylcholine balance hypothesis [64] is quite effective in this instance in that DA blockade or cholinergic stimulation have similar effects. A cholinergic deficit or compensatory decrease in cholinergic function to the decreased DA input during aging might then result in shorter catalepsy durations. As an initial test of this hypothesis we examined pilocarpine-induced catalepsy in C57BL/6J mice at 4, 9, and 24 months of age. The results were clearly consistent with this hypothesis. The old- and middle-aged animals were significantly less sensitive to pilocarpine than were the older mice. It would be interesting in this context to determine whether chronic L-Dopa treatment would reinstate the sensitivity to cholinergic agents as might be expected if the original deficit were a functional compensation for the falling DA levels. Cholinergic receptor binding is decreased with age [20] and could as easily be a compensatory change as a pathological one in some areas of the CNS.
RESPONSE OF AGING RODENTS TO CHRONIC NEUROLEPTICS
Chronic administration of the neuroleptic would, of course, be more analagous to treatment regimes in the human. We therefore evaluated the catalepsy resulting from 0.6 mg/kg haloperidol before and after daily intraperitoneal injections of 1.2 mg/kg haloperidol. Again, on first exposure to the drug we obtained an age-related decrease in catalepsy duration. However, following the chronic haloperidol treatment, old animals showed much longer catalepsy durations than young- or middle-aged mice. These data must be considered preliminary because of possible confounds with agerelated alterations in drug metabolism and, more importantly, subsequently discovered interactions of the stress of daily injection with the cataleptic response (Randall, in preparation). Phenomenologically, however, the data are quite stable and reproducible. In chronic neuroleptic regimes the aging mice show enormously elevated catalepsy scores, while young and middle-aged mice show a moderate tolerance to the effect. The absence of tolerance in the aging mice is a tentative finding, but did lead to a more direct experiment on compensation for chronic pharmacological blockade in aging mice. During this treatment a number of compensatory events may occur, one of the most important of which is increased sensitivity to DA agonists in the striatum, ie. supersensitivity. The evidence from behavioral [8, 72, 73], biochemical [5, 14, 42], and electrophysiological [58] experiments leaves little doubt that increases in receptor number in striatum occur as a result of denervation or chronic neuroleptic treatment with most agents. If this is a compensatory response to the neuroleptic, then the absence or diminished efficacy of this sensitization might help explain the absence of tolerance in the aging animals. Five-, 12- and 24-to-26-month-old C57BL/6J mice were given 1.2 mg/kg haloperidol, intraperitoneally or in the drinking water, for 21 days. Seven days following the drug regime they were given 2.0 mg/kg apomorphine and stereotyped behavior was scored using a 7 point rating scale and a timesampling procedure. Observations (20 sec duration) were made every six minutes during a 1-hour test session. Young- and middle-aged mice showed a significant elevation of stereotypy scores as a result of the chronic drug treatment. The old chronic-haloperidol-treated animals, however, were identical to the old vehicle-treated groups. The next day the animals were sacrificed and 3H-spiroperidol binding evaluated. The Bmax for young and middle-aged animals was elevated by approximately 30% in the chronicdrug treated animals, whereas no elevation was observed in the aging mice. No changes were observed in Kd values either as a result of age or drug treatments, suggesting that the absence of supersensitivity was not a result of residual haloperidol and less rapid elimination of the chronic drug by the older animals. At least one compensatory response to neuroleptic treatment, then appears to be diminished with age. It is not clear whether a more severe DA denervation, higher doses of the neuroleptic or lesion of the DA containing pathway, would have resulted in receptor supersensitivity in the aging mice. Certainly, very long term haloperidol treatment in rats produces more extreme and prolonged effects on receptor binding, stereotypic behavior and DA sensitive adenylcyclase [9,46]. The best available data [27] and our own initial experiments with intrastriatal 6-hydroxydopamine in aging mice suggest that the aging animal is still capable of
AGING--NIGRO-STRIATAL SYSTEM supersensitivity to a sufficient stimulus. It is quite likely, then, that the difference is one of threshold of the response--a more severe stimulus is required to initiate the onset of supersensitivity in the aging animal. The diminished receptor supersensitivity during aging may be a relatively general phenomenon. Studies on B-adrenergic receptor regulation in 3- and 24-month-old rats gave strikingly similar results to this study. Old rats had fewer B-receptors in cerebellum, pineal, and striatum and failed to increase receptors following chronic treatments (reserpine, constant light) which produced typical increases of binding sites in the young [75]. These results suggest that the same aspects of receptor regulation are selectively impaired in pharmacologically and anatomically distinct dopaminergic and noradrenergic systems. Deficits in catecholaminergic receptor regulation with age thus may be widely distributed.
EFFECTS OF CHRONIC BROMOCRIPTINE
This experiment suggests that an underlying deficit may be unmasked by pharmacological challenge--chronic blockade of DA receptors. While the aging rodent may provide an adequate model for increased incidence of Parkinson's-like dysfunction to neuroleptics in the elderly, it is in apparent contradiction to the elevated incidence of tardive dyskinesia in the elderly [13,28]. This occasional side-effect to very long-term neuroleptic treatment is thought to result from overcompensatory supersensitivity of DA receptors leading to an essentially hyperdopaminergic state [30, 32, 33]. Particularly early in the disorder, the dyskinesia is most prominent in the orobuccal area. It may, in more severe cases invade the trunk and extremities [15]. If the elderly are less likely to develop DA receptor supersensitivity, then why are they more likely to show this disorder? One experiment [60] may provide at least partial solution to this problem. BALB/cJ and CBA/J mice differ considerably in the numbers of tyrosine hydroxylase reactive neurons, the CBA/J having 75% of those present in the BALB/cJ [4, 53, 54]. Since we had observed strain differences in the induction of DA supersensitivity, we also examined the subsensitivity response to 7 days of daily subcutaneous injections of 15.0 mg/kg bromocriptine, a putative DA agonist [10,16]. CBA/J and C57BL/6J mice both responded to the chronic bromocriptine with a diminution of stereotype ratings and a 15% decrease in striatal 3H-spiroperidol binding. The BALB/cJ, however, at an apomorphine dose of 2.0 mg/kg displayed a behavioral syndrome we have not seen in any strain at any dose of apomorphine. While the coherent stereotypic syndrome as measured by strict adherence to the rating scale had disappeared entirely, they responded to the apomorphine with high levels of activity and a peculiar orobuccal abnormality of repeated and severe tongue protrusion. Since the behavior is induced by apomorphine and blocked by haloperidol [51] it is likely to be mediated by DA receptor activation, probably in the striatum, although the appropriate experiments remain to be done. It should be emphasized that this enhancement of some component of DA-stimulated behavior is accompanied by a 15% reduction in striatal 3H-spiroperidol binding. This is, of course, similar to the relatively large literature on "agonist induced supersensitivity" [31], although in this strain of mouse the behavior is quite different qualitatively than the normal stereotypic pattern.
179 Our current interpretation of this phenomenon is similar to that proposed by Muller and Seeman [44] for the more general phenomenon of agonist-induced supersensitivity. We believe the oral dyskinetic effects in the BALB/cJ mice to be the result of disruption of a normal regulatory mechanism in the nigro-striatal pathway. The understanding of regulation of the activity in this neuronal pathway has changed dramatically in the last few years, on essentially all levels of analysis---neurochemical, electrophysiological, and behavioral. The most fundamental change being the proliferation of dopamine receptors of different types and different anatomical location, some of which may serve a regulatory function. There is currently reasonable evidence for five DA receptors differing in receptor characteristics and location [29]. (1) A post-synaptic receptor on the soma or dendrites of cells in the striatum which is coupled with adenyl cyclase [17, 22, 26] and which corresponds most closely to the "classic" DA receptor. (2) A receptor located on frontal cortical afferents, not coupled with adenyl cyclase and which apparently modulates glutamate release from these axons [55,56]. (3) A presynaptic receptor on dopamine terminals in striatum which has highly potent effects on striatal tyrosine hydroxylase activity and possibly on dopamine release. This receptor is apparently not coupled with adenyl cyclase [45]. (4) Receptors on DA cell bodies themselves in the substantia nigra, which are not coupled with adenyl cyclase and have a highly potent inhibitory effect on firing rate [50,61]. (5) Receptors which are located on the axons or terminals of the striato-nigral pathway. These receptors are coupled with adenyl cyclase and are anatomically in a position to modify the effect of the GABA and/or Substance P striato-nigral pathways [50]. When a dopaminergically active drug is administered chronically, it is likely to have effects on several of these receptor types, depending on the spectrum of action of the particular agent employed, There is good evidence for supersensitivity of the pre-synaptic DA receptor as well as the post-synaptic striatal receptor [21,65]. The oral dyskinesias in the BALB/cJ mice may be the result of a relatively selective subsensitization of the pre-synaptic receptor. Bromocriptine is highly effective in the production of agonist induced supersensitivity in other experimental preparations. The enhanced stereotypic behavior in one study was associated with the disappearance (tolerance) during the chronic treatment, of the drug's inhibitory effect on wheel running in rats [62]. In this experiment, the possible subsensitivity of the pre-synaptic receptor is suggested by the absence of locomotor inhibition during any part of the apomorphine time-course. In addition, we had observed that CBA/J mice were very sensitive to the soporific effects of apomorphine. While 10 of 12 control animals were apparently asleep by the end of the test session, only one of 12 of the chronicbromocriptine treated CBA/J's showed this response. It should be mentioned, however, that bromocriptine has complex "mixed agonist-antagonist" effects at the dopamine receptor [24,63]. In a similar manner, elderly humans may show increased risk of tardive dyskinesia as a result of regulatory insufficiency. Even though supersensitivity may be delayed to some extent or be of lesser magnitude relative to the younger patient, the elderly may be less able to compensate for this when it occurs. The inadequacy of intranigral regulatory receptors or of other possibly compensatory neurotransmitter systems (eg. acetylcholine) may be responsible for an "un-
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2.0 mg/kg HALOPERIDOL
PRE DRUG
POST DRUG 6 0 MIN.
80 uv I .05 see. FIG. 1. Striatal response to a cataleptic dose of haloperidol in the 57BL/6J mouse. Figure represents a sample raw tracing of on-going electrical activity from a bipolar electrode (l mm tip separation) in rostral striatum before and after 2 mg/kg haloperidol (IP). All recordings were done in freely moving animals.
damped swing" from the hypodopaminergic state of druginduced Parkinsonism to the hyperdopaminergic state present in tardive dyskinesia. ELECTROPHYSIOLOGICALRESPONSE TO DOPAMINERGICDRUGS The understanding and possible reversal of aging deficits associated with the nigro-striatal pathway may be highly dependent on the understanding of the mechanisms by which stability or homeostasis is maintained. These mechanisms are certainly complex, involving several putative neurotransmitter systems and the several dopamine receptors discussed above. In addition, the high probability o f dendritic release of DA in the substantia nigra [23,35] altering the firing rate of nigral DA neurons (eg. [76]) increases the complexity of these interactions considerably. Undoubtedly other "non-classical" forms of neuronal communication remain to be discovered. One major obstacle in the analysis of nigrostriatal control systems is the absence of a metric or measurement instrument for the final output of striatal DA influence. This quantity is a function of firing rate of DA neurons, local synaptic modulation of DA release per action potential, receptor sensitivity, integration of the DA influence with other striatal neurotransmitters, and possibly more subtle quantities such as synchrony of firing in the nigral DA neurons. Most neurochemical and electrophysiological measurements are, directly or indirectly, indices of the mechanisms of control, not the controlled quantity itself. The firing rate of DA neurons, for example, is not the level of DA stimulation itself, but one
mechanism by which it is maintained. This is most obvious in the case of receptor blockade where the DA influence in the striatum is minimal, but the firing rate of DA neurons is elevated. Ultimately we must understand the open component of the system as well as closed negative feedback components. If this system is to modulate behavioral activation or to reliably convey any information it must respond, directly or indirectly, to other neurotransmitter systems and ultimately to environmental demands. Thus the set point of the regulation or the gain of feedback loops must be sensitive to events external to the system. On the simple basis of examining generalized aspects of D A input to the striatum, we have begun investigating ongoing electrical activity recorded from gross bipolar electrodes in anterior striatum of C57BL/6J mice [37]. Without a rigidly stratified system to eliminate sampling error in single unit recording, this appeared to be a reasonable place to start. The ongoing activity from this electrode is shown in the top portion of Figs. 1 and 2. It has a predominant frequency of 10--12 Hz, and in the alert animal a peak-to-peak amplitude of approximately 100 /xV, which, of course, will vary with electrode placement in individual preparations. Following injection of 3 mg/kg haloperidol we observed a striking increase in amplitude of this signal with some apparent slowing of frequency. This was calculated by power spectral analysis to be a 250% increase in total power from 1 to 15 Hz. More recently we have used a zero crossing analysis on these data and observe an 80% increase in mean deviation of the signal from baseline with a 30% decrease in number of zero crossings. Figure 1 shows a raw record of the
A G I N G - - N I G R O - S T R I A T A L SYSTEM
181
5.0 mg/kg APOMORPHINE PRE DRUG
POST DRUG 30 MIN.
POST DRUG 90
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FIG. 2. Biphasic effect of a stereotypic dose of apomorphine on the striatal gross potential (see Fig. 1). Record was taken 30 or 90 min following intraperitoneal injection of 3.0 mg/kg apomorphine.
recording before and after 2.0 mg/kg haloperidol. Figure 3 additionally shows the time course of the increase in amplitude following this same treatment. Stereotypic doses of apomorphine, on the other hand, during the stereotypic phase produce an approximate halving of the amplitude. We were surprised to observe a very potent rebound effect beginning roughly ninety minutes after administration of this dose of apomorphine. The striatal potential increased in amplitude to a similar extent as the response following 3 mg/kg. Subsequent power calculations revealed a 272% increase in total power of the signal. On the basis of this haloperidol-like record, we attempted to induce catalepsy in these animals and a reliable and strongly cataleptic response was obtained. The electrophysiologicai rebound is present in virtually all animals and is invariably accompanied by catalepsy. This biphasic effect of apomorphine is shown in Fig. 2. In retrospect the interpretation of the rebound effect in terms of the current literature is obvious. Since the "autoregulatory r e c e p t o r " is thought to be at least an order of magnitude more s e n s i t i v e to some agonists [61], certain periods of time following injection will be characterized by sufficiently high blood (or brain) levels of the drug to inhibit DA containing nigral cells, but insufficient to effect the striatai post-synaptic receptor. The decreased locomotor activity following low doses of apomorphine is thought to be a
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FIG. 3. Time-course of the effect of haloperidol on the amplitude of the striatal gross potential. Amplitude was defined as meanmaximum inter-zero-crossing deviation from baseline. Each point represents 6 to 10, 10 sec acquisition sweeps.
182
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FIG. 4. Hypothesized time-course of the effect of a stereotypic dose of apomorphine on preand post-synaptic receptor activation. See text for explanation. similar phenomenon [67-69, 71]. The time-course of bromocriptine is much longer than that of apomorphine which is rapidly distributed and metabolized. Thus, during the early part of the time course, the drug behaves essentially as an antagonist, by reducing the release of endogenous dopamine. Apomorphine, too, should have a similar portion to the dose-response curve, but as a result of the pharmacokinetics of the drug would be so brief as to be unnoticed in most experimental preparations. As a result of the exponential nature of the elimination of the drug, however, the period of intermediate drug level might be considerably longer following the stereotypic phase, rather than preceding it. Figure 4 depicts this situation schematically. On the bottom panel of the graph is shown the blood level of the drug in arbitrary units calculated from a simple three compartment kinetic model. The thresholds for effect on the two receptor types are approximated. The middle panel depicts activation of the autoregulatory receptor, which with a stereotypic dose of the drug would quite rapidly reach near maximal levels. This level of activation of the receptor would, of course, continue for a relatively long period of time, i.e. the drug would have to fall to very low levels before activation of this receptor would decline. The top panel depicts the postsynaptic receptor activation expected from this model. A very short phase of decreases in DA receptor activation would be followed by a longer phase of supernormal stimulation resulting in stereotyped behavior. Following this period, however, would be a much longer period of subnormal activation as a result of the drug level falling below that
necessary for post-synaptic effects. During this period of time we observe the high magnitude striatal potential and catalepsy inducibility. If this interpretation is correct then injection of very low doses of apomorphine ought to produce the haloperidol-like striatal record and catalepsy during the time period in which higher doses of the drug produce stereotypic behavior. Intraperitoneal injection of 0.05 mg/kg apomorphine produced a striatal record and behavioral profile identical to that of the rebound from the higher dose. The time course was, however, slightly delayed from that of the stereotypic behavior reaching a maximal effect at approximately 30 minutes while in our laboratory we usually see maximal stereotypy approximately 10 minutes earlier. SUMMARY The available data indicate that the physiological or behavioral effects of age-related alterations in nigro-striatal function may not be evident without some environmental or pharmacological perturbation. At least one compensatory mechanism, the development of receptor supersensitivity, may be compromised with age. Additionally, evidence from other experiments indicates that fluctuations in shorter-term control mechanisms, eg. DA auto-receptors, may have hamor influences on the functional characteristics of nigrostriatal function. Electrophysiological measurements of striatal activity following pharmacological manipulation may provide a metric for analyzing these control systems.
AGING--NIGRO-STRIATAL
SYSTEM
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Functional Aging of the Nigro-Striatal System PATRICK K. RANDALL
Andrus Gerontology Center, University o f Southern California, Los Angeles, CA 90007
RANDALL, P. K. Functional aging of the nigro-striatal system. PEPTIDES 1: Suppl. 1, 177-184, 1980.--A number of movement disorders in the elderly are suggestive of age-associated extrapyramidal dysfunction. In addition, the onsets of two major movement disorders of better-known extrapyramidal pathophysiological locus, Huntington's chorea and Parkinson's disease, are highly age related. While slight to moderate declines in a number of presynaptic dopaminergic markers in the stfiatum have generally supported the view of declining dopaminergic function in the aged, a clear picture of the relationship between these biochemical parameters and function of this important extrapyramidal pathway has yet to emerge. Recent results from a number of laboratories representing combined behavioral, electrophysiological, and biochemical approaches have allowed a more analytical approach to the problem. First, age-related changes in this pathway appear to be coordinated between the pre- and post-synaptic components of the system. Dopamine receptor binding and dopamine-activated adenyl cyclase decrease at an earlier age and to a greater extent than do the presynaptic markers of the dopamine neurons themselves. Second, functional disorder of the nigro-striatal system may be more regulatory in nature than simple neuronal loss would suggest. Aging C57BL/6J mice, for example, did not show behavioral or biochemical supersensitivity following a chronic haloperidol regime which resulted in both phenomena in young- and middle-aged mice. Regulatory decreases in receptor number and response to dopamine agonists appear to be intact in the aging mouse. Lastly, the increased vulnerability of the elderly to seemingly incompatible motoric side-effects to the neuroleptic drugs indirectly suggest alterations in the control system parameters of the nigro-striatal pathway. Electrophysiologicalapproaches are now opening this avenue of investigation. Nigro-striatal system
Functional aging
IT is now generally agreed that a slight to moderate reduction of the biochemical markers of the dopaminergic nigrostriatal pathway occurs during aging, both in the human and in the laboratory rodent [1, 7, 18, 19, 27, 40, 41, 47]. While the specific degree of the DA loss is still a matter of some controversy, it is clear that its magnitude is less than is generally necessary in disease states or experimental preparations to produce functional impairments on physiological or behavioral levels. Post-mortem material from Parkinson's patients, for example, suggests a DA depletion of greater than 80% before functional impairment occurs [36]. Similarly, most of the severe behavioral consequences of lesions of the DA containing pathway are not apparent in animals with depletions of less than 80-90% [66]. It is apparent, then, that the nigro-striatal pathway has a high compensatory capability for the normalization of function in the face of all but the most severe damage. There is, however, evidence that given the appropriate context, eg. disease state or pharmacological challenge, age-related changes in the nigrostriatal pathway may be of functional significance. The well-known increase in the symptomatology of Parkinson's disease with age is suggestive of an interaction between a normal aging process and an underlying pathophysiological lesion of as yet unknown origin [19]. Additionally, the aged are much more likely to develop two, often severe, extrapyramidal side-effects to neuroleptic drugs [2, 12, 13, 28]. The increased incidence of drug-induced extrapyramidal dysfunction overlaps consid-
erably with the age distribution of onset of Parkinson's symptomatology [2]. The high degree of association between these side-effects to the major tranquilizers and age strongly suggests an increased vulnerability to pharmacological blockade of striatal DA receptors---even in a population with no obvious pathology of the nigro-striatal system. If placed in functional context, the aging deficits in nigro-striatal function must be considered relative to the adequacy of compensatory mechanisms rather than a simple loss of neuronal elements or biochemical machinery. Pharmacological challenge may reveal compensated aging deficits much as it does in Parkinson's disease or Huntington's chorea [34]. Environmental challenge in the form of severe task requirements may also reveal underlying deficits. Marshall and Berrios [39], for example, have recently reported that aging deficits in swimming behavior in rats are diminished by apomorphine. In this experiment, the swimming paradigm itself may provide sufficient environmental stress to unmask the underlying nigrostriatal deficit. Also arguing against the view of simple presynaptic loss of DA function is the apparent coordinated aging of the system as a whole, including non-dopaminergic elements. Recent evidence from a number of laboratories have suggested that impairments of function in the cells post-synaptic to the nigrostriatal pathway may be more severe and occur earlier than those in the nigro-striatal pathway itself. Losses as great as 50% have been reported in the binding of 3Hhaloperidol and 3H-spiroperidoi in the striatum of old mice
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and rats [27, 38, 57]. Dopamine-sensitive adenyl-cyclase has been reported to show substantial losses at relatively early ages [25, 38, 48, 56].
MOTORIC RESPONSE OF AGING RODENTS TO ACUTE DRUG TREATMENT
Our initial work in this area was simply to determine whether the aging rodent, as well as the human, shows increased vulnerability to DA blockade. A convenient test of motoric sensitivity to neuroleptic drugs in the rodent is the induction of catalepsy. This response, thought to result from blockade of striatal DA receptors [11], consists of a sustained immobility following a slight postural displacement. The cataleptic response may last from several seconds to approximately 20 minutes and is usually inducible within 20 minutes of intraperitoneal haloperidol. The time course of the drug is several hours. In this experiment we simply placed the animal, head downward, on a 45° ramp of wire mesh and gently pulled the tail. This procedure reliably produces an immobility roughly proportional to the dose of the neuroleptic. Contrary to our expectations, 24-month-old C57BL/6J mice were less, rather than more, sensitive to the extrapyramidal effects of the drug than were 4-month-old mice, ie. they showed shorter, rather than longer, immobility durations. A progressive change across the life-span was suggested by intermediate values in 12-month-old animals. The short duration of catalepsy in aging mice and rats on first exposure to haloperidol or spiroperidol is a reliable finding in our laboratory. It is unlikely that the aging animals metabolize the drug more rapidly than the young, making differential drug metabolism an improbable explanation. In addition, the motoric requirements of the response with this procedure are slight. In other experiments (see below) old mice show profoundly longer cataleptic durations, so that motoric inability to perform the response is also an unlikely interpretation. The diminished response to the neuroleptic in aging animals is potentially explainable by a deficit (or perhaps compensatory change) in another opposing system, which might mask any dopaminergic deficit. The catalepsy produced by neuroleptic drugs can be blocked by cholinergic antagonists, and cholinergic agonists produce a catalepsy quite similar to that produced by DA blockade [3,78]. The dopamine-acetylcholine balance hypothesis [64] is quite effective in this instance in that DA blockade or cholinergic stimulation have similar effects. A cholinergic deficit or compensatory decrease in cholinergic function to the decreased DA input during aging might then result in shorter catalepsy durations. As an initial test of this hypothesis we examined pilocarpine-induced catalepsy in C57BL/6J mice at 4, 9, and 24 months of age. The results were clearly consistent with this hypothesis. The old- and middle-aged animals were significantly less sensitive to pilocarpine than were the older mice. It would be interesting in this context to determine whether chronic L-Dopa treatment would reinstate the sensitivity to cholinergic agents as might be expected if the original deficit were a functional compensation for the falling DA levels. Cholinergic receptor binding is decreased with age [20] and could as easily be a compensatory change as a pathological one in some areas of the CNS.
RESPONSE OF AGING RODENTS TO CHRONIC NEUROLEPTICS
Chronic administration of the neuroleptic would, of course, be more analagous to treatment regimes in the human. We therefore evaluated the catalepsy resulting from 0.6 mg/kg haloperidol before and after daily intraperitoneal injections of 1.2 mg/kg haloperidol. Again, on first exposure to the drug we obtained an age-related decrease in catalepsy duration. However, following the chronic haloperidol treatment, old animals showed much longer catalepsy durations than young- or middle-aged mice. These data must be considered preliminary because of possible confounds with agerelated alterations in drug metabolism and, more importantly, subsequently discovered interactions of the stress of daily injection with the cataleptic response (Randall, in preparation). Phenomenologically, however, the data are quite stable and reproducible. In chronic neuroleptic regimes the aging mice show enormously elevated catalepsy scores, while young and middle-aged mice show a moderate tolerance to the effect. The absence of tolerance in the aging mice is a tentative finding, but did lead to a more direct experiment on compensation for chronic pharmacological blockade in aging mice. During this treatment a number of compensatory events may occur, one of the most important of which is increased sensitivity to DA agonists in the striatum, ie. supersensitivity. The evidence from behavioral [8, 72, 73], biochemical [5, 14, 42], and electrophysiological [58] experiments leaves little doubt that increases in receptor number in striatum occur as a result of denervation or chronic neuroleptic treatment with most agents. If this is a compensatory response to the neuroleptic, then the absence or diminished efficacy of this sensitization might help explain the absence of tolerance in the aging animals. Five-, 12- and 24-to-26-month-old C57BL/6J mice were given 1.2 mg/kg haloperidol, intraperitoneally or in the drinking water, for 21 days. Seven days following the drug regime they were given 2.0 mg/kg apomorphine and stereotyped behavior was scored using a 7 point rating scale and a timesampling procedure. Observations (20 sec duration) were made every six minutes during a 1-hour test session. Young- and middle-aged mice showed a significant elevation of stereotypy scores as a result of the chronic drug treatment. The old chronic-haloperidol-treated animals, however, were identical to the old vehicle-treated groups. The next day the animals were sacrificed and 3H-spiroperidol binding evaluated. The Bmax for young and middle-aged animals was elevated by approximately 30% in the chronicdrug treated animals, whereas no elevation was observed in the aging mice. No changes were observed in Kd values either as a result of age or drug treatments, suggesting that the absence of supersensitivity was not a result of residual haloperidol and less rapid elimination of the chronic drug by the older animals. At least one compensatory response to neuroleptic treatment, then appears to be diminished with age. It is not clear whether a more severe DA denervation, higher doses of the neuroleptic or lesion of the DA containing pathway, would have resulted in receptor supersensitivity in the aging mice. Certainly, very long term haloperidol treatment in rats produces more extreme and prolonged effects on receptor binding, stereotypic behavior and DA sensitive adenylcyclase [9,46]. The best available data [27] and our own initial experiments with intrastriatal 6-hydroxydopamine in aging mice suggest that the aging animal is still capable of
AGING--NIGRO-STRIATAL SYSTEM supersensitivity to a sufficient stimulus. It is quite likely, then, that the difference is one of threshold of the response--a more severe stimulus is required to initiate the onset of supersensitivity in the aging animal. The diminished receptor supersensitivity during aging may be a relatively general phenomenon. Studies on B-adrenergic receptor regulation in 3- and 24-month-old rats gave strikingly similar results to this study. Old rats had fewer B-receptors in cerebellum, pineal, and striatum and failed to increase receptors following chronic treatments (reserpine, constant light) which produced typical increases of binding sites in the young [75]. These results suggest that the same aspects of receptor regulation are selectively impaired in pharmacologically and anatomically distinct dopaminergic and noradrenergic systems. Deficits in catecholaminergic receptor regulation with age thus may be widely distributed.
EFFECTS OF CHRONIC BROMOCRIPTINE
This experiment suggests that an underlying deficit may be unmasked by pharmacological challenge--chronic blockade of DA receptors. While the aging rodent may provide an adequate model for increased incidence of Parkinson's-like dysfunction to neuroleptics in the elderly, it is in apparent contradiction to the elevated incidence of tardive dyskinesia in the elderly [13,28]. This occasional side-effect to very long-term neuroleptic treatment is thought to result from overcompensatory supersensitivity of DA receptors leading to an essentially hyperdopaminergic state [30, 32, 33]. Particularly early in the disorder, the dyskinesia is most prominent in the orobuccal area. It may, in more severe cases invade the trunk and extremities [15]. If the elderly are less likely to develop DA receptor supersensitivity, then why are they more likely to show this disorder? One experiment [60] may provide at least partial solution to this problem. BALB/cJ and CBA/J mice differ considerably in the numbers of tyrosine hydroxylase reactive neurons, the CBA/J having 75% of those present in the BALB/cJ [4, 53, 54]. Since we had observed strain differences in the induction of DA supersensitivity, we also examined the subsensitivity response to 7 days of daily subcutaneous injections of 15.0 mg/kg bromocriptine, a putative DA agonist [10,16]. CBA/J and C57BL/6J mice both responded to the chronic bromocriptine with a diminution of stereotype ratings and a 15% decrease in striatal 3H-spiroperidol binding. The BALB/cJ, however, at an apomorphine dose of 2.0 mg/kg displayed a behavioral syndrome we have not seen in any strain at any dose of apomorphine. While the coherent stereotypic syndrome as measured by strict adherence to the rating scale had disappeared entirely, they responded to the apomorphine with high levels of activity and a peculiar orobuccal abnormality of repeated and severe tongue protrusion. Since the behavior is induced by apomorphine and blocked by haloperidol [51] it is likely to be mediated by DA receptor activation, probably in the striatum, although the appropriate experiments remain to be done. It should be emphasized that this enhancement of some component of DA-stimulated behavior is accompanied by a 15% reduction in striatal 3H-spiroperidol binding. This is, of course, similar to the relatively large literature on "agonist induced supersensitivity" [31], although in this strain of mouse the behavior is quite different qualitatively than the normal stereotypic pattern.
179 Our current interpretation of this phenomenon is similar to that proposed by Muller and Seeman [44] for the more general phenomenon of agonist-induced supersensitivity. We believe the oral dyskinetic effects in the BALB/cJ mice to be the result of disruption of a normal regulatory mechanism in the nigro-striatal pathway. The understanding of regulation of the activity in this neuronal pathway has changed dramatically in the last few years, on essentially all levels of analysis---neurochemical, electrophysiological, and behavioral. The most fundamental change being the proliferation of dopamine receptors of different types and different anatomical location, some of which may serve a regulatory function. There is currently reasonable evidence for five DA receptors differing in receptor characteristics and location [29]. (1) A post-synaptic receptor on the soma or dendrites of cells in the striatum which is coupled with adenyl cyclase [17, 22, 26] and which corresponds most closely to the "classic" DA receptor. (2) A receptor located on frontal cortical afferents, not coupled with adenyl cyclase and which apparently modulates glutamate release from these axons [55,56]. (3) A presynaptic receptor on dopamine terminals in striatum which has highly potent effects on striatal tyrosine hydroxylase activity and possibly on dopamine release. This receptor is apparently not coupled with adenyl cyclase [45]. (4) Receptors on DA cell bodies themselves in the substantia nigra, which are not coupled with adenyl cyclase and have a highly potent inhibitory effect on firing rate [50,61]. (5) Receptors which are located on the axons or terminals of the striato-nigral pathway. These receptors are coupled with adenyl cyclase and are anatomically in a position to modify the effect of the GABA and/or Substance P striato-nigral pathways [50]. When a dopaminergically active drug is administered chronically, it is likely to have effects on several of these receptor types, depending on the spectrum of action of the particular agent employed, There is good evidence for supersensitivity of the pre-synaptic DA receptor as well as the post-synaptic striatal receptor [21,65]. The oral dyskinesias in the BALB/cJ mice may be the result of a relatively selective subsensitization of the pre-synaptic receptor. Bromocriptine is highly effective in the production of agonist induced supersensitivity in other experimental preparations. The enhanced stereotypic behavior in one study was associated with the disappearance (tolerance) during the chronic treatment, of the drug's inhibitory effect on wheel running in rats [62]. In this experiment, the possible subsensitivity of the pre-synaptic receptor is suggested by the absence of locomotor inhibition during any part of the apomorphine time-course. In addition, we had observed that CBA/J mice were very sensitive to the soporific effects of apomorphine. While 10 of 12 control animals were apparently asleep by the end of the test session, only one of 12 of the chronicbromocriptine treated CBA/J's showed this response. It should be mentioned, however, that bromocriptine has complex "mixed agonist-antagonist" effects at the dopamine receptor [24,63]. In a similar manner, elderly humans may show increased risk of tardive dyskinesia as a result of regulatory insufficiency. Even though supersensitivity may be delayed to some extent or be of lesser magnitude relative to the younger patient, the elderly may be less able to compensate for this when it occurs. The inadequacy of intranigral regulatory receptors or of other possibly compensatory neurotransmitter systems (eg. acetylcholine) may be responsible for an "un-
180
RANDALL
2.0 mg/kg HALOPERIDOL
PRE DRUG
POST DRUG 6 0 MIN.
80 uv I .05 see. FIG. 1. Striatal response to a cataleptic dose of haloperidol in the 57BL/6J mouse. Figure represents a sample raw tracing of on-going electrical activity from a bipolar electrode (l mm tip separation) in rostral striatum before and after 2 mg/kg haloperidol (IP). All recordings were done in freely moving animals.
damped swing" from the hypodopaminergic state of druginduced Parkinsonism to the hyperdopaminergic state present in tardive dyskinesia. ELECTROPHYSIOLOGICALRESPONSE TO DOPAMINERGICDRUGS The understanding and possible reversal of aging deficits associated with the nigro-striatal pathway may be highly dependent on the understanding of the mechanisms by which stability or homeostasis is maintained. These mechanisms are certainly complex, involving several putative neurotransmitter systems and the several dopamine receptors discussed above. In addition, the high probability o f dendritic release of DA in the substantia nigra [23,35] altering the firing rate of nigral DA neurons (eg. [76]) increases the complexity of these interactions considerably. Undoubtedly other "non-classical" forms of neuronal communication remain to be discovered. One major obstacle in the analysis of nigrostriatal control systems is the absence of a metric or measurement instrument for the final output of striatal DA influence. This quantity is a function of firing rate of DA neurons, local synaptic modulation of DA release per action potential, receptor sensitivity, integration of the DA influence with other striatal neurotransmitters, and possibly more subtle quantities such as synchrony of firing in the nigral DA neurons. Most neurochemical and electrophysiological measurements are, directly or indirectly, indices of the mechanisms of control, not the controlled quantity itself. The firing rate of DA neurons, for example, is not the level of DA stimulation itself, but one
mechanism by which it is maintained. This is most obvious in the case of receptor blockade where the DA influence in the striatum is minimal, but the firing rate of DA neurons is elevated. Ultimately we must understand the open component of the system as well as closed negative feedback components. If this system is to modulate behavioral activation or to reliably convey any information it must respond, directly or indirectly, to other neurotransmitter systems and ultimately to environmental demands. Thus the set point of the regulation or the gain of feedback loops must be sensitive to events external to the system. On the simple basis of examining generalized aspects of D A input to the striatum, we have begun investigating ongoing electrical activity recorded from gross bipolar electrodes in anterior striatum of C57BL/6J mice [37]. Without a rigidly stratified system to eliminate sampling error in single unit recording, this appeared to be a reasonable place to start. The ongoing activity from this electrode is shown in the top portion of Figs. 1 and 2. It has a predominant frequency of 10--12 Hz, and in the alert animal a peak-to-peak amplitude of approximately 100 /xV, which, of course, will vary with electrode placement in individual preparations. Following injection of 3 mg/kg haloperidol we observed a striking increase in amplitude of this signal with some apparent slowing of frequency. This was calculated by power spectral analysis to be a 250% increase in total power from 1 to 15 Hz. More recently we have used a zero crossing analysis on these data and observe an 80% increase in mean deviation of the signal from baseline with a 30% decrease in number of zero crossings. Figure 1 shows a raw record of the
A G I N G - - N I G R O - S T R I A T A L SYSTEM
181
5.0 mg/kg APOMORPHINE PRE DRUG
POST DRUG 30 MIN.
POST DRUG 90
MIN. llODv L •08 wv.
FIG. 2. Biphasic effect of a stereotypic dose of apomorphine on the striatal gross potential (see Fig. 1). Record was taken 30 or 90 min following intraperitoneal injection of 3.0 mg/kg apomorphine.
recording before and after 2.0 mg/kg haloperidol. Figure 3 additionally shows the time course of the increase in amplitude following this same treatment. Stereotypic doses of apomorphine, on the other hand, during the stereotypic phase produce an approximate halving of the amplitude. We were surprised to observe a very potent rebound effect beginning roughly ninety minutes after administration of this dose of apomorphine. The striatal potential increased in amplitude to a similar extent as the response following 3 mg/kg. Subsequent power calculations revealed a 272% increase in total power of the signal. On the basis of this haloperidol-like record, we attempted to induce catalepsy in these animals and a reliable and strongly cataleptic response was obtained. The electrophysiologicai rebound is present in virtually all animals and is invariably accompanied by catalepsy. This biphasic effect of apomorphine is shown in Fig. 2. In retrospect the interpretation of the rebound effect in terms of the current literature is obvious. Since the "autoregulatory r e c e p t o r " is thought to be at least an order of magnitude more s e n s i t i v e to some agonists [61], certain periods of time following injection will be characterized by sufficiently high blood (or brain) levels of the drug to inhibit DA containing nigral cells, but insufficient to effect the striatai post-synaptic receptor. The decreased locomotor activity following low doses of apomorphine is thought to be a
80 % CHANGE 60 AMPLITUDE
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I
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I
I
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120
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POST INJECTION
FIG. 3. Time-course of the effect of haloperidol on the amplitude of the striatal gross potential. Amplitude was defined as meanmaximum inter-zero-crossing deviation from baseline. Each point represents 6 to 10, 10 sec acquisition sweeps.
182
RANDALL IC~ LLI It LL ILl
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FIG. 4. Hypothesized time-course of the effect of a stereotypic dose of apomorphine on preand post-synaptic receptor activation. See text for explanation. similar phenomenon [67-69, 71]. The time-course of bromocriptine is much longer than that of apomorphine which is rapidly distributed and metabolized. Thus, during the early part of the time course, the drug behaves essentially as an antagonist, by reducing the release of endogenous dopamine. Apomorphine, too, should have a similar portion to the dose-response curve, but as a result of the pharmacokinetics of the drug would be so brief as to be unnoticed in most experimental preparations. As a result of the exponential nature of the elimination of the drug, however, the period of intermediate drug level might be considerably longer following the stereotypic phase, rather than preceding it. Figure 4 depicts this situation schematically. On the bottom panel of the graph is shown the blood level of the drug in arbitrary units calculated from a simple three compartment kinetic model. The thresholds for effect on the two receptor types are approximated. The middle panel depicts activation of the autoregulatory receptor, which with a stereotypic dose of the drug would quite rapidly reach near maximal levels. This level of activation of the receptor would, of course, continue for a relatively long period of time, i.e. the drug would have to fall to very low levels before activation of this receptor would decline. The top panel depicts the postsynaptic receptor activation expected from this model. A very short phase of decreases in DA receptor activation would be followed by a longer phase of supernormal stimulation resulting in stereotyped behavior. Following this period, however, would be a much longer period of subnormal activation as a result of the drug level falling below that
necessary for post-synaptic effects. During this period of time we observe the high magnitude striatal potential and catalepsy inducibility. If this interpretation is correct then injection of very low doses of apomorphine ought to produce the haloperidol-like striatal record and catalepsy during the time period in which higher doses of the drug produce stereotypic behavior. Intraperitoneal injection of 0.05 mg/kg apomorphine produced a striatal record and behavioral profile identical to that of the rebound from the higher dose. The time course was, however, slightly delayed from that of the stereotypic behavior reaching a maximal effect at approximately 30 minutes while in our laboratory we usually see maximal stereotypy approximately 10 minutes earlier. SUMMARY The available data indicate that the physiological or behavioral effects of age-related alterations in nigro-striatal function may not be evident without some environmental or pharmacological perturbation. At least one compensatory mechanism, the development of receptor supersensitivity, may be compromised with age. Additionally, evidence from other experiments indicates that fluctuations in shorter-term control mechanisms, eg. DA auto-receptors, may have hamor influences on the functional characteristics of nigrostriatal function. Electrophysiological measurements of striatal activity following pharmacological manipulation may provide a metric for analyzing these control systems.
AGING--NIGRO-STRIATAL
SYSTEM
183 REFERENCES
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