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Retrograde axonal transport of β-adrenoreceptors in rat brain: Effect of reserpine
Brain Research, 300 (1984) 103-112 Elsevier
103
Retrograde Axonal Transport of fl-Adrenoreceptors in Rat Brain: Effect of Reserpine BARRY E. LEVIN
Neurology Service (127), VA Medical Center, East Orange, NJ 07019 and Department of Neurosciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103 (U.S.A.) (Accepted September 27th, 1983)
Key words: retrograde transport --fl-adrenoreceptors - - norepinephrine - - locus coeruleus - - 6-hydroxydopamine - iodocyanopindolol - - axonal transport - - reserpine
Retrograde axonal transport of fl-adrenoreceptors was assessed by measuring the accumulation of binding sites for the r-receptor ligand [125I]iodocyanopindolol([125I]ICP)distal to a unilateral 6-hydroxydopamine (6-OHDA) lesion placed in the ascendingnoradrenergic axons of the locus coeruleus. Accumulation of binding sites was linear over a 3 day period and was blocked by intracerebroventricular 6-OHDA given 1 day prior to sacrifice. A single dose of reserpine (5 mg/kg, i.p.) caused a long lasting (6-8 week) biphasic depletion of frontal cortex norepinephrine (NE) associated with increased frontal cortex binding of another r-receptor ligand, [3H]dihydroalprenolol (pH]DHA), at 7-14 days, and again at 28 days post-reserpine. Unlike the changes in cortical r-receptors, retrograde transport of [1251]ICPin presynaptic noradrenergic neurons was decreased or blocked completely at 7-14 days and at 6 weeks, and was increased to 470% and 240% of control at 21 days and 8 weeks after reserpine. Anterograde transport of [3H]DHA binding sites was measured by accumulation proximal to a 6-OHDA lesion in this pathway. This transport varied in a pattern similar to that seen for retrograde transport of [JzsI]ICPbinding sites. These data and others suggest that presynaptic r-receptors are regulated independentlyof frontal cortex r-receptors, which appear to be located primarily on postsynaptic cells. On the other hand, the regulation of both anterograde and retrograde transport appears to be interrelated since both types of transport were altered in a similar way in the face of long-term NE depletion by reserpine. INTRODUCTION Axonal transport of subcellular constituents represents one important mechanism by which n e u r o n s can regulate their metabolic status u n d e r varying conditions to which the cell is exposed. Retrograde transport of materials from the nerve terminal and axon back to the cell body appears to be one means by which signals from the periphery inform the cell body as to existing conditions at these sites. As such, various retrogradely transported intraneuronai materials appear to initiate certain changes in the synthetic priorities of the cell body in response to growth, axonal injury and regeneration 4.10,19,46. In addition, a diverse collection of extraneuronal substances seems to be taken up at the nerve terminal and retrogradely transported. These include viral nucleic acids l, antibodies 9,42, amino acids 31, toxins 18'35'45'52, neurotransmitters47 and assorted other proteins 3.17.2°,22,3°. The exact function for the pres-
ence of specific uptake and retrograde transport mechanisms for most of these extracellular substances remains largely obscure. Recently, a n u m b e r of laboratories have demonstrated the anterograde and/or retrograde transport of receptors for such substances as opiates54, cholecystokinin56 and acetylcholine (muscarinic)2t,57 in peripheral or cranial nerves. This laboratory first described the anterograde axonai transport of fladrenoreceptors in noradrenergic neurons of the locus coeruleus (LC) and showed that these appeared to be of the ill-subtype25. In a preliminary communication, Z a r b i n et al. 55 demonstrated both anterograde and retrograde transport primarily of fl2adrenoreceptors in rat sciatic nerve. The present work documents the retrograde axonal transport of fl-adrenoreceptors in noradrenergic neurons of the LC by measuring the accumulation of binding sites for the fl-adrenoreceptor ligand [t25I]iodocyanopindolol ([125I]ICP) distal to a lesion placed
Correspondence: B. E. Levin, Neurology Service (127), VA Medical Center, East Orange, NJ 07019, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
104 in the ascending LC pathway. This model system is a modification of the original nerve ligation technique introduced by Weiss and Hiscoe 51 and has been used previously to demonstrate the anterograde transport of the norepinephrine (NE) synthesizing enzymes tyrosine hydroxylase and dopamine-fl-hydroxylase24 as well as fl-adrenoreceptors 25 in rat brain. This technique has now been further used to investigate the interrelationship of the presynaptic transport of fl-receptors and the regulation of postsynaptic fl-receptor number during alterations induced by the administration of reserpine. METHODS
Animals and drug treatments Adult male Sprague-Dawley rats (200-300 g) were group-housed at 23 °C on a 12 h:12 h, lightdark cycle and fed Purina rat chow. Animals received either a single injection of 0.5 ml reserpine 5 mg/kg, i.p., (Serpasil; Ciba-Geigy) or saline. Groups of 6-12 rats so injected were then decapitated at intervals of 4 days to 8 weeks for the studies below.
Assay of fl-adrenergic receptor binding Binding of [125I]iodocyanopindolol ([125I]lCP; 2200 Ci/mmol; New England Nuclear) to hypothalamic membranes was carried out by modification of the methods of Minneman et al. 32. Hypothalamic membranes pooled from hypothalamic sections from 6-8 rats were prepared by homogenization (Polytron) in a 200-fold dilution of 154 mM NaCI, 20 mM TrisHC1 buffer, pH 7.5. Partially purified membranes were recovered in the pellet of a 40,000 g centrifugation for 20 min and resuspended in the same buffer to a final concentration of 0.2-0.8 mg/ml. Incubation of various concentrations of [125I]lCP (25-250 pM) was carried out in a final volume of 160/~1 for 15 min at 37 °C in the presence of 100 ktM phentolamine (Regitine; Ciba-Geigy) to reduce non-specific binding32. Non-specific binding was defined as that seen in the presence of 20/~M (--)-propranolol and ranged from 30 to 45%. Incubations were terminated by vacuum filtration over Whatman GF/B filters with 2 washes of ice-cold buffer. [125I]ICP activity was counted in a Nuclear Chicago y counter at 71% efficiency. Total specific binding of 30 nM [3H]dihydroalprenolol ([3H]DHA: 49 Ci/mmol; New England Nucle-
ar) to partially purified hypothalamic membranes was assessed by modification 25 of the method of Torda et al. 4s in a final volume of 160 ~1 at final protein concentrations of 1-2 mg/ml. This concentration of [3H]DHA gave near maximal binding to hypothalamic membranes. Non-specific binding was assessed in the presence of 10ktM (--)-propranolol (Ayerst Labs) and ranged from 35 to 50%. Total specific [3H]DHA binding to frontal cortex membranes was assessed by modification of the methods of Bylund and Snyder 7 as modified by Maggi et al. 29. Membranes, in a final concentration 0.5-1.0 mg/ml, were incubated in a final volume of 0.5 ml of 50 mM TrisHCI buffer, pH 7.7, with 2 nM [3H]DHA for 30 rain at 25 °C. Incubation was terminated by vacuum filtration over Whatman GF/B filters. Non-specific binding was estimated in the presence of 10 ~M (--)propranolol and ranged from 25 to 35%. All binding assays were run in triplicate at least 2 times and proteins were assayed by the method of Lowry et al.2S.
Transport of fl-receptor binding sites Retrograde axonal transport was assessed by measuring the accumulation of [125I]ICP binding sites distal to a lesion made with 2/~1 of 6-hydroxydopamineHBR (6-OHDA; 4~g/~l free base containing 0.1 ~g/~l ascorbic acid in distilled H20 ) injected into the proximal portion of the ascending locus coeruleus (LC) pathway 14,27,34 in the posterior hypothalamus (A 3.0, L 1.2, down 8.4 mm from skull surfacel6). Transport was then estimated by measuring the difference in [125I]ICP binding in 2 mm thick sections of left anterior hypothalamus (A 5-A 7) distal to these lesions as compared to binding in the comparable uninjected right side, at various times after 6-OHDA injection 2a. This left-right difference gives an estimate of the accumulation, and thus of the retrograde transport, of fl-receptor binding sites 24. [1251]ICP was used in these assays rather than [3H]DHA because of the former's greater affinity for these binding sites and higher specific activity, thus allowing the use of smaller amounts of membrane protein in the assays. All assays were done with 6 concentrations (25-250 pM) of [1251]ICP as described above in membranes pooled from 6-8 rats. Anterograde transport of fl-receptors was measured in individual rat brains by the accumulation of [3H]DHA binding sites in anterior hypothalamic sec-
105 tions (A 5-A 7) proximal to a 6-OHDA injection placed in the more rostral portion of the left ascending LC pathway (A 9.0, L 2.0, down 8.0 mm), as previously described 25, using 30 nM [3H]DHA. The effect of reserpine on cortical levels and retrograde and anterograde transport of fl-receptors was assessed by injecting groups of 6-20 rats with reserpine (5 mg/kg, i.p.) or saline, in a total volume of 0.5 ml, 7 days to 8 weeks prior to sacrifice. For retrograde transport, rats were injected in the ascending LC pathway 2 days prior to sacrifice and for anterograde transport, rats were injected in the pathway 1 day prior to sacrifice. These were the shortest times at which significant left-right differences were seen by the two methods used (see Results, Fig. 1, and ref. 25).
Assay of frontal cortex norepinephrine (NE) levels Groups of 6-8 rats were sacrificed at various intervals after reserpine or saline administration. Samples of left and right frontal cortex from each rat were pooled, homogenized by sonication in 0.1 N perchloric acid containing 10 pg/pl of dihydroxybenzylamine-HBR (free base). The supernatant of a 10,000 g centrifugation for 20 min was brought to pH 8.6 with 2 M Tris buffer and NE was adsorbed on 20 mg of alumina, washed twice with H20 and eluted in 0.1 N perchloric acid. Eluates were processed by reverse phase high performance liquid chromatography on a C18 #Bondapak, Z-module encased column (10 pm; Waters Associates) in a mobile phase of 0.1 M potassium phosphate, 4 mM sodium octyl sulfonate, 0.1 mM E D T A and 5% methanol, pH 3.0 run at 2 ml/min. Electrochemical detection was used to quantitate NE at an applied voltage of 0.72 V using a glassy-carbon electrode (BAS). Resulting chromatograms were analyzed by area under the curve on a Perkin-Elmer Sigma 10 data analyzer.
Statistics Data from experiments in which ligand binding was assessed at one concentration between left and right sides in the same animals were evaluated by paired t-test. Where results were compared between reserpine-treated and saline-treated (control) animals, evaluation was by Student's t-test for unpaired groups. Saturation isotherms were analyzed by the method of Scatchard 40.
RESULTS
Retrograde axonal transport of p251]ICP binding sites The retrograde transport of fl-adrenoreceptors was estimated by measuring the accumulation of [125I]ICP binding sites distal to a 6-OHDA lesion placed in the left ascending LC pathway in the posterior hypothalamus, when compared to the uninjected (right) side (Fig. 1). Binding increased linearly on the left side over 4 days while binding on the right remained unchanged for 3 days and then increased by 40% over control values. Saturation binding studies in right hypothalamic membranes gave: Ka = 132 + 14 pM and Bma x = 34.5 + 1.9 fmol/mg of protein. Retrograde axonal transport, expressed as the left-right difference, was first significant at 2 days after the lesion placement at 125% of the right-sided control. The left-right difference reached maximum at 3 days at 161% and then fell to 143% of the rightside values at 4 days when [125I]ICP binding on the uninjected right side had risen to 140% of its control levels. At no time did the affinity constant (Kd) for binding of [1251]ICP change significantly from control values.
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Fig. 1. Retrograde accumulation of [I251]ICP binding sites distal to a 6 - O H D A lesion in the ascending LC pathway. Groups of 6-12 rats were injected with 6 - O H D A in the left pathway in the posterior hypothalamus 1-4 days prior to sacrifice. Membranes pooled from 2 mm thick hypothalamic hemi-sections (6-8 rats), taken distal to the injection sites (left, C)) and the comparable, uninjected (right, Q) side were incubated with various concentrations of [125I]ICP (25-250 pM). Transport ( I ) is expressed as the difference between left and right sides. All data points represent the mean + S.E. (vertical bars) of Bmax values as estimated from triplicate determinations analyzed by the method of Scatchard 4°. * P < 0.05 or less when left- and right-sided Bmax values were compared.
106 was injected with 6 - O H D A in the left ascending LC pathway 2 days before, and with intracerebroventricular 6 - O H D A (250/~g in 20 ~1 saline) 1 day before
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Fig. 2. Saturation isotherm and Scatchard plot (inset) of [125I]ICP binding to hypothalamic membranes pooled from 6-12 rats injected with 6-OHDA in the left (©) LC pathway 2 days before, versus the uninjected (right) side (0). B = [~25I]ICP bound in fmol/mg protein; F = free concentration of ligand in nM. Data points represent the mean of triplicate determinations from 2-3 assays each. Left side: K a = 140 + 16 p M ; Bma x = 38.4 + 2.3 fmol/mg protein; right side: Ka = 124 + 11 pM; Bma x = 30.5 + 1.4 fmol/mg protein; P < /).025 for left-right difference in Bmax values calculated from the Scatchard plot38.
trol values. Levels then fell again to a nadir of 55% of control at 6 weeks and returned to control levels again by 8 weeks after reserpine injection. Total specific binding of [3H]DHA to frontal cortex fl-receptors followed a roughly inverse pattern to that seen for NE levels (Fig. 3) for the first 4 weeks following reserpine. Binding increased to 230% and 196% of control at 7 and 14 days respectively, returned to
fect of reserpine upon retrograde transport since accumulation was still increasing linearly at this time
control levels at 21 days and then rose again at 28 days to 167% of control. However, at 6 weeks after reserpine administration the inverse relationship between cortical NE levels and [3H]DHA binding broke down as both levels and binding were reduced to half of control values. The effect of reserpine on [125I]ICP binding to hy-
(Fig. 1). To show that this accumulation was indeed occurring in noradrenergic neurons, a group of 6 rats
pothalamic m e m b r a n e s (Fig. 4) was somewhat different than that seen for [3H]DHA binding to frontal
The 2 day time period following LC pathway lesions (Fig. 2) was used in all future studies on the ef-
TABLE I
Blockade of retrograde transport of [1251]ICPbinding sites"by intracerebroventricular 6-OHDA Two groups of 6-8 rats were injected with 2 ktl 6-OHDA in the left LC bundle 2 days before sacrifice. One group was also injected with intracerebroventricular 6-OHDA (i.c.v., 6-OHDA; 250/~g in 20/~1 saline) 1 day before sacrifice to destroy noradrenergic neurons of the LC. Norepinephrine levels were measured in frontal cortex (ng/g wet weight tissue) and total specific binding of 100 pM [1251]ICP to injected (left) and uninjected (right) hypothalamic membranes taken distal to the 2-day LC bundle lesion was assessed in fmol/mg protein. Data are mean + S.E. Transport = accumulation of [125I]ICPbinding sites distal to the left-sided lesion determined by subtracting binding on the right from the left sides.
Control i.c.v.,6-OHDA
Cortex NE
Hypothalamic [1251]1CPbinding
(ng/g)
Left
Right
330 + 24 88 _+ 11"
5.15 _+ 0.73 3.86 + 0.50
3.74 + 1/.32 4.15 _+ 0.17
* P < 0.01 when comparable values in 6-OHDA-treated rats were compared to controls.
Transport 1.42 _+ 0.45 ---0.29 + 0.41"
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Fig. 3. Effect of reserpine on frontal cortex NE levels and [3H]DHA binding. Groups of 10-20 rats were injected with saline (0 days = control) or reserpine (5 mg/kg i.p.) and sacrificed at various time intervals (7 days-8 weeks) after reserpine injection. Total specific fl-adrenoreceptor binding in fmol/mg membrane protein was assessed using 2 nM [3H]DHA. NE values are #tg/g wet weight tissue. Values represent mean + S.E. (vertical bars). * P < 0.05, ** P < 0.001 when values from reserpine-treated rats were compared to saline-injected controls,
cortex. Binding decreased to 60% of control at 7 days rose to 214% at 21 days and r e t u r n e d to control levels by 6 weeks after reserpine injection. This general pattern of binding to hypothalamic m e m b r a n e s was a p p r o x i m a t e l y m i r r o r e d for the first 4 weeks after reserpine by the accumulation of [12sI]ICP binding sites (Bmax) distal to a left-sided lesion in the LC pathway (Fig. 4). This accumulation ( r e t r o g r a d e transport) fell gradually to a nadir at 14 days to 15% of control and then increased over the next 7 days and reached 470% of control by 21 days post-reserpine. Retrograde transport then decreased over the next 3 weeks, so that at 6 weeks it was completely abolished. A secondary increase to 240% of control occurred at 8 weeks and by 9 weeks after reserpine administration, r e t r o g r a d e transport had r e t u r n e d again to control levels (not shown in Fig. 4). A t no time after reserpine injection did the affinity for [125I]ICP binding (Ka) change distal to the lesion site on the left side, suggesting that r e t r o g r a d e l y trans-
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Fig. 4. Effect of reserpine on the binding of [125I]ICP to hypothalamic membranes and the retrograde transport of fl-recep-
tor sites. Groups of 6-12 rats were sacrificed 7 days to 8 weeks after 5 mg/kg, i.p. injections of reserpine. Additionally, rats received 6-OHDA in the left ascending LC pathway 2 days prior to sacrifice. Data points represent the mean + S.E, (vertical bars) percent of control Bmax values as determined by Scatchard analysis of saturation isotherms from incubation of various concentrations (25-250 pM) of [125I]ICPwith hypothalamic membranes from injected (left) and uninjected (right) sides. 'Hypothalamic' values are from the uninjected right side where control values were: Ka = 132 + 14 pM; Bmax = 34.5 -+ 1.9 , fmol/mg protein. Retrograde transport represents the leftright difference where saline-injected control values were: 7.9 + 0.8 fmol/mg protein, * P < 0.05 or less where values from reserpine-treated rats were compared to saline-injected controls.
p o r t e d receptors showed no alteration in their affinity state from control. It was possible that the increases in frontal cortex binding of [3H]DHA at 7-14 days and 28 days postreserpine were due, to some degree, to changes in the n u m b e r of presynaptic fl-receptors undergoing retrograde and anterograde transport in the presynaptic noradrenergic axons innervating the frontal cortex. Since the total n u m b e r of receptors measured should reflect the algebraic sum of receptors transported into and out of the terminals, plus those present on postsynaptic cells, a n t e r o g r a d e transport of flreceptors was also examined at various time intervals after the injection of reserpine (Table II). A n t e r o grade transport of fl-receptors (accumulation of [3H]DHA binding sites proximal to a 6 - O H D A lesion placed in the left ascending LC pathway 1 day prior
108 TABLE II Anterograde axonal transport of [3H]DHA binding sites Groups of 6-19 rats were injected with saline (control) or reserpine (5 mg/kg, i.p.) at various intervals (7 days-8 weeks) before sacrifice and with 6-OHDA in the left ascending LC pathway 1 day prior to death. Axonal transport was assessed by the difference in accumulation of total specific binding of 30 nM [3H]DHA proximal to the lesion site (left) compared to the comparable uninjected (right) side.
Control 7 days 14 days 21 days 28days 6weeks 8weeks
{~H]DHA transport (fmol/mg protein)
Percent of control
13.(I _+ 5.3 --2.0 _+ 2.2* --3.2 _+ 4.1" 42.(I _+ 5.(/** 25.7 _+ 4.6* --5.3 _+ 2.5* 12.8 _+ 2.8
-0 0 323 198 (} 98
* P < 0.05, ** P < 0.01 when transport (accumulation) in reserpine-treated animals was compared to controls where data represent the mean + S.E.
to sacrifice) was altered in a pattern very similar to that seen for retrograde transport following reserpine administration. A n t e r o g r a d e transport was completely blocked from 7 to 14 days after reserpine, rose to 320% of control at 21 days, decreased to complete blockade at 6 weeks and returned to control levels by 8 weeks post-reserpine. Thus, the presynaptic retrograde and a n t e r o g r a d e transport of fl-receptors a p p e a r e d to be independently regulated when c o m p a r e d to frontal cortex fl-receptors (which were presumably located on postsynaptic cells) in response to alterations in N E content p r o d u c e d by reserpine administration. DISCUSSION The present studies confirm the previous observation that anterograde axonal transport of fl-adrenoreceptors occurs in presynaptic noradrenergic neurons in the rat brain 25. Additionally, retrograde transport of these receptors in the same neurons also appears to be present since the linear accumulation of binding sites for the fl-adrenergic ligand [125I]ICP was shown to occur over a 3 day period distal to a lesion placed in the pathway. This accumulation could be blocked by destruction of these noradrenergic axons by intraventricular 6 - O H D A given 1 day prior to sacrifice, confirming the presynaptic location of this
transport. Although no attempt was m a d e here to define the subtype of fl-receptor, it is assumed that retrogradely transported, like anterogradely transported fl-receptors, were also of the ill-subtype25. It must be pointed out that the m e a s u r e m e n t of accumulation of receptor binding sites proximal or distal to an axonal lesion may be influenced by certain experimental artifacts. For example, the methods used here obviously require that the axon be injured. As a consequence, a certain amount of accumulated material will turn a r o u n d at the lesion site and return towards its site of origin 4,5.53. This makes calculation of actual transport rate difficult, although the measurement of total accumulation of a substance gives a fairly good indication of true transport if the time between the axonal injury and the m e a s u r e m e n t of that substance's accumulation is kept short. For this reason, a 2 day period was used for retrograde, and a 1 day period for anterograde transport, i.e. times at which left-right differences were significant and accumulation of receptors was still occurring in a linear fashion 24. This minimized both the effects of turnaround at the lesion site, and the effects of axonal injury on cell metabolism and axonal transport. A n o t h er reason for using this shorter time period of 2 days is that, after 3-4 days, the levels of [1251]ICP binding began to increase in the uninjected right hypothalamus. This later change appears to be due to some generalized, but at yet unexplained, p h e n o m e n o n in which unilateral lesions can affect function in contralateral brain noradrenergic neurons 26,3s. Similar changes in contralaterai hypothalamic levels of N E and of dopamine-fl-hydroxylase and tyrosine hydroxylase activities also occur after c o m p a r a b l e unilateral lesions in the ascending LC pathway (unpublished results). Reserpine had a surprisingly long lasting effect on both the presynaptic anterograde and retrograde transport of fl-receptors, as well as on frontal cortex levels of N E and fl-receptor number. It is uncertain what mechanism adequately explains the long duration of changes evoked by a single dose of reserpine. It is interesting that the effect on cortical N E levels was biphasic, showing two periods of decline separated by a transient return to control levels. Similar biphasic and triphasic changes in the anterograde transport and cell body synthesis of various subclasses of proteins 23 and NE synthesizing en-
109 zymes 2,24,36,37,39,58 have been documented in this
same model system over a 3 week period. It is unclear whether the even longer term effects of reserpine seen here were related to a chain of events initially triggered by the depletion of brain NE caused by the reserpine-induced destruction of NE storage vesiclesS,50, or to the other well-described, non-specific effects of reserpine upon cell metabolism15. 33. Since it is presumed that the neuronal cell body must change its metabolic priorities to synthesize new vesicles and NE-synthesizing enzymes, it is perhaps not surprising that the initial effects of reserpine might initiate a series of long lasting alterations in the metabolic function of the noradrenergic cell. Furthermore, since reserpine was shown here, and has been previously described to affect what appear to be primarily postsynaptic fl-receptors in the frontal cortex 41 innervated by noradrenergic LC neurons (see below), these postsynaptic cortical cells might also be expected to reciprocally influence the presynaptic noradrenergic neuron by means of feedback regulation of some sort. Such a feedback system has been well described in the nigro-striatal system 6. A major finding of this study was the demonstration the fl-adrenergic receptors in presynaptic noradrenergic neurons and fl-adrenoreceptors on cortical cells innervated by these neurons were independently regulated in response to reserpine administration. Several lines of evidence point to a postsynaptic location for most of the fl-receptors undergoing change in response to reserpine here. First, it has been shown that the total number of cortical fl-receptors actually increases when presynaptic noradrenergic innervation is removed 13,41,44,49. Second, cortical fl-receptors and fl-receptor-mediated adenylate cyclase either develop normally or are increased in the absence of presynaptic noradrenergic input in neonatal rats ll and in cortical cells raised in tissue culture 12. We have also found that destruction of presynaptic noradrenergic neurons with intraventricular 6OHDA, given 1 day prior to sacrifice, did not alter the increased number of cortical fl-receptors seen at 7 and 28 days after reserpine administration (Levin, B.E. and Biegon, A., unpublished observation). Finally, the present study showed that, while the patterns of anterograde and retrograde transport of presynaptic fl-receptors tended to parallel each other after reserpine administration, the changes in cortical fl-re-
ceptor binding followed an almost totally different pattern. Another interesting observation was that the inverse relationship between frontal cortical binding of [3H]DHA and levels of NE appeared to become dissociated at 6 weeks post-reserpine. At that time, both parameters were reduced to half of control levels and presynaptic retrograde and anterograde transport of fl-receptors was completely blocked. While unexplained, this finding suggests that the secondary decrease in cortical fl-receptor binding seen at this time was regulated by factors other than presynaptic levels of NE. From the preceding discussion it is obvious that the numerical contribution of presynaptic fl-receptors located on noradrenergic terminals in the cerebral cortex to the total population of cortical fl-receptors must be extremely small. Where anterograde transport of fl-receptors was completely blocked following reserpine (7-14 days), retrograde transport was still present to some degree. In opposite manner, the 220% increase above baseline seen for anterograde receptor transport at 21 days post-reserpine was matched by an even greater relative increase of 370% in retrograde transport. Finally, following complete blockade of both anterograde and retrograde flreceptor transport at 6 weeks, retrograde transport rose to 240% of control at 8 weeks post-reserpine at a time when anterograde transport had returned to control levels. Therefore, in almost every,case, the relative increases in retrograde transport were always greater than those for anterograde transport. However, under control conditions, the relative proportion of fl-receptors moving in an anterograde direction (25% per day of the intrinsic population of receptors) 25 is twice that of those moving in a retrograde direction (26% per 2 days; see Figs. 1 and 2). Presumably then, the greater anterograde transport of receptors was balanced by a smaller retrograde transport away from, plus the degradation of receptors at the nerve terminals. Therefore, under conditions of metabolic alterations of the neuronal milieu, such as was seen in the current studies, these three factors appear capable of changing in such a manner as to maintain an equilibrium of receptors at the presynaptic terminals. A final observation was that hypothalamic binding of fl-receptors did not follow the same pattern of changes in response to reserpine as did cortical fl-re-
110 ceptors. This was not simply due to differences in the ligands since the pattern of right hypothalamic binding of [3H]DHA in the anterograde transport experiments was similar to that seen for [125I]ICP binding in Fig. 4 (data not shown). While hypothalamic N E levels were not measured, there are at least two reasons why regulation of cortical and hypothalamic fl-receptors might not be expected to be similar. First, the hypothalamus receives nodradrenergic input from both the LC and from more caudally placed N E neurons in the brainstem, whereas the frontal cortex receives primarily LC input aone 27.34. Since reserpine may affect anatomically discrete groups of neurons containing the same neurotransmitter differently 36, regulation of fl-receptor binding in areas receiving differing noradrenergic input might be expected to reflect this fact. Second, the hypothalamus contains approximately equal numbers of ill- and fl2-adrenoreceptors 25 while the frontal cortex contains primarily ill-receptors32. While it is not known whether reserpine differentially affects the two receptor subtypes, it is quite possible that such differences do exist and would therefore be reflected in the pattern of receptor binding seen using non-specific fl-receptor ligands such as [3H]DHA or [125I]ICP.
REFERENCES l Bak, I. J., Markham, C. H., Cook, M. L. and Stevens, J. G., Intraaxonal transport of herpes simplex virus in the rat central nervous system, Brain Research, 136 (1977) 415-429. 2 Black, I. B., Increased tyrosine hydroxylase activity in frontal cortex and cerebellum after reserpine, Brain Research, 95 (1975) 170-176. 3 Borges, L. F. and Sidman, R. L., Axonal transport of lectins in the peripheral nervous system, J. Neurosci., 2 (1982) 647-653. 4 Brimijoin, S., Transport and turnover of dopamine-fl-hydroxylase (EC 1.14.2.1) in sympathetic nerves of the rat, J. Neurochem., 19 (1972) 2183-2193. 5 Brimijoin, S. and Helland, L., Rapid retrograde transport of dopamine-fl-hydroxylase as examined by the stop-flow technique, Brain Research, 102 (1976) 217-228. 6 Bunney, B. S. and Aghajanian, G. K., D-Amphetamine induced inhibition of central dopaminergic neurons: mediation by a striato-nigral feedback pathway, Science, 192 (1976) 391-393. 7 Bylund, D. B. and Snyder, S. H., Beta-adrenergic receptor binding in membrane preparations from mammalian brain, Molec. Pharmacol., 12 (1976) 568-580. 8 Carlsson, A., Rosengren, E., Bertler, A. and Nelsson, J., The effect of reserpine on the metabolism of catechola-
In conclusion, the present study has d e m o n s t r a t e d the presence of retrograde axonal transport of fladrenergic receptor binding sites in rat brain noradrenergic neurons which occurred independently of the regulation of presumed postsynaptic cortical flreceptors in response to reserpine administration. F u r t h e r m o r e , the anterograde and retrograde transport of receptors and other subcellular constituents has again been shown to be one of several important mechanisms by which the neuron can regulate its metabolic activity in response to various perturbations of its baseline functioning. ACKNOWLEDGEMENTS This work was s u p p o r t e d by the Medical Research Service of the Veterans Administration. W e wish to thank A y e r s t L a b o r a t o r i e s for supplying ( - - ) - p r o pranolol, and Ciba-Geigy Pharmaceuticals for the phentolamine (Regitine) and reserpine (Serpasil). We gratefully acknowledge the expert technical assistance of E d w a r d A g y k u m and Mark Schachman, and secretarial assistance of A n t o i n e t t e Colitti.
mines. In S. Garattini and U. Ghetti (Eds.), Psychotropic Drugs, Elsevier, Amsterdam, 1957, pp. 363-372. 9 Fillenz, M., Gagnon, C., Stoeckel, K. and Thoenen, H., Selective uptake and retrograde axonal transport of dopamine-fl-hydroxylase antibodies in peripheral adrenergic neurons, Brain Research, 114 (1976) 293-303. 10 Goedert, M., Stoeckel, K. and Otten U., Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 5895-5898. 11 Harden, T. K., Wolfe, B. B., Sporn, J. R., Poulos, B. K. and Molinoff, P. B., Effects of 6-hydroxydopamine on the development of the beta adrenergic receptor/adenylate cyclase system in rat cerebral cortex, J. Pharmacol. exp. Ther., 203 (1977) 132-143. 12 Harden, T. K. and McCarthy, K. D., Identification of the beta adrenergic receptor subtype on astroglia purified from rat brain, J. Pharmacol. exp. Ther., 222 (1982) 600-605. 13 Harik, S. I., Duckrow, R. B., LaManna, J. C., Rosenthal, M., Sharma, V. K. and Banerjee, S. P., Cerebral compensation for chronic noradrenergic denervation induced by locus coeruleus lesion: recovery of receptor binding, isoproterenol-induced adenylate cyclase activity, and oxidative metabolism, J. Neurosci., 1 (1981)641-649. 14 Jones, B. E. and Moore, R. Y., Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study, Brain Research, 127 (1977) 23-53.
111 15 Kirpekar, S. M. and Lewis, J. J., Some effects of reserpine and hydralazine upon tissue respiration and the concentration of adenosine nucleotides in certain tissues, Brit. J. Pharmacol. Chemother., 14 (1959) 40-45. 16 Koenig, J. F. R. and Klippel, R. A., The Rat Brain, William and Wilkins, Baltimore, MD, 1963. 17 Kristensson, K., Retrograde transport of macromolecules in axons, Ann. Rev. Pharmacol. Toxicol., 18 (1978) 91-110. 18 Kristensson, K. and Olsson, T., Uptake and retrograde axonal transport of horseradish peroxidase in botulinum-intoxicated mice, Brain Research, 155 (1978) 118--123. 19 Kristensson, K. and Sj6strand, J., Retrograde transport of protein tracer in the rabbit hypoglossal nerve during regeneration, Brain Research, 45 (1972) 175-181. 20 Kuypers, H. G. J. M., Catsman-Berrevoets, C. E. and Padt, R. E., Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6 (1977) 127-135. 21 Laduron, P., Axoplasmic transport of muscarinic receptors, Nature (Lond.), 286 (1980) 287-288. 22 LaVail, J. H. and LaVail, M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study, J. comp. Neurol., 157 (1974) 303-358. 23 Levin, B. E., Effects of reserpine on fast, intermediate and slow axonal transport of proteins in rat locus coeruleus neurons, Brain Research, 189 (1980) 495-504. 24 Levin, B. E., Reserpine effect on the axonal transport of dopamine-fl-hydroxylase and tyrosine hydroxylase in rat brain, Exp. Neurol., 72 (1981) 99-112. 25 Levin, B. E., Presynaptic location and axonal transport of fll-adrenoreceptors in the rat brain, Science, 217 (1982) 555-557. 26 Levin, B. E., Alterations of norepinephrine metabolism in rat locus coeruleus neurons in response to axonal injury, Brain Research, in press. 27 Lindvall, O. and Bj6rklund, A., The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method, Acta physiol, scand., 412 (1974) 1-48. 28 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 29 Maggi, A., U'Prichard, D. C. and Enna, S. J., Differential effects of antidepressant treatment on brain monoaminergic receptors, Europ. J. Pharmacol., 61 (1980) 91-98. 30 Malmgren, L., Olsson, Y., Olsson, T. and Kristensson, K., Uptake and retrograde axonal transport of various exogenous macromolecules in normal and crushed hypoglossal nerves, Brain Research, 153 (1978) 477-493. 31 Meibach, R. C. and Siegel, A., Efferent connections of the septal area in the rat: an analysis utilizing retrograde and anterograde transport methods, Brain Research, 119 (1977) 1-20. 32 Minneman, K. P., Hegstrand, L. R. and Molinoff, P. B., Simultaneous determination of beta 1- and beta 2-adrenergic receptors in tissues containing both receptor subtypes, Molec. Pharmacol., 16 (1979) 34-46. 33 Patel, A. J., Bendek, G. and Balazs, R., Effect of reserpine on cell proliferation in the developing rat brain: a biochemical study, Brain Research, 129 (1977) 283-297. 34 Pickel, V. M., Segal, M. and Bloom, F. E., A radioautographic study of the efferent pathways of the nucleus locus
coeruleus, J. comp. Neurol., 155 (1974) 15--41. 35 Price, D. L., Griffin, J., Young, A., Peck, K. and Stocks, A., Tetanus toxin: direct evidence for retrograde intraaxonal transport, Science, 188 (1975) 945-947. 36 Reis, D. J., Joh, T. H. and Ross, R. A., Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopamine-fl-hydroxylase in catecholamine neuronal systems in rat brain, J. Pharmacol. exp. Ther., 193 (1975) 775-784. 37 Reis, D. J., Joh, T. H., Ross, R. A. and Pickel, V. M., Reserpine selectively increases tyrosine hydroxylase and dopamine-fl-hydroxylase enzyme protein in central noradrenergic neurons, Brain Research, 81 (1974) 380-386. 38 Robinson, R. G. and Stitt, T. G., Intracortical 6-hydroxydopamine induces an asymmetrical behavioral response in the rat brain, Brain Research, 213 (1981) 387-395. 39 Ross, R. A., Joh, T. H. and Reis, I~. J., Increase in the relative rate of synthesis of dopamine-fl-hydroxylase in the nucleus locus coeruleus elicited by reserpine, J. Neurochem., 31 (1978) 1491-1500. 40 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N. Y. Acad. Sci., 51 (1949) 660-672. 41 Sharma, V. K., Harik, S. I., Busto, R. and Banerjee, S. P., Effects of noradrenaline depletion on adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum, Exp. Neurol., 72 (1981) 179--194. 42 Silver, M. A. and Jacobowitz, D. M., Specific uptake and retrograde flow of antibody to dopamine-fl-hydroxylase by central nervous system noradrenergic neurons in vivo, Brain Research, 167 (1979) 65-75. 43 Singer, P. A., Mehler, S. and Fernandez, H. L., Blockade of retrograde axonal transport delays the onset of metabolic and morphologic changes induced by axotomy, J. Neurosci., 2 (1982) 1299--1306. 44 Sporn, J. R., Harden, T. K., Wolfe, B. B. and Molinoff, P. B., fl-Adrenergic receptor involvement in 6-hydroxydopamine-induced supersensitivity in rat cerebral cortex, Science, 194 (1976) 624-626. 45 Stoeckel, K., Schwab, M. and Thoenen, H., Role of gangliosides in the uptake and retrograde axonal transport of cholera and tetanus toxin as compared to nerve growth factor and wheat germ agglutinin, Brain Research, 132 (1977) 273--285. 46 Stoeckel, K. and Thoenen, H., Retrograde axonal transport of nerve growth factor: specificity and biological importance, Brain Research, 85 (1975) 337-341. 47 Streit, P., Knecht, E. and Cuenod, M., Transmitter-specific retrograde labeling in the striato-nigral and raphe-nigral pathways, Science, 205 (1979) 306-308. 48 Torda, T., Yamaguchi, I., Hirata, F., Kopin, I. J. and Axelrod, J., Mepacrine treatment prevents immobilization-induced desensitization of beta-adrenergic receptors in rat hypothalamus and brain stem, Brain Research, 205 (1981) 441--444. 49 U'Prichard, D. C., Reisine, T. D., Yamamura, S., Mason, S. T., Fibiger, H. C., Ehlert, F. and Yamamura, H. I., Differential supersensitivity of fl-receptor subtypes in rat cortex and cerebellum after central noradrenergic denervation, Life Sci., 26 (1980) 355-364. 50 Wakade, A. R., A comparison of rates of depletion and recovery of noradrenaline stores of peripheral and central noradrenergic neurons after reserpine administration: importance of neuronal activity, Brit. J. Pharmacol., 68 (1980) 93-98. 51 Weiss, P. and Hiscoe, H., Experiments on the mechanism
112 of nerve growth, J. exp. Zool., 107 (1948) 315-395. 52 Wiley, R. G., Blessing, W. W. and Reis, D. J., Suicide transport: destruction of neurons by retrograde transport of ricin, abrin, and modeccin, Science, 216 (1982) 889-890. 53 Wooten, G. F., Subcellular distribution and rapid axonal transport of dopamine-fl-hydroxylase, Brain Research, 55 (1973) 491--494. 54 Young, III, W. C., Wamsley, J. K., Zarbin, M. A. and Kuhar, M. J., Opioid receptors undergo axonal flow, Science, 210 (1980) 76--77. 55 Zarbin, M. A., Palacios, J. M., Wamsley, J. K. and Kuhar, M. J., Axonal transport of beta-adrenergic receptors in rat sciatic nerve, Soc. Neurosci. Abstr., 8 (1982) 829.
56 Zarbin, M. A., Wamsley, J. K., Innis, R. B. and Kuhar, M. J., Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve, Life Sci., 29 (1981) 697-705. 57 Zarbin, M. A., Wamsley, J. K. and Kuhar, M. J., Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity, J. Neurosci., 2 (1982) 934-941. 58 Zigmond, R. E., Tyrosine hydroxylase activity in noradrenergic neurons of the locus coeruleus after reserpine administration: sequential increase in cell bodies and nerve terminals, J. Neurochem., 32 (1979) 23-29.
103
Retrograde Axonal Transport of fl-Adrenoreceptors in Rat Brain: Effect of Reserpine BARRY E. LEVIN
Neurology Service (127), VA Medical Center, East Orange, NJ 07019 and Department of Neurosciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103 (U.S.A.) (Accepted September 27th, 1983)
Key words: retrograde transport --fl-adrenoreceptors - - norepinephrine - - locus coeruleus - - 6-hydroxydopamine - iodocyanopindolol - - axonal transport - - reserpine
Retrograde axonal transport of fl-adrenoreceptors was assessed by measuring the accumulation of binding sites for the r-receptor ligand [125I]iodocyanopindolol([125I]ICP)distal to a unilateral 6-hydroxydopamine (6-OHDA) lesion placed in the ascendingnoradrenergic axons of the locus coeruleus. Accumulation of binding sites was linear over a 3 day period and was blocked by intracerebroventricular 6-OHDA given 1 day prior to sacrifice. A single dose of reserpine (5 mg/kg, i.p.) caused a long lasting (6-8 week) biphasic depletion of frontal cortex norepinephrine (NE) associated with increased frontal cortex binding of another r-receptor ligand, [3H]dihydroalprenolol (pH]DHA), at 7-14 days, and again at 28 days post-reserpine. Unlike the changes in cortical r-receptors, retrograde transport of [1251]ICPin presynaptic noradrenergic neurons was decreased or blocked completely at 7-14 days and at 6 weeks, and was increased to 470% and 240% of control at 21 days and 8 weeks after reserpine. Anterograde transport of [3H]DHA binding sites was measured by accumulation proximal to a 6-OHDA lesion in this pathway. This transport varied in a pattern similar to that seen for retrograde transport of [JzsI]ICPbinding sites. These data and others suggest that presynaptic r-receptors are regulated independentlyof frontal cortex r-receptors, which appear to be located primarily on postsynaptic cells. On the other hand, the regulation of both anterograde and retrograde transport appears to be interrelated since both types of transport were altered in a similar way in the face of long-term NE depletion by reserpine. INTRODUCTION Axonal transport of subcellular constituents represents one important mechanism by which n e u r o n s can regulate their metabolic status u n d e r varying conditions to which the cell is exposed. Retrograde transport of materials from the nerve terminal and axon back to the cell body appears to be one means by which signals from the periphery inform the cell body as to existing conditions at these sites. As such, various retrogradely transported intraneuronai materials appear to initiate certain changes in the synthetic priorities of the cell body in response to growth, axonal injury and regeneration 4.10,19,46. In addition, a diverse collection of extraneuronal substances seems to be taken up at the nerve terminal and retrogradely transported. These include viral nucleic acids l, antibodies 9,42, amino acids 31, toxins 18'35'45'52, neurotransmitters47 and assorted other proteins 3.17.2°,22,3°. The exact function for the pres-
ence of specific uptake and retrograde transport mechanisms for most of these extracellular substances remains largely obscure. Recently, a n u m b e r of laboratories have demonstrated the anterograde and/or retrograde transport of receptors for such substances as opiates54, cholecystokinin56 and acetylcholine (muscarinic)2t,57 in peripheral or cranial nerves. This laboratory first described the anterograde axonai transport of fladrenoreceptors in noradrenergic neurons of the locus coeruleus (LC) and showed that these appeared to be of the ill-subtype25. In a preliminary communication, Z a r b i n et al. 55 demonstrated both anterograde and retrograde transport primarily of fl2adrenoreceptors in rat sciatic nerve. The present work documents the retrograde axonal transport of fl-adrenoreceptors in noradrenergic neurons of the LC by measuring the accumulation of binding sites for the fl-adrenoreceptor ligand [t25I]iodocyanopindolol ([125I]ICP) distal to a lesion placed
Correspondence: B. E. Levin, Neurology Service (127), VA Medical Center, East Orange, NJ 07019, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
104 in the ascending LC pathway. This model system is a modification of the original nerve ligation technique introduced by Weiss and Hiscoe 51 and has been used previously to demonstrate the anterograde transport of the norepinephrine (NE) synthesizing enzymes tyrosine hydroxylase and dopamine-fl-hydroxylase24 as well as fl-adrenoreceptors 25 in rat brain. This technique has now been further used to investigate the interrelationship of the presynaptic transport of fl-receptors and the regulation of postsynaptic fl-receptor number during alterations induced by the administration of reserpine. METHODS
Animals and drug treatments Adult male Sprague-Dawley rats (200-300 g) were group-housed at 23 °C on a 12 h:12 h, lightdark cycle and fed Purina rat chow. Animals received either a single injection of 0.5 ml reserpine 5 mg/kg, i.p., (Serpasil; Ciba-Geigy) or saline. Groups of 6-12 rats so injected were then decapitated at intervals of 4 days to 8 weeks for the studies below.
Assay of fl-adrenergic receptor binding Binding of [125I]iodocyanopindolol ([125I]lCP; 2200 Ci/mmol; New England Nuclear) to hypothalamic membranes was carried out by modification of the methods of Minneman et al. 32. Hypothalamic membranes pooled from hypothalamic sections from 6-8 rats were prepared by homogenization (Polytron) in a 200-fold dilution of 154 mM NaCI, 20 mM TrisHC1 buffer, pH 7.5. Partially purified membranes were recovered in the pellet of a 40,000 g centrifugation for 20 min and resuspended in the same buffer to a final concentration of 0.2-0.8 mg/ml. Incubation of various concentrations of [125I]lCP (25-250 pM) was carried out in a final volume of 160/~1 for 15 min at 37 °C in the presence of 100 ktM phentolamine (Regitine; Ciba-Geigy) to reduce non-specific binding32. Non-specific binding was defined as that seen in the presence of 20/~M (--)-propranolol and ranged from 30 to 45%. Incubations were terminated by vacuum filtration over Whatman GF/B filters with 2 washes of ice-cold buffer. [125I]ICP activity was counted in a Nuclear Chicago y counter at 71% efficiency. Total specific binding of 30 nM [3H]dihydroalprenolol ([3H]DHA: 49 Ci/mmol; New England Nucle-
ar) to partially purified hypothalamic membranes was assessed by modification 25 of the method of Torda et al. 4s in a final volume of 160 ~1 at final protein concentrations of 1-2 mg/ml. This concentration of [3H]DHA gave near maximal binding to hypothalamic membranes. Non-specific binding was assessed in the presence of 10ktM (--)-propranolol (Ayerst Labs) and ranged from 35 to 50%. Total specific [3H]DHA binding to frontal cortex membranes was assessed by modification of the methods of Bylund and Snyder 7 as modified by Maggi et al. 29. Membranes, in a final concentration 0.5-1.0 mg/ml, were incubated in a final volume of 0.5 ml of 50 mM TrisHCI buffer, pH 7.7, with 2 nM [3H]DHA for 30 rain at 25 °C. Incubation was terminated by vacuum filtration over Whatman GF/B filters. Non-specific binding was estimated in the presence of 10 ~M (--)propranolol and ranged from 25 to 35%. All binding assays were run in triplicate at least 2 times and proteins were assayed by the method of Lowry et al.2S.
Transport of fl-receptor binding sites Retrograde axonal transport was assessed by measuring the accumulation of [125I]ICP binding sites distal to a lesion made with 2/~1 of 6-hydroxydopamineHBR (6-OHDA; 4~g/~l free base containing 0.1 ~g/~l ascorbic acid in distilled H20 ) injected into the proximal portion of the ascending locus coeruleus (LC) pathway 14,27,34 in the posterior hypothalamus (A 3.0, L 1.2, down 8.4 mm from skull surfacel6). Transport was then estimated by measuring the difference in [125I]ICP binding in 2 mm thick sections of left anterior hypothalamus (A 5-A 7) distal to these lesions as compared to binding in the comparable uninjected right side, at various times after 6-OHDA injection 2a. This left-right difference gives an estimate of the accumulation, and thus of the retrograde transport, of fl-receptor binding sites 24. [1251]ICP was used in these assays rather than [3H]DHA because of the former's greater affinity for these binding sites and higher specific activity, thus allowing the use of smaller amounts of membrane protein in the assays. All assays were done with 6 concentrations (25-250 pM) of [1251]ICP as described above in membranes pooled from 6-8 rats. Anterograde transport of fl-receptors was measured in individual rat brains by the accumulation of [3H]DHA binding sites in anterior hypothalamic sec-
105 tions (A 5-A 7) proximal to a 6-OHDA injection placed in the more rostral portion of the left ascending LC pathway (A 9.0, L 2.0, down 8.0 mm), as previously described 25, using 30 nM [3H]DHA. The effect of reserpine on cortical levels and retrograde and anterograde transport of fl-receptors was assessed by injecting groups of 6-20 rats with reserpine (5 mg/kg, i.p.) or saline, in a total volume of 0.5 ml, 7 days to 8 weeks prior to sacrifice. For retrograde transport, rats were injected in the ascending LC pathway 2 days prior to sacrifice and for anterograde transport, rats were injected in the pathway 1 day prior to sacrifice. These were the shortest times at which significant left-right differences were seen by the two methods used (see Results, Fig. 1, and ref. 25).
Assay of frontal cortex norepinephrine (NE) levels Groups of 6-8 rats were sacrificed at various intervals after reserpine or saline administration. Samples of left and right frontal cortex from each rat were pooled, homogenized by sonication in 0.1 N perchloric acid containing 10 pg/pl of dihydroxybenzylamine-HBR (free base). The supernatant of a 10,000 g centrifugation for 20 min was brought to pH 8.6 with 2 M Tris buffer and NE was adsorbed on 20 mg of alumina, washed twice with H20 and eluted in 0.1 N perchloric acid. Eluates were processed by reverse phase high performance liquid chromatography on a C18 #Bondapak, Z-module encased column (10 pm; Waters Associates) in a mobile phase of 0.1 M potassium phosphate, 4 mM sodium octyl sulfonate, 0.1 mM E D T A and 5% methanol, pH 3.0 run at 2 ml/min. Electrochemical detection was used to quantitate NE at an applied voltage of 0.72 V using a glassy-carbon electrode (BAS). Resulting chromatograms were analyzed by area under the curve on a Perkin-Elmer Sigma 10 data analyzer.
Statistics Data from experiments in which ligand binding was assessed at one concentration between left and right sides in the same animals were evaluated by paired t-test. Where results were compared between reserpine-treated and saline-treated (control) animals, evaluation was by Student's t-test for unpaired groups. Saturation isotherms were analyzed by the method of Scatchard 40.
RESULTS
Retrograde axonal transport of p251]ICP binding sites The retrograde transport of fl-adrenoreceptors was estimated by measuring the accumulation of [125I]ICP binding sites distal to a 6-OHDA lesion placed in the left ascending LC pathway in the posterior hypothalamus, when compared to the uninjected (right) side (Fig. 1). Binding increased linearly on the left side over 4 days while binding on the right remained unchanged for 3 days and then increased by 40% over control values. Saturation binding studies in right hypothalamic membranes gave: Ka = 132 + 14 pM and Bma x = 34.5 + 1.9 fmol/mg of protein. Retrograde axonal transport, expressed as the left-right difference, was first significant at 2 days after the lesion placement at 125% of the right-sided control. The left-right difference reached maximum at 3 days at 161% and then fell to 143% of the rightside values at 4 days when [125I]ICP binding on the uninjected right side had risen to 140% of its control levels. At no time did the affinity constant (Kd) for binding of [1251]ICP change significantly from control values.
"~ 60 E
0 LEFT
RIGHT • TRANSPORT
•
~ so ~
4o
o
5o
Z
IO I
I
I
I
I
2
3
4
DAYS
Fig. 1. Retrograde accumulation of [I251]ICP binding sites distal to a 6 - O H D A lesion in the ascending LC pathway. Groups of 6-12 rats were injected with 6 - O H D A in the left pathway in the posterior hypothalamus 1-4 days prior to sacrifice. Membranes pooled from 2 mm thick hypothalamic hemi-sections (6-8 rats), taken distal to the injection sites (left, C)) and the comparable, uninjected (right, Q) side were incubated with various concentrations of [125I]ICP (25-250 pM). Transport ( I ) is expressed as the difference between left and right sides. All data points represent the mean + S.E. (vertical bars) of Bmax values as estimated from triplicate determinations analyzed by the method of Scatchard 4°. * P < 0.05 or less when left- and right-sided Bmax values were compared.
106 was injected with 6 - O H D A in the left ascending LC pathway 2 days before, and with intracerebroventricular 6 - O H D A (250/~g in 20 ~1 saline) 1 day before
30
~-~ E -6 E
O
LEFT
•
RIGHT
/
-
sacrifice (Table I). The ventricular injection of 6-
_--e
O H D A reduced NE levels to 27% of control. This completely abolished the accumulation of binding
20
/•/
sites expected distal to the LC pathway lesion site when total specific binding was measured using 100 pM [125I]ICP.
/
O Z O CO IO
Effect o f reserpine on frontal cortex N E levels and fireceptor number and axonal transport
j
/,,; ?I
"~-;%
'~I
Administration of a single dose of reserpine (5 mg/kg, i.p.) reduced frontal cortex NE levels to 48%
I
,,o ,,o':bJo
/'
of control by 7 days (levels were 10% of control at 3
B i
50
i
IOO
i
i
i
150
200
250
days) after injection (Fig. 3). Levels remained depressed until 21 days when they returned to con-
[ItSl]-ICP (pM)
Fig. 2. Saturation isotherm and Scatchard plot (inset) of [125I]ICP binding to hypothalamic membranes pooled from 6-12 rats injected with 6-OHDA in the left (©) LC pathway 2 days before, versus the uninjected (right) side (0). B = [~25I]ICP bound in fmol/mg protein; F = free concentration of ligand in nM. Data points represent the mean of triplicate determinations from 2-3 assays each. Left side: K a = 140 + 16 p M ; Bma x = 38.4 + 2.3 fmol/mg protein; right side: Ka = 124 + 11 pM; Bma x = 30.5 + 1.4 fmol/mg protein; P < /).025 for left-right difference in Bmax values calculated from the Scatchard plot38.
trol values. Levels then fell again to a nadir of 55% of control at 6 weeks and returned to control levels again by 8 weeks after reserpine injection. Total specific binding of [3H]DHA to frontal cortex fl-receptors followed a roughly inverse pattern to that seen for NE levels (Fig. 3) for the first 4 weeks following reserpine. Binding increased to 230% and 196% of control at 7 and 14 days respectively, returned to
fect of reserpine upon retrograde transport since accumulation was still increasing linearly at this time
control levels at 21 days and then rose again at 28 days to 167% of control. However, at 6 weeks after reserpine administration the inverse relationship between cortical NE levels and [3H]DHA binding broke down as both levels and binding were reduced to half of control values. The effect of reserpine on [125I]ICP binding to hy-
(Fig. 1). To show that this accumulation was indeed occurring in noradrenergic neurons, a group of 6 rats
pothalamic m e m b r a n e s (Fig. 4) was somewhat different than that seen for [3H]DHA binding to frontal
The 2 day time period following LC pathway lesions (Fig. 2) was used in all future studies on the ef-
TABLE I
Blockade of retrograde transport of [1251]ICPbinding sites"by intracerebroventricular 6-OHDA Two groups of 6-8 rats were injected with 2 ktl 6-OHDA in the left LC bundle 2 days before sacrifice. One group was also injected with intracerebroventricular 6-OHDA (i.c.v., 6-OHDA; 250/~g in 20/~1 saline) 1 day before sacrifice to destroy noradrenergic neurons of the LC. Norepinephrine levels were measured in frontal cortex (ng/g wet weight tissue) and total specific binding of 100 pM [1251]ICP to injected (left) and uninjected (right) hypothalamic membranes taken distal to the 2-day LC bundle lesion was assessed in fmol/mg protein. Data are mean + S.E. Transport = accumulation of [125I]ICPbinding sites distal to the left-sided lesion determined by subtracting binding on the right from the left sides.
Control i.c.v.,6-OHDA
Cortex NE
Hypothalamic [1251]1CPbinding
(ng/g)
Left
Right
330 + 24 88 _+ 11"
5.15 _+ 0.73 3.86 + 0.50
3.74 + 1/.32 4.15 _+ 0.17
* P < 0.01 when comparable values in 6-OHDA-treated rats were compared to controls.
Transport 1.42 _+ 0.45 ---0.29 + 0.41"
107
400
3°°I HYPOTHALA MUS llt
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Fig. 3. Effect of reserpine on frontal cortex NE levels and [3H]DHA binding. Groups of 10-20 rats were injected with saline (0 days = control) or reserpine (5 mg/kg i.p.) and sacrificed at various time intervals (7 days-8 weeks) after reserpine injection. Total specific fl-adrenoreceptor binding in fmol/mg membrane protein was assessed using 2 nM [3H]DHA. NE values are #tg/g wet weight tissue. Values represent mean + S.E. (vertical bars). * P < 0.05, ** P < 0.001 when values from reserpine-treated rats were compared to saline-injected controls,
cortex. Binding decreased to 60% of control at 7 days rose to 214% at 21 days and r e t u r n e d to control levels by 6 weeks after reserpine injection. This general pattern of binding to hypothalamic m e m b r a n e s was a p p r o x i m a t e l y m i r r o r e d for the first 4 weeks after reserpine by the accumulation of [12sI]ICP binding sites (Bmax) distal to a left-sided lesion in the LC pathway (Fig. 4). This accumulation ( r e t r o g r a d e transport) fell gradually to a nadir at 14 days to 15% of control and then increased over the next 7 days and reached 470% of control by 21 days post-reserpine. Retrograde transport then decreased over the next 3 weeks, so that at 6 weeks it was completely abolished. A secondary increase to 240% of control occurred at 8 weeks and by 9 weeks after reserpine administration, r e t r o g r a d e transport had r e t u r n e d again to control levels (not shown in Fig. 4). A t no time after reserpine injection did the affinity for [125I]ICP binding (Ka) change distal to the lesion site on the left side, suggesting that r e t r o g r a d e l y trans-
14
DAYS
i
21
i
2e
'
'
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WEEKS
Fig. 4. Effect of reserpine on the binding of [125I]ICP to hypothalamic membranes and the retrograde transport of fl-recep-
tor sites. Groups of 6-12 rats were sacrificed 7 days to 8 weeks after 5 mg/kg, i.p. injections of reserpine. Additionally, rats received 6-OHDA in the left ascending LC pathway 2 days prior to sacrifice. Data points represent the mean + S.E, (vertical bars) percent of control Bmax values as determined by Scatchard analysis of saturation isotherms from incubation of various concentrations (25-250 pM) of [125I]ICPwith hypothalamic membranes from injected (left) and uninjected (right) sides. 'Hypothalamic' values are from the uninjected right side where control values were: Ka = 132 + 14 pM; Bmax = 34.5 -+ 1.9 , fmol/mg protein. Retrograde transport represents the leftright difference where saline-injected control values were: 7.9 + 0.8 fmol/mg protein, * P < 0.05 or less where values from reserpine-treated rats were compared to saline-injected controls.
p o r t e d receptors showed no alteration in their affinity state from control. It was possible that the increases in frontal cortex binding of [3H]DHA at 7-14 days and 28 days postreserpine were due, to some degree, to changes in the n u m b e r of presynaptic fl-receptors undergoing retrograde and anterograde transport in the presynaptic noradrenergic axons innervating the frontal cortex. Since the total n u m b e r of receptors measured should reflect the algebraic sum of receptors transported into and out of the terminals, plus those present on postsynaptic cells, a n t e r o g r a d e transport of flreceptors was also examined at various time intervals after the injection of reserpine (Table II). A n t e r o grade transport of fl-receptors (accumulation of [3H]DHA binding sites proximal to a 6 - O H D A lesion placed in the left ascending LC pathway 1 day prior
108 TABLE II Anterograde axonal transport of [3H]DHA binding sites Groups of 6-19 rats were injected with saline (control) or reserpine (5 mg/kg, i.p.) at various intervals (7 days-8 weeks) before sacrifice and with 6-OHDA in the left ascending LC pathway 1 day prior to death. Axonal transport was assessed by the difference in accumulation of total specific binding of 30 nM [3H]DHA proximal to the lesion site (left) compared to the comparable uninjected (right) side.
Control 7 days 14 days 21 days 28days 6weeks 8weeks
{~H]DHA transport (fmol/mg protein)
Percent of control
13.(I _+ 5.3 --2.0 _+ 2.2* --3.2 _+ 4.1" 42.(I _+ 5.(/** 25.7 _+ 4.6* --5.3 _+ 2.5* 12.8 _+ 2.8
-0 0 323 198 (} 98
* P < 0.05, ** P < 0.01 when transport (accumulation) in reserpine-treated animals was compared to controls where data represent the mean + S.E.
to sacrifice) was altered in a pattern very similar to that seen for retrograde transport following reserpine administration. A n t e r o g r a d e transport was completely blocked from 7 to 14 days after reserpine, rose to 320% of control at 21 days, decreased to complete blockade at 6 weeks and returned to control levels by 8 weeks post-reserpine. Thus, the presynaptic retrograde and a n t e r o g r a d e transport of fl-receptors a p p e a r e d to be independently regulated when c o m p a r e d to frontal cortex fl-receptors (which were presumably located on postsynaptic cells) in response to alterations in N E content p r o d u c e d by reserpine administration. DISCUSSION The present studies confirm the previous observation that anterograde axonal transport of fl-adrenoreceptors occurs in presynaptic noradrenergic neurons in the rat brain 25. Additionally, retrograde transport of these receptors in the same neurons also appears to be present since the linear accumulation of binding sites for the fl-adrenergic ligand [125I]ICP was shown to occur over a 3 day period distal to a lesion placed in the pathway. This accumulation could be blocked by destruction of these noradrenergic axons by intraventricular 6 - O H D A given 1 day prior to sacrifice, confirming the presynaptic location of this
transport. Although no attempt was m a d e here to define the subtype of fl-receptor, it is assumed that retrogradely transported, like anterogradely transported fl-receptors, were also of the ill-subtype25. It must be pointed out that the m e a s u r e m e n t of accumulation of receptor binding sites proximal or distal to an axonal lesion may be influenced by certain experimental artifacts. For example, the methods used here obviously require that the axon be injured. As a consequence, a certain amount of accumulated material will turn a r o u n d at the lesion site and return towards its site of origin 4,5.53. This makes calculation of actual transport rate difficult, although the measurement of total accumulation of a substance gives a fairly good indication of true transport if the time between the axonal injury and the m e a s u r e m e n t of that substance's accumulation is kept short. For this reason, a 2 day period was used for retrograde, and a 1 day period for anterograde transport, i.e. times at which left-right differences were significant and accumulation of receptors was still occurring in a linear fashion 24. This minimized both the effects of turnaround at the lesion site, and the effects of axonal injury on cell metabolism and axonal transport. A n o t h er reason for using this shorter time period of 2 days is that, after 3-4 days, the levels of [1251]ICP binding began to increase in the uninjected right hypothalamus. This later change appears to be due to some generalized, but at yet unexplained, p h e n o m e n o n in which unilateral lesions can affect function in contralateral brain noradrenergic neurons 26,3s. Similar changes in contralaterai hypothalamic levels of N E and of dopamine-fl-hydroxylase and tyrosine hydroxylase activities also occur after c o m p a r a b l e unilateral lesions in the ascending LC pathway (unpublished results). Reserpine had a surprisingly long lasting effect on both the presynaptic anterograde and retrograde transport of fl-receptors, as well as on frontal cortex levels of N E and fl-receptor number. It is uncertain what mechanism adequately explains the long duration of changes evoked by a single dose of reserpine. It is interesting that the effect on cortical N E levels was biphasic, showing two periods of decline separated by a transient return to control levels. Similar biphasic and triphasic changes in the anterograde transport and cell body synthesis of various subclasses of proteins 23 and NE synthesizing en-
109 zymes 2,24,36,37,39,58 have been documented in this
same model system over a 3 week period. It is unclear whether the even longer term effects of reserpine seen here were related to a chain of events initially triggered by the depletion of brain NE caused by the reserpine-induced destruction of NE storage vesiclesS,50, or to the other well-described, non-specific effects of reserpine upon cell metabolism15. 33. Since it is presumed that the neuronal cell body must change its metabolic priorities to synthesize new vesicles and NE-synthesizing enzymes, it is perhaps not surprising that the initial effects of reserpine might initiate a series of long lasting alterations in the metabolic function of the noradrenergic cell. Furthermore, since reserpine was shown here, and has been previously described to affect what appear to be primarily postsynaptic fl-receptors in the frontal cortex 41 innervated by noradrenergic LC neurons (see below), these postsynaptic cortical cells might also be expected to reciprocally influence the presynaptic noradrenergic neuron by means of feedback regulation of some sort. Such a feedback system has been well described in the nigro-striatal system 6. A major finding of this study was the demonstration the fl-adrenergic receptors in presynaptic noradrenergic neurons and fl-adrenoreceptors on cortical cells innervated by these neurons were independently regulated in response to reserpine administration. Several lines of evidence point to a postsynaptic location for most of the fl-receptors undergoing change in response to reserpine here. First, it has been shown that the total number of cortical fl-receptors actually increases when presynaptic noradrenergic innervation is removed 13,41,44,49. Second, cortical fl-receptors and fl-receptor-mediated adenylate cyclase either develop normally or are increased in the absence of presynaptic noradrenergic input in neonatal rats ll and in cortical cells raised in tissue culture 12. We have also found that destruction of presynaptic noradrenergic neurons with intraventricular 6OHDA, given 1 day prior to sacrifice, did not alter the increased number of cortical fl-receptors seen at 7 and 28 days after reserpine administration (Levin, B.E. and Biegon, A., unpublished observation). Finally, the present study showed that, while the patterns of anterograde and retrograde transport of presynaptic fl-receptors tended to parallel each other after reserpine administration, the changes in cortical fl-re-
ceptor binding followed an almost totally different pattern. Another interesting observation was that the inverse relationship between frontal cortical binding of [3H]DHA and levels of NE appeared to become dissociated at 6 weeks post-reserpine. At that time, both parameters were reduced to half of control levels and presynaptic retrograde and anterograde transport of fl-receptors was completely blocked. While unexplained, this finding suggests that the secondary decrease in cortical fl-receptor binding seen at this time was regulated by factors other than presynaptic levels of NE. From the preceding discussion it is obvious that the numerical contribution of presynaptic fl-receptors located on noradrenergic terminals in the cerebral cortex to the total population of cortical fl-receptors must be extremely small. Where anterograde transport of fl-receptors was completely blocked following reserpine (7-14 days), retrograde transport was still present to some degree. In opposite manner, the 220% increase above baseline seen for anterograde receptor transport at 21 days post-reserpine was matched by an even greater relative increase of 370% in retrograde transport. Finally, following complete blockade of both anterograde and retrograde flreceptor transport at 6 weeks, retrograde transport rose to 240% of control at 8 weeks post-reserpine at a time when anterograde transport had returned to control levels. Therefore, in almost every,case, the relative increases in retrograde transport were always greater than those for anterograde transport. However, under control conditions, the relative proportion of fl-receptors moving in an anterograde direction (25% per day of the intrinsic population of receptors) 25 is twice that of those moving in a retrograde direction (26% per 2 days; see Figs. 1 and 2). Presumably then, the greater anterograde transport of receptors was balanced by a smaller retrograde transport away from, plus the degradation of receptors at the nerve terminals. Therefore, under conditions of metabolic alterations of the neuronal milieu, such as was seen in the current studies, these three factors appear capable of changing in such a manner as to maintain an equilibrium of receptors at the presynaptic terminals. A final observation was that hypothalamic binding of fl-receptors did not follow the same pattern of changes in response to reserpine as did cortical fl-re-
110 ceptors. This was not simply due to differences in the ligands since the pattern of right hypothalamic binding of [3H]DHA in the anterograde transport experiments was similar to that seen for [125I]ICP binding in Fig. 4 (data not shown). While hypothalamic N E levels were not measured, there are at least two reasons why regulation of cortical and hypothalamic fl-receptors might not be expected to be similar. First, the hypothalamus receives nodradrenergic input from both the LC and from more caudally placed N E neurons in the brainstem, whereas the frontal cortex receives primarily LC input aone 27.34. Since reserpine may affect anatomically discrete groups of neurons containing the same neurotransmitter differently 36, regulation of fl-receptor binding in areas receiving differing noradrenergic input might be expected to reflect this fact. Second, the hypothalamus contains approximately equal numbers of ill- and fl2-adrenoreceptors 25 while the frontal cortex contains primarily ill-receptors32. While it is not known whether reserpine differentially affects the two receptor subtypes, it is quite possible that such differences do exist and would therefore be reflected in the pattern of receptor binding seen using non-specific fl-receptor ligands such as [3H]DHA or [125I]ICP.
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